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https://openalex.org/W2077795443
https://escholarship.org/content/qt93j0d05v/qt93j0d05v.pdf?t=n4fgx4
English
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Sensitivity of tracer transports and stratospheric ozone to sea surface temperature patterns in the doubled CO<sub>2</sub> climate
Journal of geophysical research
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cc-by
12,533
Peer reviewed Peer reviewed UC Irvine Faculty Publications Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ UC Irvine Faculty Publications Title Sensitivity of tracer transports and stratospheric ozone to sea surface temperature patterns in the doubled CO 2 climate Permalink https://escholarship.org/uc/item/93j0d05v Journal Journal of Geophysical Research, 107(D24) ISSN 0148-0227 Author Rind, David Publication Date 2002 DOI 10.1029/2002JD002483 Copyright Information This work is made available under the terms of a Creative Commons Attribution License availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine Faculty Publications Title Sensitivity of tracer transports and stratospheric ozone to sea surface temperature patterns in the doubled CO 2 climate Permalink https://escholarship.org/uc/item/93j0d05v Journal Journal of Geophysical Research, 107(D24) ISSN 0148-0227 Author Rind, David Publication Date 2002 DOI 10.1029/2002JD002483 Copyright Information This work is made available under the terms of a Creative Commons Attribution License availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine Faculty Publications Title Sensitivity of tracer transports and stratospheric ozone to sea surface temperature patterns in the doubled CO 2 climate Permalink https://escholarship.org/uc/item/93j0d05v Journal Journal of Geophysical Research, 107(D24) ISSN 0148-0227 Author Rind, David Publication Date 2002 DOI 10.1029/2002JD002483 Copyright Information This work is made available under the terms of a Creative Commons Attribution Lice availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine Faculty Publications Title Sensitivity of tracer transports and stratospheric ozone to sea surface temperature patterns in the doubled CO 2 climate Permalink https://escholarship.org/uc/item/93j0d05v Journal Journal of Geophysical Research, 107(D24) ISSN 0148-0227 Author Rind, David Publication Date 2002 DOI 10.1029/2002JD002483 Copyright Information This work is made available under the terms of a Creative Commons Attribution Lice availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed Michael Prather Earth Systems Science Department, University of California, Irvine, Irvine, California, USA Received 26 April 2002; revised 30 July 2002; accepted 1 August 2002; published 28 December 2002. [1] Two sets of sea surface temperature/sea ice changes are used to test the sensitivity of tracer transport to the pattern of warming in the doubled CO2 climate. One set (2CO2WT) has greater tropical and high latitude sea surface temperature changes than the other (2CO2), although both fall within the range of plausible response. Simulations were done both with and without interactive ozone. Results show that the SST pattern affects the circulation change throughout the troposphere and middle atmosphere; the ozone interaction affects primarily the upper stratosphere, but through wave-mean flow interaction has effects that extend down into the upper troposphere. Both experiments feature increased tropospheric/stratospheric exchange at low latitudes and greater vertical mixing within the troposphere; only the WT experiments result in increased interhemispheric transport and a more direct circulation in the high latitude stratosphere. Ozone increases in the upper stratosphere and decreases in the lower stratosphere in all the simulations, with greater transport of high latitude ozone into the troposphere in the WT runs. At sea level there is a more positive phase of the Arctic Oscillation (AO)-type oscillation, and this is also true at 100 mbar, but there is no significance in the middle troposphere and the sign is different in the middle stratosphere. Many of these results differ from those generated in older versions of the GISS GCMAM despite the same SST forcing due to differences in control run characteristics, which has implications for model intercomparison experiments. INDEX TERMS: 0341 Atmospheric Composition and Structure: p p Middle atmosphere—constituent transport and chemistry (3334); 0368 Atmospheric Composition and Structure: Troposphere—constituent transport and chemistry; 1620 Global Change: Climate dynamics (3309); 3334 Meteorology and Atmospheric Dynamics: Middle atmosphere dynamics (0341, 0342); KEYWORDS: tracer transports and climate, stratosphere and climate change, climate change and ozone, future SSTs and tracer transports Citation: Rind, D., J. Lerner, J. Perwitz, C. McLinden, and M. Prather, Sensitivity of tracer transports and stratospheric ozone to sea surface temperature patterns in the doubled CO2 climate, J. Geophys. Res., 107(D24), 4800, doi:10.1029/2002JD002483, 2002. Copyright 2002 by the American Geophysical Union. 0148-0227/02/2002JD002483 Sensitivity of tracer transports and stratospheric ozone to sea surface temperature patterns in the doubled CO2 climate David Rind, Jean Lerner, and Judith Perlwitz Goddard Institute for Space Studies at Columbia University, New York, New York, USA David Rind, Jean Lerner, and Judith Perlwitz Goddard Institute for Space Studies at Columbia University, New York, New York, USA David Rind, Jean Lerner, and Judith Perlwitz Goddard Institute for Space Studies at Columbia University, New York, New York, USA Chris McLinden Air Quality Research Branch, Meteorological Service of Canada, Downsview, Ontario, Canada Chris McLinden Air Quality Research Branch, Meteorological Service of Canada, Downsview, Ontario, Canada Powered by the California Digital Library University of California Powered by the California Digital Library University of California eScholarship.org JOURNAL OF GEOPHYSICAL RESEARCH, VOL. 107, NO. D24, 4800, doi:10.1029/2002JD002483, 200 2. Model and Experiments These limitations are removed here, as we use two of the sea surface temperature/sea ice changes in the same (newer) version of the model with on-line tracers, and compare their effects directly. [4] These comparisons were based on the overt meteoro- logical responses, and they did not explicitly calculate what the effect would be on the tracer transports themselves. Furthermore, the latest experiment had used a newer version of the GISS Global Climate/Middle Atmosphere Model (GCMAM), so model dependence could not be separated from the effect of different sea surface temperature patterns. These limitations are removed here, as we use two of the sea surface temperature/sea ice changes in the same (newer) version of the model with on-line tracers, and compare their effects directly. [5] To the extent that altered depictions of tropospheric climate change produce different dynamical responses extending into the stratosphere, the effect of the doubled CO2 climate on stratospheric ozone and its transport into the troposphere might also be different. Our previous investigations of the response of ozone to increased CO2 [Rind et al., 1998; Shindell et al., 1998] all used the older version of the GCMAM [Rind et al., 1988]; how would it differ with the newer model, and how would it depend on the sea surface temperature changes? Furthermore, the older approach did not allow the ozone changes to be advected, a limitation which precluded investigation of changes in stratospheric/tropospheric transport. These questions are addressed briefly in this paper, and more extensively in a companion paper (C.A. McLinden et al., manuscript in preparation, 2002) as the linearized ozone photochemistry scheme (LINOZ) [McLinden et al., 2000], including online transports, is incorporated into these experiments. [9] Although the second-order moments advection scheme [Prather, 1986] captures finer resolution effects than would be apparent from the given 4  5 horizontal resolution, this coarse grid is not likely to capture the physical intricacies of stratosphere/troposphere exchange associated with fine-scale tropopause folds. Overall, the troposphere/stratospheric exchange is similar in this model to the previous 4  5 versions of the stratospheric model, which were shown to be produce generally realistic magni- tudes of transport [Rind et al., 1999]. The model’s large- scale and eddy processes appear to be producing overall [6] Finally, the resulting changes in ozone distributions themselves might alter atmospheric dynamics. That issue is investigated by running the doubled CO2/LINOZ simula- tions in two ways. 1. Introduction used in that experiment, the subtropical residual circulation intensified in the upper troposphere/lower stratosphere, increasing the rate of tropical transfer of tropospheric air mass and constituents into the stratosphere by about 30%. Transport from the stratosphere to troposphere at higher latitudes also increased as the high latitude residual circu- lation intensified, so that the stratospheric residence time of bomb 14C decreased by about 11%. Within the troposphere, interhemispheric transport was reduced by 5% due to decreased Hadley Cell intensity; greater poleward transport occurred within the northern hemisphere due to eddy/Ferrel Cell changes; and greater vertical transport took place from low levels to the middle and upper troposphere via increased penetrating and overall convective mass fluxes. [2] The possibility of increased atmospheric CO2 affect- ing atmospheric dynamics has many ramifications, includ- ing effects such as changes in the phase of the Arctic Oscillation (AO) [e.g., Shindell et al., 1999; Fyfe et al., 1999; Monahan et al., 2000; Perlwitz et al., 2000], and altered distributions of tropospheric and stratospheric trace gases. In a recent publication [Rind et al., 2001], we investigated the impact of the doubled CO2 climate on various tracer transports. In the doubled CO2 climate change 25 - 1 25 - 1 ACL RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS ACL 25 - 2 In the discussion (section 7) we comment on which results depended on the SST field, and which were affected by the stratospheric ozone changes; we also include a discussion of potential inter-model differences by comparison to the results given by Rind et al. [1998, 2001]. A summary of the main results is given in the conclusions (section 8). ACL 25 - 2 [3] In that publication, and in work by Rind et al. [2002], the generality of these doubled CO2 results were assessed by comparing the meteorological changes in the latest simulation to those generated with different sea surface temperature changes [Rind et al., 1990, 1998]. The two effects that were the most robust were the increase in the subtropical residual circulation, and the increase in pene- trating convection to the upper troposphere, although the magnitude of their increase depended on the degree of tropical warming. Increases in transport from the surface to the middle troposphere depended on the convection parameterization, while changes in the Hadley circulation and the residual circulation in the polar stratosphere depended on the latitudinal distributions of sea surface temperature change. Hadley Cell changes with increasing CO2 have been known to vary from model to model (e.g., Dai et al. [2001] with the NCAR CCM found the Hadley Cell weakened by 10% by 2090, Ramstein et al. [1998] with the LMD model also found a 10% weakening with doubled CO2, while Senior [1995] found an increased Hadley cell with the Hadley Centre model run at 250 km resolution, and no change at 500 km resolution). atmospheric and tracer transport response depend on the altered sea surface temperatures, and which are affected by the ozone response. p [7] Following the introduction and model/experiment description (section 2), the different foci of this paper are presented sequentially: meteorological effects of the differ- ent SSTs (section 3), the effect on tracer transports (section 4), and on ozone (section 5), followed by the dynamical influence of the interactive ozone response (section 6). In the discussion (section 7) we comment on which results depended on the SST field, and which were affected by the stratospheric ozone changes; we also include a discussion of potential inter-model differences by comparison to the results given by Rind et al. [1998, 2001]. A summary of the main results is given in the conclusions (section 8). [7] Following the introduction and model/experiment description (section 2), the different foci of this paper are presented sequentially: meteorological effects of the differ- ent SSTs (section 3), the effect on tracer transports (section 4), and on ozone (section 5), followed by the dynamical influence of the interactive ozone response (section 6). 2. Model and Experiments [8] The model used for these simulations is the GISS GCMAM, model II0 with some alterations. In previous studies, several specific model weaknesses were identified with the previous 31-layer version of the model [Douglass et al., 1999; Pawson et al., 2000]. Chief among these was a weaker than observed subtropical barrier to tracer transport in the stratosphere, insufficient vertical resolution in the lower stratosphere, reduced stratospheric winter variability, and reduced vertical transport to the upper stratosphere. In comparison to the previous version [e.g., Pawson et al., 2000], the model now has 53 layers in the vertical, in conjunction with 4  5 resolution. The increased vertical layering provides for 500 m resolution in the middle to upper troposphere, 0.5 to 1 km resolution in the lower stratosphere, sufficient to resolve important gradients in that region [Douglass et al., 1999], and 2 to 2.5 km in the upper stratosphere. This layering can be compared with the 1.5 to 2 km resolution between 2 and 20 km used in the previous, 31-layer version of the model. The fourth order momentum scheme [Rind and Lerner, 1996] used in the 31-layer model was found to produce a tendency for spurious polar east- erlies in the upper troposphere, which had the effect of limiting interannual variability, and also altering planetary wave refraction so as to reduce the stratospheric jet inten- sity. It is replaced in this model by a second order momen- tum scheme, which cured both of these problems. These changes also ameliorated the leakiness of the subtropical barrier, and the smaller than observed transport into the upper stratosphere. However, the problems still remain to some extent and may quantitatively affect the results; the mean age of air at 20 km varies from 1.5 years in the tropics to 3.5 years at 60N, compared with 1 year in the tropics to 4 years at 60N in observations [Andrews et al., 2001]. [4] These comparisons were based on the overt meteoro- logical responses, and they did not explicitly calculate what the effect would be on the tracer transports themselves. Furthermore, the latest experiment had used a newer version of the GISS Global Climate/Middle Atmosphere Model (GCMAM), so model dependence could not be separated from the effect of different sea surface temperature patterns. RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS ACL 25 - 2 ACL 25 - 2 ACL 25 - 3 replicas of real-world processes without all the details; the climate change effect of such differences can only be gauged by future experiments with finer resolution models. [10] For the doubled CO2 experiments, we continue the approach used by Rind et al. [2001] and input altered sea surface temperature/sea ice fields from previous equilibrium experiments. The atmospheric CO2 is doubled as well. The first set of experiments (called ‘‘2 CO2’’) uses the same doubled CO2 changes in sea surface temperatures as given by Rind et al. [2001], which were originally generated by a nine-layer version of the GISS model II0. The control runs and the experiments are run both with and without the ozone changes interacting with the radiation field (see Table 1). The second set of experiments uses the sea surface temperatures/sea ice field generated by an equilibrium run of the older version of the GISS GCMAM [Rind et al., 1998]. In both cases, fixed dynamical ocean transport in conjunction with a mixed layer ocean (a ‘‘q-flux ocean’’) was used in the original simulations to allow the sea surface temperatures to change. ACL 25 - 3 ACL 25 - 3 Table 1. Simulations Used in This Study SSTs/Sea Ice Ozone Control current noninteractive Control (I) current interactive 2CO2 Rind et al. [2001] noninteractive 2CO2 (I) Rind et al. [2001] interactive 2CO2WT Rind et al. [1998] noninteractive 2CO2WT(I) Rind et al. [1998] interactive 2CO2WT is slightly higher than the IPCC nominal range of 1.5–4.5C (as shown in Table 2). [13] In addition to ozone, the other online tracers used were those discussed by Rind et al. [2001]: CO2, SF6, Rn222, CFC-11, CH4, N2O, and bomb 14C. The sources/ sinks of each of these tracers is given by Rind et al. [2001]; the stratospheric photochemical sinks for CFC-11, CH4 and N2O were calculated in a consistent manner with the LINOZ photochemistry. [14] The control runs and experiments were integrated for 12 years with results reported for 10 year averages. In addition, we investigated whether the differences seen during five years in the first half of the simulations were similar to those during the last half, and discuss only those which had consistent changes for the two time periods. A complete list of the control runs and doubled CO2 simu- lations is given in Table 1. replicas of real-world processes without all the details; the climate change effect of such differences can only be gauged by future experiments with finer resolution models. [10] For the doubled CO2 experiments, we continue the approach used by Rind et al. [2001] and input altered sea surface temperature/sea ice fields from previous equilibrium experiments. The atmospheric CO2 is doubled as well. The first set of experiments (called ‘‘2 CO2’’) uses the same doubled CO2 changes in sea surface temperatures as given by Rind et al. [2001], which were originally generated by a nine-layer version of the GISS model II0. The control runs and the experiments are run both with and without the ozone changes interacting with the radiation field (see Table 1). The second set of experiments uses the sea surface temperatures/sea ice field generated by an equilibrium run of the older version of the GISS GCMAM [Rind et al., 1998]. In both cases, fixed dynamical ocean transport in conjunction with a mixed layer ocean (a ‘‘q-flux ocean’’) was used in the original simulations to allow the sea surface temperatures to change. 2. Model and Experiments In the first, the model is run with a climatological ozone field for atmospheric radiation purpo- ses, and in those experiments the on-line ozone is a passive tracer. In the second, we substitute the LINOZ-produced ozone values in the radiation routine, so the doubled CO2 ozone changes interact with the climate. The complete set of experiments allows us to separate which features of the RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS ACL 25 - 3 3. Meteorological Effects of the Different Sea Surface Temperature Patterns [15] The different sets of sea surface temperatures help generate different meteorological responses. We concentrate on those phenomena deemed responsible for changes in transports, and the dynamics discussion is focused on the Northern Hemisphere. To indicate the robustness of the conclusions, and/or the impact of the interactive ozone, we Figure 1a. The marine surface temperature (input sea surface temperature plus calculated sea ice temperature) anomaly in the two experiments ((top) 2CO2 and (bottom) 2CO2WT). [11] The marine surface (sea surface plus sea ice) temper- ature differences for the individual experiments are shown in Figure 1, with the resulting surface air temperature changes (for the noninteractive experiments) shown in Figure 1b. Since the sea surface temperatures from Rind et al. [1998] featured a warmer tropical temperature response, experiments which used those values are called 2CO2 WT (for warmer tropics), although the runs also feature warmer high latitude temperatures. g p [12] Which SST pattern is more realistic? The differences are largely associated with a change in the cloud cover parameterization and cloud feedbacks [Rind et al., 2002]. The tropical warming in the two simulations in general varies between 2.5 and 4C (Figure 1a). Yao and Del Genio [1999] show that such variations can be generated by differences in the parameterized cloud feedback within the range of current uncertainty. The smallest response occurs when thick anvil clouds arise in association with tropical convection (reducing solar insolation reaching the surface) and no cloud optical thickness feedback is allowed. The greatest warming occurs with the reverse situation: Optical thickness is predicted (and decreases in the tropics as climate warms due to water depletion by precipitation), while anvil clouds are not included. Yao and Del Genio [1999] argue that the optical thickness feedback is sup- ported by observations, while the realism of the anvil detrainment feedback is more difficult to assess. That would suggest that a higher tropical sensitivity might be more realistic if one had to choose between the two, although numerous processes (including other cloud parameteriza- tions) affect the overall and tropical sensitivity. At this point no conclusion can be definitive. The overall warming in Figure 1a. The marine surface temperature (input sea surface temperature plus calculated sea ice temperature) anomaly in the two experiments ((top) 2CO2 and (bottom) 2CO2WT). RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS ACL Figure 1b. 3. Meteorological Effects of the Different Sea Surface Temperature Patterns As in Figure 1a except for surface air temperature changes calculated in the experiments. cooled considerably. Given the magnitude of the imbalance, we estimate that this model has an equilibrium climate sensitivity for doubled CO2 of about 2.65C, or about 0.66C/Wm2. [17] Of relevance for interhemispheric transport in the troposphere, the Hadley Cell during Northern Hemisphere winter is intensified in the 2CO2WT runs, with little change in 2CO2. Of relevance for intrahemispheric transport, plan- etary longwave energy increases substantially in the 2CO2WT runs, although eddy kinetic energy in general shows only small changes; again there is little change in either of these quantities in the 2CO2 simulations. Concern- ing vertical mixing within the troposphere, penetrative convective mass flux increases in all the doubled CO2 simulations, more so in 2CO2WT, though total convective mass flux decreases somewhat in all the experiments, again more so in the WT runs. With increased penetrative con- vection, there is the possibility of reducing the time-aver- aged instability, decreasing more shallow convection. [18] Shown in Figure 2 are the temperature changes in the different experiments, with tropospheric warming and stra- tospheric cooling as expected. The prime difference is that the WT experiments have greater warming in the tropical upper troposphere (and at the tropical tropopause), consis- tent with their warmer sea surface temperatures. In addition, the WT runs have greater warming at high latitudes during winter (Figure 1b). Given in Figures 3a and 3b are the resulting changes in zonal wind and Eliassen-Palm (EP) fluxes (the zonally averaged wave energy fluxes normalized by the zonal wind). The WT runs have greater relative west wind increases from 45S to 45N in the upper troposphere and lower stratosphere; over this range of latitudes, all of the doubled CO2 simulations show relative equatorward prop- agation of EP fluxes in conjunction with these wind changes. At the highest latitudes, however, only 2CO2 shows west wind increases in the troposphere during North- ern Hemisphere winter, as the greater warming in the WT runs leads to greater relative east winds, and poleward wave refraction. The decrease in high latitude temperature gra- dient imposed in WT at the surface (Figure 1b) extended up to some 6.5 km altitude, reducing the zonal wind shear throughout that region. Figure 1b. As in Figure 1a except for surface air temperature changes calculated in the experiments. often show results from both the noninteractive and inter- active runs. 3. Meteorological Effects of the Different Sea Surface Temperature Patterns [16] Given in Table 2 are the annual, global average quantities of the relevant parameters (except for the peak stream function values, which are shown for the two solstice seasons). As an indication of the consistency of the results, we also show the average difference in the respective quantities over the two different 5-year periods (years 2–6 versus years 8–12). The 2CO2WT runs are considerably warmer than the 2CO2 simulations. The neg- ative value of the net radiation at the model top for all the doubled CO2 simulations show that had the sea surface temperatures been allowed to adjust, the runs would have [19] The EP flux divergence changes for the different simulations are shown in Figure 4. Associated with the relative equatorward EP flux refraction change, increased convergence occurs in the lower stratosphere in both hemi- Table 2. Annual Atmospheric Features in the Different Experiments Unit Control Control (I) 2CO2 2CO2 (I) 2CO2 WT 2CO2 WT(I) 5-Year  Global surf temp C 12.39 12.35 15.67 15.67 17.13 17.11 0.007 Sea ice coverage % 4.4 4.4 2.6 2.6 2.3 2.3 0 Air temperature C 25.33 25.30 22.62 22.55 20.87 20.87 0.013 Water vapor mm 20.2 20.1 24.6 24.6 27.4 27.3 0.03 Cloud cover % 68.8 68.7 66.3 66.2 65.3 65.1 0.03 Planetary albedo % 32.33 32.27 30.28 30.39 30.18 30.30 0.08 Net radiation at model top Wm2 0.1 0.4 1.1 1.5 3.5 3.9 0.016 Moist convective mass flux 109 Kgs1 1554 1568 1546 1550 1474 1483 1.5 Moist control mass flux at 195 mbar 109 Kgs1 141.3 145.5 222.8 232.0 278.7 290.0 0.6 Eddy kinetic energy 104 Jm2 64.9 65.0 63.5 64.2 67.2 67.5 0.27 EKE, wave numbers 1–4 1017 J 647 645 639 651 697 704 9.67 Peak D-F stream function 109 Kgs1 175 178 173 180 208 213 3 Peak J-A stream function 109 Kgs1 203 199 199 200 197 202 1.67 ACL 25 - 5 ACL 25 - 5 RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS Figure 2. Temperature changes in the different experiments, averaged over the last 10 years. Shown are the results for the annual average and the two solstice seasons for (top) 2CO2, (second row) 2CO2(I), (third row) 2CO2WT and (fourth row) 2CO2WT(I). Figure 2. Temperature changes in the different experiments, averaged over the last 10 years. 3. Meteorological Effects of the Different Sea Surface Temperature Patterns Shown are the results for the annual average and the two solstice seasons for (top) 2CO2, (second row) 2CO2(I), (third row) 2CO2WT and (fourth row) 2CO2WT(I). RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS ACL 25 - 6 ACL 25 - 6 spheres from 45S to 45N, more so in the WT runs. In addition, the wind changes in the upper troposphere/lower stratosphere alter the wave refraction and lead to greater EP poleward of 45N. In the extratropical troposphere, EP flux convergences are associated with the east wind change a the highest latitudes while divergences arise with the wes Figure 3. Change in the zonal winds (shading/contours) and EP fluxes (arrows) for the two solstice seasons in the different experiments (a) 2CO2 and 2CO2(I) and (b) 2CO2WT and 2CO2WT(I). urs) and EP fluxes (arrows) for the two solstice O2(I) and (b) 2CO2WT and 2CO2WT(I). Figure 3. Change in the zonal winds (shading/contours) and EP fluxes (arrows) for the two solstice seasons in the different experiments (a) 2CO2 and 2CO2(I) and (b) 2CO2WT and 2CO2WT(I). Figure 3. Change in the zonal winds (shading/contour seasons in the different experiments (a) 2CO2 and 2CO2 Figure 3. Change in the zonal winds (shading/contours) and EP fluxes (arrows) for the two solstice seasons in the different experiments (a) 2CO2 and 2CO2(I) and (b) 2CO2WT and 2CO2WT(I). poleward of 45N. In the extratropical troposphere, EP flux convergences are associated with the east wind change at the highest latitudes, while divergences arise with the west wind change at upper midlatitudes. These effects are also spheres from 45S to 45N, more so in the WT runs. In addition, the wind changes in the upper troposphere/lower stratosphere alter the wave refraction, and lead to greater EP flux convergences in the WT runs above 10 mbar primarily RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS ACL 25 - 7 greatest in the WT runs, and also somewhat larger in the interactive runs of each set. [20] Changes in the transformed Eulerian (residual) cir- culation are presented in Figure 5. The intensified EP flux convergences drive an increased residual circulation at low latitudes in all the doubled CO2 simulations, more so in the WT runs. 3. Meteorological Effects of the Different Sea Surface Temperature Patterns in the midstratosphere, while in 2CO2 there is greater cyclonicity like the positive phase of the Arctic Oscillation. [22] However, from the perspective of the sea level pressure (Figure 6, right) the results are very different. Now it appears qualitatively as if all the runs are producing the positive phase of the AO and North Atlantic Oscillation, the latter with lower pressure in the vicinity of the Icelandic Low, and relatively higher pressure near Portugal. This result illustrates that in a climate change situation one cannot necessarily expect the recent barotropic nature of AO response [Thompson et al., 2000] to prevail. We return to this point in the discussion section. in the midstratosphere, while in 2CO2 there is greater cyclonicity like the positive phase of the Arctic Oscillation. [21] These differences in high latitude response provide for a different pattern of geopotential response. The WT runs have increased 10 mbar heights around the pole in the winter hemisphere (Figure 6, left), while the 2CO2 runs have smaller changes or decreases. These results are con- sistent not only with the amount of high latitude warming, but also the EP flux convergence changes at high latitudes above 10 mbar with their generation of the residual circu- lation, i.e., EP flux convergence, a more direct circulation, and subsidence warming in the WT runs. From this per- spective, the WT runs have a change toward the negative phase of the Arctic Oscillation (AO) (weaker polar vortex) 3. Meteorological Effects of the Different Sea Surface Temperature Patterns At higher latitudes during Northern Hemisphere winter, the intensified EP flux convergences shown in Figure 4 above 10 mbar help the 2CO2WT runs drive a more direct circulation from 10 to 100 mbar via the down- Figure 3. (continued) Figure 3. (c greatest in the WT runs, and also somewhat larger in the interactive runs of each set. latitudes in all the doubled CO2 simulations, more so in the WT runs. At higher latitudes during Northern Hemisphere Figure 3. (continued) Figure 3. (continued) latitudes in all the doubled CO2 simulations, more so in the WT runs. At higher latitudes during Northern Hemisphere winter, the intensified EP flux convergences shown in Figure 4 above 10 mbar help the 2CO2WT runs drive a more direct circulation from 10 to 100 mbar via the down- greatest in the WT runs, and also somewhat larger in the interactive runs of each set. greatest in the WT runs, and also somewhat larger in the interactive runs of each set. [20] Changes in the transformed Eulerian (residual) cir- culation are presented in Figure 5. The intensified EP flux convergences drive an increased residual circulation at low ACL 25 - 8 25 - 8 ACL RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS Figure 4. Change in the EP flux divergence in the different experiments. Figure 4. Change in the EP flux divergence in the different experiments. in the midstratosphere, while in 2CO2 there is greater cyclonicity like the positive phase of the Arctic Oscillation. [22] However, from the perspective of the sea level pressure (Figure 6, right) the results are very different. Now it appears qualitatively as if all the runs are producing the positive phase of the AO and North Atlantic Oscillation, the latter with lower pressure in the vicinity of the Icelandic Low, and relatively higher pressure near Portugal. This result illustrates that in a climate change situation one cannot necessarily expect the recent barotropic nature of AO response [Thompson et al., 2000] to prevail. We return to this point in the discussion section. ward control principle [Haynes et al., 1991]. In contrast, the circulation is somewhat more indirect in the same region in 2CO2. In the troposphere, the more direct circulation at the highest latitudes follows the EP flux convergences, both being greatest in the WT simulations. Figure 6. Change in the (left) 10-mbar heights and (right) sea level pressure for December–January– February. Figure 6. Change in the (left) 10-mbar heights and (right) sea level pressure for December–January– February. 4. Changes in Transports [23] In this section, we present changes in the transports as discussed by Rind et al. [2001]. While 2CO2 uses the RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS ACL 25 - 9 ACL 25 - 9 Figure 5. Change in the transformed Eulerian (residual) stream function in the different experiments. A positive value indicates a relative counterclockwise change, to a more indirect (direct) circulation in the Northern Hemisphere (Southern Hemisphere). Figure 5. Change in the transformed Eulerian (residual) stream function in the different experiments. A positive value indicates a relative counterclockwise change, to a more indirect (direct) circulation in the Northern Hemisphere (Southern Hemisphere). RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS ACL 25 - 10 ACL ACL 25 - 11 ACL 25 - 11 ACL 25 - 11 Table 3. Interhemispheric Exchange Times (Years) in the Different Simulations SF6 CFC-11 CH4 Control 1.20 1.10 1.01 Control(I) 1.20 1.10 1.00 2CO2 1.21 1.15 1.00 2CO2(I) 1.20 1.13 1.00 2CO2WT 1.07 0.97 0.89 2CO2WT(I) 1.07 0.96 0.87 5-Year  0.04 0.007 0.007 Table 3. Interhemispheric Exchange Times (Years) in the Different Simulations simulations, while the increased downward transport in the subtropics is also evident. The global change is thus much less than the tropical upwelling component. 4.6. Transport Within the Stratosphere [30] Evident in both Figures 8a and 8b is a two-cell distribution for the annual average change in the Northern Hemisphere stratosphere, with relative upwelling at the pole and equator, and downwelling around 45N. Quantitatively the extratropical downwelling is greater in the WT runs as a result of the greater EP flux convergences above 10 mbar. The transport change is somewhat similar in the Southern Hemisphere except that the downwelling extends below 10 mbar only in the WT runs. 4.2. Intrahemispheric Transport [25] For the same species shown in Table 3, there is greater southward transport within the Northern Hemisphere by a few percent (relative to the hemispheric tracer mass) in 2CO2WT, due both to the intensified Hadley Cell in North- ern Hemisphere winter and the somewhat greater planetary longwave energy. There is little change in 2CO2. 4.5. Vertical Transport From the Stratosphere to the Troposphere [29] The change in the residual stream function shown in Figure 5 can be interpreted to imply that in all the doubled CO2 simulations, there is increased downward transport through the Northern Hemisphere lowermost stratosphere at high latitudes and at midlatitudes (around 45N) relative to the control run values (seen to some extent in Figure 7). To illustrate this more directly, given in Figure 8a is the change in SF6 transport due to the large-scale dynamics (hence not including convection) for 2CO2; results for 2CO2WT given in Figure 8b show qualitatively similar features in the extratropics. The circulation change is especially effective for bomb 14C which has its peak initial source around 50 mbar. Globally, downward transport increases by some 3–4% through 100 mbar, and by up to 10% through 240 mbar in the 2CO2WT experiments. This results in a shorter residence time for bomb 14C in the stratosphere, with the effects greatest in the 2CO2WT runs; averaged over 6 years, there is 5.5% less 14C in the atmosphere, with a decreased atmospheric residence time of 15–20%. Changes are smaller, but in the same direction, in the 2CO2 runs. As noted in the meteorology discussion, this in turn results from the greater relative EP flux convergences in the extratropics in driving a more direct circulation, especially in winter (Figure 5). fied Hadley Cell during Northern Hemisphere winter (Table 2) in the WT experiments. 4.3. Vertical Transport Within the Troposphere [26] With respect to the distribution of short-lived species such as Rn222, increased convection diminishes tropical gradients between low levels and the upper troposphere in all of the doubled CO2 runs, and diminishes the gradients between the surface and midtroposphere in 2CO2WT (despite the overall decrease in convective mass flux indicated in Table 2). At high latitudes, the increased warmth provides for an additional radon source, from previously frozen ground, and results in increased vertical gradients away from the surface, more so in the warmer (WT) simulations. 4.1. Interhemispheric Transport same sea surface temperatures and sea ice changes as in that paper, this model has 53 layers (as compared to 31), and several of the physics routines (boundary layer, momentum advection, radiation) have been changed. The degree to which the boundary conditions can enforce the same response in these two related, but somewhat different models, will be addressed in the discussion section. [24] Given in Table 3 are the interhemispheric exchange times for three of the species in the different simulations, as well as the differences between the two five year time periods. There is little change in the 2CO2 simulation, but interhemispheric exchange times decrease by 10–15% in 2CO2WT. This is qualitatively consistent with the intensi- [24] Given in Table 3 are the interhemispheric exchange times for three of the species in the different simulations, as well as the differences between the two five year time periods. There is little change in the 2CO2 simulation, but interhemispheric exchange times decrease by 10–15% in 2CO2WT. This is qualitatively consistent with the intensi- RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS 5. Changes in Ozone Distribution [32] The results presented in the previous section can be combined with the changes in ozone photochemistry to understand how ozone changed in these doubled CO2 simulations. Shown in Figure 10 are the percentage changes in ozone on the annual average in the different experiments. Increases in the tropical upper stratosphere are associated with the colder temperatures due to doubled CO2 and they do not depend on the different SST patterns, being similar in 2CO2 and 2CO2WT. The interactive runs have less ozone increase there, because as the ozone increases, the temper- ature does too, relatively, reducing the photochemical source. Therefore, in the upper stratosphere, while the ozone increase maximizes in the tropics in the noninteractive runs, the changes are of comparable value at low and high latitudes in the interactive simulations. [35] In a related development, below about 30 mbar, and extending down through the middle troposphere, the inter- active runs have greater EP flux convergences at high latitudes, greater relative east winds, and warmer temper- atures during Northern Hemisphere winter (Figures 2–4). This effect is the result of their weaker subtropical west wind increase in the upper stratosphere diverting less wave energy upward, allowing for greater convergences below. This greater high latitude warming results in a greater increase in 10 mbar height at the pole (more negative phase of the AO) in the interactive runs (most obvious in Figure 6 for the 2CO2 runs), an effect which extends down into the upper troposphere (see also Table 5, discussed below). [33] Elsewhere, the SSTs do affect the ozone distribution. Ozone decreases in the tropical lower stratosphere, partly due to reduced UV penetration and chemistry, partly the result of increased tropical upwelling bringing up low tropospheric ozone concentrations (note that the only tropo- spheric ozone in these simulations is that which arises from stratospheric transport). This latter effect is greater with the warmer tropical SSTs. 2CO2WT also features greater ozone increases at high latitudes and in the troposphere, due to its increased poleward and downward transport, as occurred for other species (e.g., methane, in Figure 9). The result is an increase in midtropospheric ozone from the stratospheric source in the 2CO2WT simulations, and a decrease in the 2CO2 runs (and for methane, with a tropospheric source, the result is just the opposite). 4.4. Vertical Transport From the Troposphere to the Stratosphere 4.4. Vertical Transport From the Troposphere to the Stratosphere [27] The ratios of upward transport from 20N to 20S between experiment and control for levels in the upper troposphere and lower stratosphere are given in Tables 4a and 4b. In the 2CO2 runs, upward transport increases by 10–30% for all the species at all the levels, associated with the intensified residual circulation at these altitudes (Figure 5). The transport increases by even larger percentages in the 2CO2WT simulations. [31] The change in CH4 distribution in the different runs is used to illustrate the effect of the altered circulation in Figure 9. Where air upwells, methane increases; regions of decrease in the Northern Hemisphere lower stratosphere and Southern Hemisphere middle stratosphere are associated with the relative downwelling. The stratospheric methane increase with greater flux from the troposphere is larger in the 2CO2WT experiments, which had the greater residual circulation change. Note that the increased interhemispheric transport in the troposphere (reduced Northern Hemisphere [28] Also shown is the ratio of the global transports (in brackets), and the values are much lower than the tropical component. This is because the intensified tropical circula- tion cells lead to increased downward transport as well. Shown in Figure 7 is the change in vertical transport of CH4 through 100 mbar for the four experiments; the other species show similar characteristics. The increased upward transport near the equator is apparent in all runs, greater with the WT Table 4a. Ratio of Vertical Transport Between 2CO2 and Control in the Tropics (20N to 20S) and Global (in Brackets) Pressure, mbar Run CO2 N2O CFC-11 CH4 5-Year  84 2CO2 1.19 [1.03] 1.19 [1.11] 1.18 [1.12] 1.19 [1.10] 0.032 [0.04] 2CO2(I) 1.16 [1.01] 1.17 [1.07] 1.16 [1.10] 1.16 [1.06] 0.005 [0.05] 100 2CO2 1.25 [1.03] 1.25 [1.11] 1.24 [1.11] 1.25 [1.09] 0.033 [0.04] 2CO2(I) 1.28 [1.02] 1.28 [1.07] 1.27 [1.09] 1.28 [1.05] 0.004 [0.05] 117 2CO2 1.20 [1.02] 1.20 [1.11] 1.18 [1.10] 1.19 [1.08] 0.005 [0.13] 2CO2(I) 1.27 [0.99] 1.27 [1.07] 1.26 [1.08] 1.27 [1.03] 0.029 [0.05] 135 2CO2 1.11 [0.97] 1.11 [1.12] 1.10 [1.08] 1.11 [1.06] 0.045 [0.04] 2CO2(I) 1.19 [0.95] 1.19 [1.08] 1.18 [1.06] 1.18 [1.00] 0.027 [0.06] RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS values relative to Southern Hemisphere) is also visible in the figure for the WT experiment. 4.4. Vertical Transport From the Troposphere to the Stratosphere gences) at low latitudes in the middle and upper stratosphere (Figure 4), and thus affects the tropical residual circulation Table 4b. Ratio of Vertical Transport Between 2CO2WT Experiments and Control in the Tropics (20N to 20S) and Global (in Brackets) Pressure, mbar Run CO2 N2O CFC-11 CH4 5-Year  84 2CO2WT 1.55 [1.05] 1.55 [1.26] 1.52 [1.26] 1.54 [1.22] 0.060 [0.09] 2CO2WT(I) 1.56 [1.04] 1.56 [1.17] 1.55 [1.21] 1.56 [1.15] 0.035 [0.08] 100 2CO2WT 1.74 [1.04] 1.74 [1.26] 1.69 [1.24] 1.73 [1.20] 0.036 [0.08] 2CO2WT(I) 1.82 [1.01] 1.83 [1.17] 1.79 1.19] 1.82 [1.12] 0.066 [0.04] 117 2CO2WT 1.64 [0.96] 1.64 [1.26] 1.60 [1.22] 1.63 [1.15] 0.011 [0.08] 2CO2WT(I) 1.74 [0.92] 1.74 [1.17] 1.70 [1.16] 1.73 [1.07] 0.066 [0.07] 135 2CO2WT 1.47 [0.87] 1.47 [1.26] 1.43 [1.19] 1.45 [1.05] 0.020 [0.06] 2CO2WT(I) 1.54 [0.83] 1.54 [1.17] 1.51 [1.14] 1.53 [0.97] 0.063 [0.07] Table 4b. Ratio of Vertical Transport Between 2CO2WT Experiments and Control in the Tropics (20N to 20S) and Global (in Brackets) ansport Between 2CO2WT Experiments and Control in the Tropics (20N to 20S) and Global (in values relative to Southern Hemisphere) is also visible in the figure for the WT experiment. gences) at low latitudes in the middle and upper stratosphere (Figure 4), and thus affects the tropical residual circulation in the lower stratosphere and upper troposphere. Hence the increase in this circulation is slightly weaker in the non- interactive runs below 100 mbar, as can be seen from the tracer transports in Tables 4a and 4b. However, the down- ward transport is also weaker (Figure 7), and so the global transport upward is actually greater in the noninteractive runs (Tables 4a and 4b). 5. Changes in Ozone Distribution [36] We can summarize the results presented above by showing how the age of air changed in the various experi- ments (Figure 11), as deduced from the SF6 tracer (as given by Rind et al. [2001]). The decrease in age of air throughout the stratosphere is greatest in the 2CO2WT runs with their intensified residual circulation, and the decrease is greater in the noninteractive runs associated with their greater net global upward transport (Table 4, again not due to greater tropical upwelling). The noticeable increase in interhemi- spheric transport in the 2CO2WT runs is evident in the troposphere as younger ages in the Southern Hemisphere, Figure 7. Change in annual vertical transport of CH4 through 100 mbar in the different experiments. RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS and to a lesser extent this appears to be true for portions of the troposphere in the 2CO2 runs as well, although that was not obvious from the hemispheric results given in Table 3. The difference relates to the increased age in the Southern Hemisphere tropical upper troposphere for the 2CO2 runs which, as can be seen from Figure 8a, is associated with an upper tropospheric vortex spanning the equator, with decreased age of air in the same region in the Northern Hemisphere. The effect is entirely missing in the 2CO2WT runs which have a coherent upward mass transport from low levels just south of the equator (Figure 8b). This distinction is related to the SST peak change south of the equator in the WT runs (Figure 1a). Greater downward transport from the stratosphere at extratropical northern latitudes in the 2CO2WT runs is indicated by the relative increase in age of air, especially relative to the strong decreases seen in the 2CO2 simulations. [39] The SST pattern, in particular the degree of tropical warmth, affects the heating in the upper troposphere and its latitudinal temperature gradient, the winds in the upper troposphere and lower stratosphere, wave energy refraction and convergence in the stratosphere, and hence the circu- lation and winds throughout the middle atmosphere. Clearly, to know what will happen in the Middle Atmosphere we need to know the pattern of prospective SST change. [40] The ozone response affects the magnitude of temper- ature change in the upper stratosphere, and the resulting latitudinal temperature gradient; this in turn affects wave energy propagation from the lower stratosphere, and wave energy convergences and circulation in the lower strato- sphere, with effects which extend down into the upper troposphere. These dynamical responses illustrate how cli- mate perturbations affecting the stratosphere can influence tropospheric processes due to wave-mean flow interactions. With respect to both the meteorology and the change in transports, the SST changes generally dominate the ozone influence, although the effects are comparable in some stratospheric regions. 6. Dynamical Effect of Interactive Ozone [34] The net effect of the greater tropical ozone increase in the noninteractive runs is to provide for warmer temper- atures in the tropical upper stratosphere, by about 2C. The warmer temperatures are then associated with a greater west wind increase in the subtropical stratosphere (e.g., see the 1 mbar wind change in Figures 3a and 3b); this affects the wave refraction and results in greater EP flux convergences (or smaller divergences) in the extratropical middle and upper stratosphere (e.g., between 10 and 1 mbar during Northern Hemisphere winter in Figure 4), and a more direct extratropical residual circulation in both winter hemispheres (Figure 5). This alteration in wave refraction pattern also produces greater EP flux divergences (or smaller conver- Figure 7. Change in annual vertical transport of CH4 through 100 mbar in the different experiments. RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS 7. Discussion [37] In this section we discuss a number of issues raised by these results as well as some popular issues associated with the response to increasing CO2 levels. RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS ACL 25 - 13 ACL 25 - 13 Figure 8. Schematic of the annual change in transport of SF6 by the large-scale dynamics (not including convection) for (a) 2CO2 and (b) 2CO2WT. Shaded region indicates the lowermost stratosphere (potential temperature less than 380 K, and potential vorticity greater than 2  105 k mbar1s1 outside the tropics and above 100 mbar in the tropics). To illustrate the altered pathways of tracer transport, all vectors are of unit length except in the tropical troposphere where the exceptionally large changes are shown as exaggerated arrows. Separate log pressure scales are used above and below 100 mbar for presentation purposes. Figure 8. Schematic of the annual change in transport of SF6 by the large-scale dynamics (not including convection) for (a) 2CO2 and (b) 2CO2WT. Shaded region indicates the lowermost stratosphere (potential temperature less than 380 K, and potential vorticity greater than 2  105 k mbar1s1 outside the tropics and above 100 mbar in the tropics). To illustrate the altered pathways of tracer transport, all vectors are of unit length except in the tropical troposphere where the exceptionally large changes are shown as exaggerated arrows. Separate log pressure scales are used above and below 100 mbar for presentation purposes. and to a lesser extent this appears to be true for portions of the troposphere in the 2CO2 runs as well, although that was not obvious from the hemispheric results given in Table 3. The difference relates to the increased age in the Southern Hemisphere tropical upper troposphere for the 2CO2 runs which, as can be seen from Figure 8a, is associated with an upper tropospheric vortex spanning the equator, with decreased age of air in the same region in the Northern Hemisphere. The effect is entirely missing in the 2CO2WT runs which have a coherent upward mass transport from low levels just south of the equator (Figure 8b). This distinction is related to the SST peak change south of the equator in the WT runs (Figure 1a). Greater downward transport from the stratosphere at extratropical northern latitudes in the 2CO2WT runs is indicated by the relative increase in age of air, especially relative to the strong decreases seen in the 2CO2 simulations. the 2CO2 and 2CO2WT runs), and what is influenced by the ozone changes (the differences between the interactive and noninteractive runs). 7.2. Intermodel Differences [38] The range of experiments allows us to address the issue of what in the troposphere/middle atmosphere system is influenced by the SST patterns (the differences between [41] A number of these results are consistent with expectations [Rind et al., 2001, 2002]. The upper tropo- RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS ACL Figure 9. Change in annual average methane in the different experiments. Figure 9. Change in annual average methane in the different experiments. sphere/stratosphere tropical residual circulation did increase in both climate simulations, and the magnitude of increase was greater with warmer sea surface temper- atures. These results were expected due to the influence of the tropical sea surface temperature warming on temper- atures in the tropical upper troposphere, and the subse- quent effect on west winds and planetary wave refraction at those altitudes. They support the assumption that increased transport from the troposphere to the strato- sphere would be a robust result with different amounts of tropical warming. However, the tracer transport changes showed that the tropical upwelling is only part of the story, for the global increase in transport into the stratosphere is much less than the tropical change. The difference is associated with increased downwelling in the subtropics that accompanied the intensified tropical residual circula- RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS ACL 25 - 15 RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS ACL 25 - 15 Figure 10. Change in annual average ozone (in percent) in the different experiments. Note that the tropospheric ozone and its change arises from stratospheric transport. Figure 10. Change in annual average ozone (in percent) in the different experiments. Note that the tropospheric ozone and its change arises from stratospheric transport. tion. This recycling of air within the 30N to 30S regime may be sensitive to the greater ‘‘leakiness’’ of the tropical pipe in the model compared to the real world. faster. The warmer tropical sea surface temperatures also led to a greater increase in penetrative convection and the corresponding increased transport of tracers to the upper troposphere, though that effect occurred in all the doubled CO2 runs. RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS Table 5. AO-Type Responses During December–Februarya UNIT 2CO2 2CO2 (I) 2CO2WT 2CO2WT(I) SLP 32N–52N minus 60N–80N mbar 2.28 (1.14) 1.98 4.91b [3.88] 5.43b 500 mbar Ht 32N–52N minus 60N–80N m 14 (41) 15 8 [7] 9 100 mbar Ht 32N–52N minus 60N–80N m 76c (19) 12 103b [142] 45d 10 mbar Ht 32N–52N minus 60N–80N m 196 (58) 57 96 [162] 220d aResults from Rind et al. [1998, 2001] are shown (without significance testing) in brackets or parentheses, respectively, for comparison. bSignificant at 99% level. cSignificant at 95% level. dSignificant at 90% level. potential energy associated with hemispheric scales [Rind et al., 1998]. of the model is somewhat less sensitive to SST patterns, with weaker surface winds, surface fluxes and resulting precipitation; hence it responded more weakly to the altered SST fields it was given (e.g., no Hadley Cell change). The newer model also had greater penetrative convection rela- tive to convection at other levels, so when climate warmed, and the penetrative convection increased, it was easier to stabilize the atmosphere and reduce convection in general. Furthermore with the greater increase in penetrating con- vection in the newer model, warming of the upper tropo- sphere was greater; combined with the unchanged Hadley circulation this resulted in a greater latitudinal temperature gradient, stronger west wind increases and a more positive AO phase (discussed further below). The tendency of climate change simulations to mimic characteristics of the control run has been noted previously [Mitchell et al., 1987; Rind, 1988; Senior, 1995]. [43] However, a number of the results were unexpected, relating primarily to changes associated with different versions of the GISS GCMAM. Rind et al. [2001] use the same marine boundary conditions as used in 2CO2 but with an earlier version of the model II0 GCMAM. In that simulation the Hadley Cell decreased in intensity, as did interhemispheric transport, by 5%; in contrast, in this 2CO2 experiment there were negligible changes in both quantities. Also, in the earlier simulation, total convective mass flux increased, as shown in Table 2, while this model produced a small decrease. [44] The differing Hadley Cell response in the two models also likely influenced the high latitude response, by altering the magnitude of dynamical heat convergence in the midlatitude troposphere [Rind et al., 2002]. RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS Heating at this latitude, compared with heating at high latitudes, affects the latitudinal temperature gradient and zonal wind change in the upper troposphere, and hence planetary wave refrac- tion. Rind et al. [2001, their Figure 3] show that the reduction in Hadley Cell intensity had minimized the midlatitude heating leading to decreased west winds in the upper troposphere at higher latitudes, relative poleward planetary wave refraction and higher sea level pressures at higher latitudes. In contrast, in the 2CO2 run here, during winter west winds increase at the higher latitudes in the upper troposphere, EP flux divergences occur and tropo- spheric subpolar lows are intensified. Why were these changes model dependent? [48] The 2CO2WT experiment should have produced simulations that resembled the results discussed by Rind et al. [1998] with the coarser resolution model, given that the sea surface temperature/sea ice changes of that run (from model II) were used in this one. The GCMAM II0 model has weaker convective fluxes due to reduced mass associated with each convective event, and hence the warming in the tropical upper troposphere is about 25% less than it was in the previous model. Consequently, the zonal wind increase in the upper troposphere is not as strong, and the magnitude of the wave refraction, EP flux divergence and residual circulation changes are somewhat reduced, but the overall pattern is similar. However, the AO phase changes differ with altitude, as discussed below. g p [45] We can address that question by comparing the current model to the version used by Rind et al. [2001]. The intermodel differences include the greater vertical resolution in the current model, both in the boundary layer (6 layers to 4 in the first 200 mbar) and overall (53 layers to 31 layers), and alterations in the model code, primarily in the boundary layer. To determine which component of the model changes affected each of the different results, we ran a control run version with reduced boundary layer and vertical resolution but with the newer 53-layer model code. [46] The results showed that increasing the vertical reso- lution, including that in the boundary layer, reduced the latent heat and total surface fluxes, and with it the precip- itation. Changes in the model code resulted in the newer model having weaker surface winds and often weaker eddy mixing in the boundary layer under unstable conditions. 7.2. Intermodel Differences And with greater tropical warming there was increased generation of planetary wave energy at the longest wavelengths, consistent with the increase in available [42] The Hadley Circulation response also matched expectations, at least in the sense that with the warmer tropical sea surface temperatures, the circulation would increase, and interhemispheric transport would become RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS The newer version ACL 25 - 17 RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS T simulations, the values are larger than that, with tratopause peak values of 15–20%, perhaps due to temperature structure of these ozone differences produ some additional cooling in the tropical lower stratosp Figure 11. Percentage change in age of air in the various experiments as calculated from the SF6 surface concentrations. Figure 11. Percentage change in age of air in the various experiments as calculated from the SF6 surface concentrations. 2CO2WT simulations, the values are larger than that, with tropical stratopause peak values of 15–20%, perhaps due to the different ozone photochemical code. The ozone loss in the tropical lower stratosphere is 5–10 times larger in the new run, undoubtedly influenced by the ozone advection, with the intensified upwelling exaggerating the lower strato- sphere loss. High latitude ozone gains are also larger when ozone advection is included. The radiative impact on the temperature structure of these ozone differences produces some additional cooling in the tropical lower stratosphere and slightly greater warming at high latitudes. RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS Increased vertical resolution resulted in greater amounts of penetrating convection to high levels and decreased fluxes to low levels. [45] We can address that question by comparing the current model to the version used by Rind et al. [2001]. The intermodel differences include the greater vertical resolution in the current model, both in the boundary layer (6 layers to 4 in the first 200 mbar) and overall (53 layers to 31 layers), and alterations in the model code, primarily in the boundary layer. To determine which component of the model changes affected each of the different results, we ran a control run version with reduced boundary layer and vertical resolution but with the newer 53-layer model code. , [49] Given these intermodel differences for their current climate simulation, it would be reasonable to have the most confidence in those simulations that produce the most realistic current depictions. The reduced convective fluxes in GCMAM II0 are likely to be more realistic than the excessive values of the coarse grid model as deduced from radon distributions (at least in comparison to other models’ radon results [Rind and Lerner, 1996]). Hence in that respect (although not with respect to sensitivity to SSTs) 2CO2WT should be given preference over the results from Rind et al. [1998]. The weaker surface response of 2CO2 is not necessarily an improvement, for example, the surface winds in the model were already too weak (as deduced from modeled soil dust [Tegen and Miller, 1998]). Hence the conclusions from 2CO2 are not necessarily to be preferred over those given by Rind et al. [2001] where they differ. [46] The results showed that increasing the vertical reso- lution, including that in the boundary layer, reduced the latent heat and total surface fluxes, and with it the precip- itation. Changes in the model code resulted in the newer model having weaker surface winds and often weaker eddy mixing in the boundary layer under unstable conditions. Increased vertical resolution resulted in greater amounts of penetrating convection to high levels and decreased fluxes to low levels. [50] The interactive ozone changes calculated by Rind et al. [1998], with the ozone anomaly scheme described by Shindell et al. [1998] that did not include ozone advection, peaked at 12% in the tropical stratopause region. In the [47] The control run differences provide explanations for the intermodel differences noted above. 7.3. AO Changes [51] Many, but not all model simulations have shown an increased positive AO phase with increasing atmospheric CO2 [Shindell et al., 1999; Paeth et al., 1999; Zorita and RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS ACL 25 - 18 ACL increased by 3–4C in the 2CO2 experiments, and 4–6C in the 2CO2WT experiments. At 164 mbar in the tropics specific humidity increased by 9%, in the 2CO2 runs and increased by 45% in 2CO2WT. Nevertheless, colder temper- atures just above the averaged tropopause associated with the increased vertical velocities and localized penetrating convection enhanced condensation, removing all the excess moisture and more. Specific humidity decreased throughout the stratosphere, with reductions of greater than 20% between 100 and 30 mbar. Gonzalez-Rouco, 2000; Gillett et al., 2002a]. All the sim- ulations shown here have decreased high latitude sea level pressures as well, as is evident in Figure 6. To assess whether these changes correspond to an altered phase of the Arctic Oscillation, we show in Table 5 the differences in sea level pressure or geopotential heights between approx- imately 30N–50N and 60N–80N (characteristic of the AO sea level pressure gradient), along with the significance of the results using a two-tailed T-test on the interannual variation for 10 modeled years. With the warmer tropical sea surface temperatures, at sea level a change to the positive phase of the AO-type oscillation is significant, and this is also true at 100 mbar, but there is no significance in the middle troposphere, and the sign is different in the middle stratosphere. 8. Conclusions These effects are weaker or missing entirely in the other simulations.  In all of the doubled CO2 simulations, there is a more direct circulation at lower latitudes, a more indirect circulation at midlatitudes in the troposphere and higher latitudes in the stratosphere, and a more direct circulation at high latitudes in the troposphere and lower stratosphere. All of these circulation changes are greater in the WT experiments.  Ozone increases in the upper stratosphere and decreases in the lower stratosphere in all of the doubled CO2 simulations. In the WT experiments ozone increases at high latitudes as well, an effect which extends down into the troposphere, while the other simulations show decreases in high latitude ozone in the midtroposphere. [54] We can also compare the results from the earlier version of the current model (shown in parentheses in Table 5) [Rind et al., 2001] to the 2CO2 results. The earlier model featured a relative negative AO phase in the sea level pressure field and throughout the troposphere, in contrast to the newer run with the same SST field. This model difference is also influenced by the different Hadley Cell convergences at midlatitudes, as discussed earlier in this section. g p p  The interactive runs show the smallest ozone increase in the tropics, as warmer temperatures reduce photochemi- cal production. By altering the ozone gradients, the interactive runs alter the temperature gradients, winds and refractive properties of planetary waves, with some effect on the circulation extending down into the troposphere.  At sea level there is a change to the positive phase of the AO-type oscillation and this is also true at 100 mbar, but there is no significance in the middle troposphere, and the sign is different in the middle stratosphere. 8. Conclusions [56] Model simulations with doubled atmospheric CO2 and two sets of sea surface temperatures (2CO2 and 2CO2WT) were used in GCM simulations with eight online tracers. Also included was a linearized ozone photochem- istry run both interactively (I) and noninteractively with the radiation. The primary results from these experiments are summarized as follows: [52] The pattern of the EP flux change in all the experi- ments features significant tropospheric divergences at upper middle latitudes (providing a west wind acceleration), with convergences at high latitudes (for an east wind acceler- ation), as in Figures 3 and 4. This wind change is realized with lower pressure circa 70N.  The simulations with the greater increase in tropical sea surface temperatures (2CO2WT, 2CO2WT (I)) caused an amplified tropical residual circulation response, including increased interhemispheric transport and an exaggerated increase in transport out of the tropical troposphere into the stratosphere. In the other simulations (2CO2, 2CO2(I)) interhemispheric transport did not change, and the increase in transport out of the tropical troposphere was smaller.  The simulations with the greater increase in tropical sea surface temperatures (2CO2WT, 2CO2WT (I)) caused an amplified tropical residual circulation response, including increased interhemispheric transport and an exaggerated increase in transport out of the tropical troposphere into the stratosphere. In the other simulations (2CO2, 2CO2(I)) interhemispheric transport did not change, and the increase in transport out of the tropical troposphere was smaller. p [53] The increased positive phase alters sign by the middle stratosphere in 2CO2WT, which then features a weaker polar vortex (see also Figure 6), a result also found by Butchart et al. [2000] and Gillett et al. [2002b]. We also show in Table 5 (in brackets) the results from the previous simulation with these SSTs in the coarse grid model, in which the phase remained positive in the middle strato- sphere; that is, the polar vortex was stronger. As can be seen in Figure 2, the temperature change in 2CO2WT at high northern latitudes in the lower stratosphere exceeds that in the middle troposphere, and also exceeds that at midlati- tudes, helping to change the phase of the AO. In contrast, the older model did not have such amplified warming in the polar lower stratosphere, and the positive AO phase change was maintained into the stratosphere (i.e., a more barotropic response). 8. Conclusions This temperature difference is consistent with the zonal wind change, which in the current simulations fea- tures weaker west winds at high latitudes, with relative poleward wave refraction, while the older model had stronger west winds and relative equatorward wave refrac- tion at most high latitudes [Rind et al., 1998, Figure 5]. The greater convective fluxes and tropical response in the older model led to greater energy convergence from the Hadley Cell change at lower midlatitudes in the upper troposphere, helping to produce these differences. [53] The increased positive phase alters sign by the middle stratosphere in 2CO2WT, which then features a weaker polar vortex (see also Figure 6), a result also found by Butchart et al. [2000] and Gillett et al. [2002b]. We also show in Table 5 (in brackets) the results from the previous simulation with these SSTs in the coarse grid model, in which the phase remained positive in the middle strato- sphere; that is, the polar vortex was stronger. As can be seen in Figure 2, the temperature change in 2CO2WT at high northern latitudes in the lower stratosphere exceeds that in the middle troposphere, and also exceeds that at midlati- tudes, helping to change the phase of the AO. In contrast, the older model did not have such amplified warming in the polar lower stratosphere, and the positive AO phase change was maintained into the stratosphere (i.e., a more barotropic response). This temperature difference is consistent with the zonal wind change, which in the current simulations fea- tures weaker west winds at high latitudes, with relative poleward wave refraction, while the older model had stronger west winds and relative equatorward wave refrac- tion at most high latitudes [Rind et al., 1998, Figure 5]. The greater convective fluxes and tropical response in the older model led to greater energy convergence from the Hadley Cell change at lower midlatitudes in the upper troposphere, helping to produce these differences.  At high latitudes, zonal winds become more easterly in the WT runs due to a combination of greater surface warming and increased EP flux convergences in the stratosphere. These effects are weaker or missing entirely in the other simulations.  At high latitudes, zonal winds become more easterly in the WT runs due to a combination of greater surface warming and increased EP flux convergences in the stratosphere. References Andrews, A. E., et al., Mean ages of stratospheric air derived from in situ observations of CO2, CH4, and N2O, J. Geophys. Res., 106, 32,295– 32,314, 2001. Rind, D., J. Lerner, K. Shah, and R. Suozzo, Use of on-line tracers as a diagnostic tool in general circulation model development, 2, Transport between the troposphere and stratosphere, J. Geophys. Res., 104, 9151– 9167, 1999. Butchart, N., J. Austin, J. R. Knight, A. A. Scaife, and M. L. Gallani, The response of the stratospheric climate to projected changes in the concen- trations of well-mixed greenhouse gases from 1992 to 2051, J. Clim., 13, 2142–2159, 2000. Rind, D., J. Lerner, and C. McLinden, Changes of tracer distributions in the doubled CO2 climate, J. Geophys. Res., 106, 28,061–28,079, 2001. Rind, D., P. Lonergan, N. K. Balachandran, and D. Shindell, 2xCO2 and solar variability influences on the troposphere through wave-mean flow interactions, J. Meteorol. Soc. Jpn., 80, 863–876, 2002. Dai, A., T. M. L. Wigley, B. A. Boville, J. T. Kiehl, and L. E. Buja, Climates of the twentieth and twenty-first centuries simulated by the NCAR climate system model, J. Clim., 14, 485–519, 2001. p Senior, C. A., The dependence of climate sensitivity on the horizontal resolution of a GCM, J. Clim., 8, 2860–2880, 1995. Douglass, A. R., M. J. Prather, T. M. Hall, S. E. Strahan, P. J. Rasch, L. C. Sparling, L. Coy, and J. M. Rodriguez, Choosing meteorological input for the global modeling initiative assessment of high-speed aircraft, J. Geo- phys. Res., 104, 27,545–27,564, 1999. Shindell, D., D. Rind, and P. Lonergan, Climate change and the middle atmosphere, IV:1, Ozone photochemical response to doubled CO2, J. Clim., 11, 895–918, 1998. p y Fyfe, J. C., G. J. Boer, and G. M. Flato, The Arctic and Antarctic oscilla- tions and their projected changes under global warming, Geophys. Res. Lett., 26, 1601–1604, 1999. Shindell, D., R. Miller, G. Schmidt, and L. Pandolfo, Simulation of recent northern winter climate trends by greenhouse-gas forcing, Nature, 399, 452–455, 1999. Gillett, N. P., M. R. Allen, R. E. McDonald, C. A. Senior, D. T. Shindell, and G. A. Schmidt, How linear is the Arctic Oscillation response to greenhouse gases?, J. Geophys. Res., 107, 4022, doi:10.1029/ 2001JD000589, 2002a. Tegen, I., and R. Miller, A general circulation model study on the inter- annual variability of soil dust aerosol, J. Geophys. Res., 103, 25,975– 25,995, 1998. Thompson, D. W., J. M. RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS RIND ET AL.: TRACER TRANSPORTS AND 2XCO2 SSTS - 19 face data sets applied to the GCMAM were also made available to the GRIPS (GCM Reality Intercomparison Project) [Pawson et al., 2000] so that different strato- spheric modeling groups could run doubled CO2 experi- ments with the same climate forcing. The goal of the project is, in effect, to compare how important intermodel differences are relative to the sea surface temperature forcings. The results presented in this paper, when com- pared with those from our earlier simulations, imply that intermodel differences outweigh the sea surface temper- ature forcings for a number of the features, and this is from a comparison of old and new GISS GCMs. We might expect the differences to be equivalent or larger among the modeling groups in general. Pawson, S., et al., The GCM-Reality Intercomparison Project for SPARC (GRIPS): Scientific issues and initial results, Bull. Am. Meteorol. Soc., 81, 781–796, 2000. , , Perlwitz, J., H. F. Graf, and R. Voss, The leading variability mode of the coupled troposphere-stratosphere winter circulation in different climate regimes, J. Geophys. Res., 105, 6915–6926, 2000. g p y Prather, M., Numerical advection by conservation of second-order mo- ments, J. Geophys. Res., 91, 6671–6681, 1986. Ramstein, G., Y. Serafini-Le Treut, H. Le Treut, M. Forichon, and S. Joussaume, Cloud processes associated with past and future climate changes, Clim. Dyn., 14, 233–247, 1998. Rind, D., Dependence of warm and cold climate depiction on climate model resolution, J. Clim., 1, 965–997, 1988. Rind, D., and J. Lerner, The use of on-line tracers as a diagnostic tool in GCM model development, J. Geophys. Res., 101, 12,667–12,683, 1996. Rind, D., R. Suozzo, N. K. Balachandran, A. Lacis, and G. L. Russell, The GISS Global Climate/Middle Atmosphere Model, I, Model structure and climatology, J. Atmos. Sci., 45, 329–370, 1988. Rind, D., R. Suozzo, N. K. Balachandran, and M. Prather, Climate change and the middle atmosphere, 1, The doubled CO2 climate, J. Atmos. Sci., 47, 475–494, 1990. [58] Acknowledgments. This work was funded by the NASA ACMAP and EOS programs. , , Rind, D., D. Shindell, P. Lonergan, and N. K. Balachandran, Climate change and the middle atmosphere, III, The doubled CO2 climate revis- ited, J. Clim., 11, 876–894, 1998. J. Lerner, J. Perlwitz, and D. Rind, Goddard Institute for Space Studies at Columbia University, 2880 Broadway, New York, N.Y. 10025, USA. (jlerner@giss.nasa.gov;judith@giss.nasa.gov;drind@giss.nasa.gov) C. McLinden, Air Quality Research Branch, Meteorological Service of Canada, 4905 Dufferin Street, Toronto, ON M3H 5T4, Canada. (chris.mclinden@ec.gc.ca) M. J. Prather, Earth System Science Department, University of California, Irvine, Irvine, CA 92697-3100, USA. (mprather@uci.edu) 7.4. Stratospheric Water Vapor Change [55] Given the increase in fluxes of tropospheric species into the stratosphere, as symbolized by the methane changes shown in Figure 8, one might have expected that (non- methane) stratospheric water vapor values would have increased as well. In addition to the transport change, tropical zonal and time-averaged tropopause temperatures [55] Given the increase in fluxes of tropospheric species into the stratosphere, as symbolized by the methane changes shown in Figure 8, one might have expected that (non- methane) stratospheric water vapor values would have increased as well. In addition to the transport change, tropical zonal and time-averaged tropopause temperatures  Many of these results differ from those generated in older versions of the GISS GCMAM despite the same SST forcing, due to differences in control run characteristics. [57] This last conclusion has implications concerning the use of altered sea surface temperatures in different GCMs as part of intercomparison projects. The two marine sur- ACL 25 - 19 [58] Acknowledgments. This work was funded by the NASA ACMAP and EOS programs. References Wallace, and G. C. Hegerl, Annular modes in the extratropical circulation, II, Trends, J. Clim., 13, 1018–1036, 2000. Gillett, N. P., M. R. Allen, and K. D. Williams, The role of stratospheric resolution in simulating the Arctic Oscillation response to greenhouse gases, Geophys. Res. Lett., 29, 1500, doi:10.1029/2001GL014444, 2002b. Yao, M.-S., and A. D. Del Genio, Effects of cloud parameterization on the simulation of climate changes in the GISS GCM, J. Clim., 12, 761–779, 1999. Haynes, P. H., C. J. Marks, M. E. McIntyre, T. G. Shepherd, and K. P. Shine, On the ‘‘downward control’’ of extratropical diabatic circulations by eddy-induced mean zonal forces, J. Atmos. Sci., 48, 651–678, 1991. Zorita, E., and F. Gonzalez-Rouco, Disagreement between predictions of the future behavior of the Arctic Oscillation as simulated in two different climate models: Implications for global warming, Geophys. Res. Lett., 27, 1755–1758, 2000. McLinden, C. A., S. C. Olsen, B. Hannegan, O. Wild, M. J. Prather, and J. Sundet, Stratospheric ozone in 3-D models: A simple chemistry and the cross-tropopause flux, J. Geophys. Res., 105, 14,653–14,665, 2000. Mitchell, J. F. B., C. A. Wilson, and W. M. Cunnington, On CO2 climate sensitivity and model dependence of results, Q. J. R. Meteorol. Soc., 113, 293–322, 1987. Monahan, A. H., J. C. Fyfe, and G. M. Flato, A regime view of Northern Hemisphere atmospheric variability and change under global warming, Geophys. Res. Lett., 27, 1139–1142, 2000. p y Paeth, H., A. Hense, R. Glowienka-Hense, S. Voss, and U. Cubasch, The North Atlantic Oscillation as an indicator for greenhouse-gas induced regional climate change, Clim. Dyn., 15, 953–960, 1999.
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The Effect of Dental Implant Drills Materials on Heat Generation in Osteotomy Sites: A Systematic Review
˜The œeuropean journal of dentistry/The european journal of dentistry
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© 2023. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. (https://creativecommons.org/licenses/by/4.0/) Thieme Medical and Scientific Publishers Pvt. Ltd., A-12, 2nd Floor, Sector 2, Noida-201301 UP, India Article published online: 2023-06-19 Article published online: 2023-06-19 Article published online: 2023-06-19 Article published online: 2023-06-19 Review Article 65 The Effect of Dental Implant Drills Materials on Heat Generation in Osteotomy Sites: A Systematic Review Shreyasi Chakraborty1 Mohammad-Adel Moufti2,3 Waad Kheder2,3 1Department of Implant Dentistry, BPP University, Birmingham, United Kingdom 2Department of Preventive and Restorative Dentistry, College of Dental Medicine University of Sharjah, Sharjah, United Arab Emirates 3Research Institute for Medical and Health Sciences, University of Sharjah, Sharjah, United Arab Emirates Address for correspondence Mohammad-Adel Moufti, BDS, Dip (OMS), MFDS (RCSEd), PhD (Ncl), College of Dental Medicine, University of Sharjah, Sharjah, P.O. Box 27272, United Arab Emirates (e-mail: mamoufti@sharjah.ac.ae). 1Department of Implant Dentistry, BPP University, Birmingham, United Kingdom 2Department of Preventive and Restorative Dentistry, College of Dental Medicine University of Sharjah, Sharjah, United Arab Emirates 3Research Institute for Medical and Health Sciences, University of Sharjah, Sharjah, United Arab Emirates Eur J Dent 2024;18:65–72. DOI https://doi.org/ 10.1055/s-0043-1768472. ISSN 1305-7456. 1Department of Implant Dentistry, BPP University, Birmingham, United Kingdom 2Department of Preventive and Restorative Dentistry, College of Dental Medicine University of Sharjah, Sharjah, United Arab Emirates 3Research Institute for Medical and Health Sciences, University of Sharjah, Sharjah, United Arab Emirates article published online June 19, 2023 Address for correspondence Mohammad-Adel Moufti, BDS, Dip (OMS), MFDS (RCSEd), PhD (Ncl), College of Dental Medicine, University of Sharjah, Sharjah, P.O. Box 27272, United Arab Emirates (e-mail: mamoufti@sharjah.ac.ae). Address for correspondence Mohammad-Adel Moufti, BDS, Dip (OMS), MFDS (RCSEd), PhD (Ncl), College of Dental Medicine, University of Sharjah, Sharjah, P.O. Box 27272, United Arab Emirates (e-mail: mamoufti@sharjah.ac.ae). article published online June 19, 2023 DOI https://doi.org/ 10.1055/s-0043-1768472. ISSN 1305-7456. Introduction preparation?” was formulated based on predefined patient, intervention, comparison, and outcome (PICO) elements that are explained below. The search was undertaken in Medline/PubMed Central, Science Direct, and Google Scholar using the following keywords and Boolean operators: “im- plant site preparation” OR “osteotomy” OR “bone drill” AND “ceramic drill” OR “zirconia drill” OR “zirconium drill” OR “zirconia oxide drill” OR “Zircon based drill” OR “Diamond Like Carbon coated drill” OR “DLC coated drills” OR “black diamond drill” OR “titanium nitride coated metal drills” OR “tungsten carbide coated metal drill” OR “tin coated drill” OR “stainless steel drill” OR “stainless steel drill” OR “titanium drill” AND “heat generation” OR “thermal change” OR “ther- mal osteonecrosis” OR “thermal variation” OR “intrabony temperature” OR “temperature change.” The selection of articles/studies followed the standardized staged process of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) as illustrated in ►Fig. 1. First, the abstracts of the initially selected articles were screened by the authors SC and WK independently based on the inclusion and exclusion criteria listed below to confirm the articles’ relevance to our study. Any disagreement was dis- cussed among the three authors and resolved. The full text of the selected articles was obtained and read in full, also by both reviewers SC and WK independently, to confirm the selection of those articles. The bibliographies of the studies were manually searched to identify any article that was not captured by the electronic search. Four articles were found relevant and selected for this review. One of the most important challenges in bone drilling during osteotomy site preparation is heat generation and thermal damages to the alveolar bone. This could be due to several biomechanical factors such as the drilling protocol (technique, force, speed, drill geometry, and irrigation),1 properties of the drills,2 and properties of the bone.3,4 Investigations have reported that temperatures of over 44°C lead to irreversible damage. Abstract The aim of this review was to examine the impact of dental implant drill materials and wear profiles on heat generation in the osteotomy sites as reported in experimental studies and to critically appraise these studies. The research question was formulated based on predefined patient, intervention, comparison, and outcome (PICO) elements. A comprehensive electronic search was undertaken in Medline/PubMed Central, Science Direct, and Google Scholar, using predetermined keywords, followed by a manual search of the bibliography of the selected articles. The selection of the studies for the critical appraisal part of our study was based on the criteria used to assess the study designs such as study aims, outcome measure, clarity of method, sample selection, randomization, allocation concealment, sample attrition, bias, method of data analysis, and external validity. Increased heat generation was observed with both ceramic and metal drills; the heat generation was proportional to drills’ wear. The literature was inconclusive regarding the association between drill material and heat generation. However, drill materials had a significant influence on the overall temper- ature increase during osteotomy. The noncoated drills showed a higher wear resis- tance, and it has been observed that using worn drills leads to more friction contact, reduced drill cutting efficiency, and increased heat generation. Eleven in vitro studies met the inclusion criteria, and showed a considerable methodological heterogeneity and confounding factors, including drill geometry, speed and load, depth and diameter, number of uses, irrigation protocol, study specimens, and the heat measur- ing device. Besides, most of the studies have a potential operator and assessor bias, and some have sponsorship bias. It is possible to conclude that the literature is not conclusive on the effect of drill materials on heat generation during osteotomy. Lack of standardization and uniformity in the study design, along with potential bias in the study methodology can be the reason for the heterogeneity of the results. Keywords ►ceramics ►dental implant ►drills ►friction ►heat ►osteotomy Heat Generation and Implant Drill Materials Chakraborty et al. 66 Critical Appraisal Due to lack of standardized critical appraisal tools for in vitro studies, our study selected appraisal criteria based on the aspects that should be assessed in this type of study designs. The aspects appraised were study aims, outcome measure, method clarity, sample selection, randomization, allocation concealment, sample attrition, bias, method of data analysis, and external validity as presented in ►Fig. 2. Results The initial electronic search identified eight articles. One article was excluded after abstract screening because it did not report the measurement of heat generation in relation to the different drill materials (►Table 1). The remaining seven articles met the inclusion criteria and were selected. An additional four articles were identified from the manual Inclusion Criteria Included in this review were studies published on the influ- ence of drill material on heat generation in osteotomy sites, studies that answer the research question, and studies including human, animal, or synthetic bone. Exclusion Criteria Case reports, case series studies, reviews, articles published earlier than the year 2000, and publications in language other than English were excluded from our review. The effect of the drill material on heat generation during osteotomy has been debated. Although some reports con- firmed this, other research claims that the tool material does not have a significant effect on heat generation during osteotomy.11 Despite the advances in the manufacturing of ceramic burs, only a limited number of studies have tried to address such an important effect, and there is still a lack of consensus when it comes to its dental applications.12 The aim of this study was to explore the experimental studies in the literature regarding the impact of drill materials and their wear profile on heat generation in osteotomy sites, and to critically appraise these studies. Introduction Drill wear is one of the important causes of the rise in temperature during osteotomy, which needs to be controlled to avoid thermal osteonecrosis.5 The elevated bone tempera- ture constitutes a major cause of early dental implant failure since it may lead to hyperemia, osteocyte degeneration, in- creased osteoclastic activity, and necrosis, which ultimately affects the regenerative capacity of the bone and consequently ends with osseointegration failure.4 Delayed healing, failure of osseointegration, and necrosis of the bone were reported during the osteotomy when the temperature exceeded 47°C for more than 1 minute.6 Therefore, for successful osseointe- gration, the osteotomy preparation in low temperature is of utmost importance. For many decades, dental implant drills were made of stainless steel because of their high cutting efficiency and durability. Ceramic drills, which are mainly composed of 80% zirconia oxide and 20% alumina oxide, were lately introduced in the market. Combining zirconia with magnesium or alumina stabilizes zirconia, resulting in better biomechanical properties.7,8 Ceramic drills have the advantage of being biocompatible, which reduces the chances of allergic responses to metal during osteotomy preparation. They show good resistance against high temperature, corrosion, and wear/abrasion.9 Thus, they are expected to generate less heat during implant site preparation.10 However, literature on the use of ceramic drills in implantology is scarce and is not conclusive on their advantages in lowering heat genera- tion during drilling. Understanding whether drill material can affect heat generation during osteotomy preparation is of high clinical relevance as it can influence the implant success and survival rate. European Journal of Dentistry Vol. 18 No. 1/2024 © 2023. The Author(s). Articles Search Strategy The research question “Is there a difference in the amount of heat generation using different drill materials for osteotomy Heat Generation and Implant Drill Materials Chakraborty et al. 67 Fig. 1 Flowchart of the articles search strategy. Fig. 1 Flowchart of the articles search strategy. the implant site has a significant impact on the success of osseointegration,3 because heat-induced bone injuries re- duce the implant primary stability, leading to implant fail- ure.17 Furthermore, if the bone is exposed during osteotomy to a temperature exceeding 47°C for more than 1 minute, irreversible cellular damage will happen, and the bone will be replaced with fibrous tissue.18 Drill materials can influ- ence the heat generation during osteotomy. Therefore, the selection of drills is of high clinical relevance as it can help in implant osseointegration and increase their success and survival rate. These studies have been performed in vitro on a variety of bone models using different temperature measurement systems (various thermocouples or infrared thermography devices). search of the bibliography of the 7 selected articles, resulting in a total of 11 articles being selected for our review (►Table 2). The article search strategy was designed to capture articles that go back to the year 2000; however, relevant studies were found only after 2010. All the included articles were in vitro studies, of which 10 were performed on bovine bone specimens except 1 study that was performed on artificially manufactured bone speci- mens. Drills used in the studies were fabricated from differ- ent materials such as stainless steel, ceramic, tungsten carbide, black diamond, and diamond like carbon-coated drills. Ten of the studies used thermocouples for tempera- ture measurement placed close to the osteotomy at different depths to measure the real-time temperature. In study number 11, the temperature was measured using an infrared thermometer. The studies evaluated other variables such as drill wear, effect of depth, and irrigation. The rotational speed and load used for osteotomy preparation was constant during the experiments and was reported in all studies. All selected articles observed an increase in the bone tempera- ture while preparing the osteotomy sites. The overall tem- perature increase varied distinctly among the studies, which could be explained by the confounding factors in each study (►Table 3). Composition of the Drills Most of the included studies compared stainless steel and ceramic materials along with some less common drill types, such as tungsten carbide carbon, titanium nitride, and other coated drills. In comparison between metal and ceramic drills, the studies have reported contradictory findings. Some found that stainless steel drills generate more heat than the ceramic drills9,11,15,19; others showed no differ- ence,12,14,20 while one study reported higher mean temper- ature with ceramic drills than metal drills.16 The effect of drill coating was also examined. One study by Allsobrook et al13 found that tungsten carbide-coated stain- less steel generated less heat compared with the stainless steel drills, while Hochscheidt et al reported that steel diamondlike carbon coating showed highest temperature than other drills fabricated from surface-treated steel, and aluminum-toughened zirconium ceramic.21 A direct com- parison between the study results is neither possible nor Articles Search Strategy Most studies reported the bone baseline temper- ature, while a few studies did not report the bone baseline temperature and only compared the maximum temperature reached by the different drill types.13–16 European Journal of Dentistry Vol. 18 No. 1/2024 © 2023. The Author(s). Discussion Osseointegration is the direct functional and structural connection between the surface of the dental implant and the living bone. Heat generation during drilling to prepare Heat Generation and Implant Drill Materials Chakraborty et al. 68 g. 2 Traffic light system summarizing critical appraisal of the included articles. g. 2 Traffic light system summarizing critical appraisal of the included articles. Fig. 2 Traffic light system summarizing critical appraisal of the included articles. Fig. 2 Traffic light system summarizing critical appraisal of the included articles. Table 1 List of excluded articles SN Study Database Article title 1 Batista Mendes et al10 PubMed Influence of implant drill materials on wear, deformation, and roughness after repeated drilling and sterilization temperature increase during osteotomy, which may be re- lated to a higher wear resistance of the noncoated drills, as explored below. Table 1 List of excluded articles European Journal of Dentistry Vol. 18 No. 1/2024 © 2023. The Author(s). Drill Wear and Number of Uses Heat Generation and Implant Drill Materials Chakraborty et al. 69 Table 2 List of included articles in a chronological order SN Author/year Database Article title 1 Sumer et al/2011 PubMed Comparison of heat generation during implant drilling using stainless steel and ceramic drills 2 Allsobrook et al/2011 Manual search Descriptive study of the longevity of dental implant surgery drills 3 Oliveira et al/2012 PubMed Thermal changes and drill wear in bovine bone during implant site preparation. A comparative in vitro study: twisted stainless steel and ceramic drills 4 Harder et al/2013 Manual search Influence of the drill material and method of cooling on the development of intrabony temperature during preparation of the site of an implant 5 Moshiri et al/2013 Manual search Evaluation the effect of drill type on heat generation in implant drilling site 6 Koo et al/2015 PubMed Effects of implant drill wear, irrigation, and drill materials on heat generation in osteotomy sites 7 Hochscheidt et al/2017 Manual search Thermal variation during osteotomy with different dental implant drills: a standardized study in bovine ribs 8 Er et al/2018 PubMed Improved dental implant drill durability and performance using heat and wear resistant protective coatings 9 Koopaie et al/2019 Science Direct Comparison of wear and temperature of zirconia and tungsten carbide tools in drilling bone: in vitro and finite element analysis 10 Scarano et al/2020 PubMed Infrared thermographic evaluation of temperature modifications induced during implant site preparation with steel vs. zirconia implant drill 11 Tur et al/2020 PubMed Thermal effects of various drill materials during implant site preparation— ceramic vs. stainless steel drills: a comparative in vitro study in a standardised bovine bone model Table 2 List of included articles in a chronological order Table 3 Overall temperature changes during osteotomy Author/Year Results Sumer et al9 The mean temperature in the water bath was 30.1°C. The mean maximum temperature with stainless steel drills was 35.05°C, whereas that with ceramic drills was 35.9°C Allsobrook et al13 The mean temperature measured over the experiment was 20.0°C. The maximum temperature reached was 27.7°C, and the temperature seldom varied 2°C from the initial temperature Oliveira et al11 Mean baseline bone temperature for the stainless steel and ceramic drills were 21.59  0.1 and 21.6  0.1°C, respectively. The mean maximum increase in temperature was 1.64  1.11°C Harder et al12 The baseline temperature was 23°C. Drill Wear and Number of Uses The mean maximum temperature recorded was 64.7°C Hochscheidt et al21 Baseline temperature was 20.32°C and the peak temperatures reached were as follows, in sequence: steel with a diamondlike carbon coating, 67.6°C at 5-mm drilling depth; aluminum-toughened zirconium ceramic, 52.1°C at 13-mm depth; and experimentally surface-treated steel, 32.0°C at 13-mm depth Er et al20 The baseline temperature was 23–24°C, with the mean maximum bone temperature at 35.5°C Koopaie et al22 Baseline temperature was 20°C. The mean temperature was 38.3°C Scarano et al15 The steel drills showed a bone temperature of 42.45  1.70°C, compared with the zirconia drills, which reported average values of 40.80  0.85°C Tur et al16 The maximum mean temperature increases for 16-mm drilling without irrigation was 27.20°C Table 2 List of included articles in a chronological order SN Author/year Database Article title 1 Sumer et al/2011 PubMed Comparison of heat generation during implant drilling using stainless steel and ceramic drills 2 Allsobrook et al/2011 Manual search Descriptive study of the longevity of dental implant surgery drills 3 Oliveira et al/2012 PubMed Thermal changes and drill wear in bovine bone during implant site preparation. A comparative in vitro study: twisted stainless steel and ceramic drills 4 Harder et al/2013 Manual search Influence of the drill material and method of cooling on the development of intrabony temperature during preparation of the site of an implant 5 Moshiri et al/2013 Manual search Evaluation the effect of drill type on heat generation in implant drilling site 6 Koo et al/2015 PubMed Effects of implant drill wear, irrigation, and drill materials on heat generation in osteotomy sites 7 Hochscheidt et al/2017 Manual search Thermal variation during osteotomy with different dental implant drills: a standardized study in bovine ribs 8 Er et al/2018 PubMed Improved dental implant drill durability and performance using heat and wear resistant protective coatings 9 Koopaie et al/2019 Science Direct Comparison of wear and temperature of zirconia and tungsten carbide tools in drilling bone: in vitro and finite element analysis 10 Scarano et al/2020 PubMed Infrared thermographic evaluation of temperature modifications induced during implant site preparation with steel vs. zirconia implant drill 11 Tur et al/2020 PubMed Thermal effects of various drill materials during implant site preparation— ceramic vs. stainless steel drills: a comparative in vitro study in a standardised bovine bone model Heat Generation and Implant Drill Materials Chakraborty et al. Drill Wear and Number of Uses Ithasbeenobservedthat using worndrillsleadstomorefriction contact, reduced drill cutting efficiency, and increased heat generation. This may increase the possibility of bone necrosis, irreversible cellular damage, and replacement of bone tissue with fat tissue.20 Therefore, the number of drill reuse is an important factor in heat generation during osteotomy. valid, as the wear resistance of pure zirconia, for instance, is different to that of zirconium oxide coating. It is apparent that drill materials had a significant influence on the overall valid, as the wear resistance of pure zirconia, for instance, is different to that of zirconium oxide coating. It is apparent that drill materials had a significant influence on the overall The number of drill uses has varied among the reviewed studies, but most of them have used the drills for 50 times, European Journal of Dentistry Vol. 18 No. 1/2024 © 2023. The Author(s). and different conclusions were reached. Oliveira et al com- pared scanning electron microscope (SEM) images of both stainless steel and zirconia drills before and following 50 uses, and identified different patterns of wear.11 The ceramic tip, contrasting with the stainless steel drill, which showed a higher tip wear in both edges. However, none of the drills appeared to present severe deformation or blunting after 50 uses. This is not consistent with Koopaie et al in which the Table 3 Overall temperature changes during osteotomy Author/Year Results Sumer et al9 The mean temperature in the water bath was 30.1°C. The mean maximum temperature with stainless steel drills was 35.05°C, whereas that with ceramic drills was 35.9°C Allsobrook et al13 The mean temperature measured over the experiment was 20.0°C. The maximum temperature reached was 27.7°C, and the temperature seldom varied 2°C from the initial temperature Oliveira et al11 Mean baseline bone temperature for the stainless steel and ceramic drills were 21.59  0.1 and 21.6  0.1°C, respectively. The mean maximum increase in temperature was 1.64  1.11°C Harder et al12 The baseline temperature was 23°C. The mean maximum intrabony temperature increased was 3.9°C Moshiri et al19 Baseline temperature not mentioned. The mean maximum temperature was 33°C Koo et al14 The mean internal temperature is 36.5°C and the surface temperature of 28°C (baseline temperature). European Journal of Dentistry Vol. 18 No. 1/2024 © 2023. The Author(s). Drill Wear and Number of Uses The mean maximum intrabony temperature increased was 3.9°C Moshiri et al19 Baseline temperature not mentioned. The mean maximum temperature was 33°C Koo et al14 The mean internal temperature is 36.5°C and the surface temperature of 28°C (baseline temperature). The mean maximum temperature recorded was 64.7°C Hochscheidt et al21 Baseline temperature was 20.32°C and the peak temperatures reached were as follows, in sequence: steel with a diamondlike carbon coating, 67.6°C at 5-mm drilling depth; aluminum-toughened zirconium ceramic, 52.1°C at 13-mm depth; and experimentally surface-treated steel, 32.0°C at 13-mm depth Er et al20 The baseline temperature was 23–24°C, with the mean maximum bone temperature at 35.5°C Koopaie et al22 Baseline temperature was 20°C. The mean temperature was 38.3°C Scarano et al15 The steel drills showed a bone temperature of 42.45  1.70°C, compared with the zirconia drills, which reported average values of 40.80  0.85°C Tur et al16 The maximum mean temperature increases for 16-mm drilling without irrigation was 27.20°C Table 3 Overall temperature changes during osteotomy and different conclusions were reached. Oliveira et al com- pared scanning electron microscope (SEM) images of both stainless steel and zirconia drills before and following 50 uses, and identified different patterns of wear.11 The ceramic drills had slightly visible alterations on one edge of the drill tip, contrasting with the stainless steel drill, which showed a higher tip wear in both edges. However, none of the drills appeared to present severe deformation or blunting after 50 uses. This is not consistent with Koopaie et al in which the overall wear of the ceramic drills after 45 uses was about half European Journal of Dentistry Vol. 18 No. 1/2024 © 2023. The Author(s). Heat Generation and Implant Drill Materials Chakraborty et al. 70 that of the tungsten carbide drills. However, the comparator in both studies was different.22 The SEM images in the third study by Allsobrook et al13 found that tungsten carbide drills display least corrosion and wear and lowest mean drilling temperatures after 20 osteotomies when compared with stainless steel drills. Interestingly Scarano et al15 found that in case of using zirconia drills, the outline of the implant bed was well defined even after 120 osteotomies. Another noteworthy finding is that by Koo et al14 who found that only the initial/pilot drills generate more heat after 50 uses. Drill Wear and Number of Uses subject, which reported higher temperature while preparing the deeper parts of the osteotomies due to ineffective cooling with irrigation. These contradictory results again raise con- cerns regarding the validity of the study methods. Study Models/Specimens The drill geometry is of critical importance to heat genera- tion, and has been investigated in many studies. Therefore, to make a valid comparison between the studies, the geometry of the drills used in the studies should be similar. Koo et al14 used drills of different shapes, which could affect the inter- pretation of the data. The other studies either have used similar drills or have not reported the design of the drills used in their experiments. No human-based articles were identified, which may be due to ethical reasons, since these experiments require an inva- sive insertion of special equipment to detect the temperature change. Most of the included articles have used bovine bone for the experiment, which has been used successfully in many studies to investigate heat generation in response to implant site preparation. However, there is no standard study model for conducting experiments on heat generation during osteotomy.6 Thus, the studies have used different parts of bovine bone like ribs, femur, and scapular bone to conduct the experiment, which was considered to have a similar density as the human bone. However, none of the studies reported the bone density type, despite the signifi- cant importance of bone density in heat generation and thermal conductivity. The included studies have only claimed that the density is comparable to that of the human bone. Irrigation during Osteotomy The use of irrigation has been proven to decrease the amount of heat generation during osteotomy. Most of the studies have used external irrigation with saline solution, but the flow rate and solution temperature are either variant or not reported, which can affect the rigor of the studies. Drill Speed and Applied Load The speed and load used during osteotomy were also not consistent among the studies. It has been established that the increase or decrease in speed and load can affect the tem- perature change during implant site preparation. Hence, the results obtained in different studies are not comparable. Furthermore, not all studies reported these parameters. For instance, Koopaie et al22 and Er et al20 did not mention the used load in the experiments, while Tur et al16 did not mention both the speed and load. There are other concerns regarding the generalizability of results obtained from dead bone. First, in addition to bone density, thermal conductivity varies significantly between live and dead bone. This can be due to the difference in cellularity, water content, and fluid movement.23 Second, dead bone can be fresh or frozen and thawed. It is of paramount importance to know the baseline temperature of the bone block prior to experiment to appreciate the exact increase in the temperature during drilling. Ten of the included articles have used bovine bone specimens, which were frozen and then thawed in a thermostat-controlled water bath or immersed in saline solution to minimize the thermophysical and mechanical changes of the proteins in the dead bones. This also maintained the internal tempera- ture of the bone specimen before the start of the experiment. Only one study by Moshiri et al19 failed to explain the procedure used to maintain the bovine bones and the baseline temperature in the experiment. European Journal of Dentistry Vol. 18 No. 1/2024 © 2023. The Author(s). Depth and Diameter of the Osteotomy The temperature was measured in the included studies at different depths and for different diameters of the final drill. Koo et al14 suggested that the initial (pilot) drill should be changed after 50 uses, as this drill leads to more heat generation because it is the first drill to cut the bone in the osteotomy compared with the following drills, but most of the studies have measured temperature increase with the final drill as it can provide accurate temperature change because the thermocouple placement needs to be closer to the prepared osteotomy wall. Similarly, the temperature was measured for one particu- lar depth in all the studies instead of measuring throughout the osteotomy and the level of depth varies in each study. Again, this raises similar doubts in the validity of comparing the studies. Few of the studies have found that the superficial part of the osteotomy cavity generates more heat compared with the deeper parts.12,15 The study by Moshiri et al19 found that the temperature was higher at the depth of 6 mm when compared with that at 3 and 9 mm. The authors observed that this result is similar to other studies stating that there will be more heat generation during the preparation of the superficial part of the osteotomy site. The studies explained this finding by stating that friction is higher in the superficial part due to the higher content of cortical bone. This conclu- sion contradicts with many previous publications on the References 1 Chauhan CJ, Shah DN, Sutaria FB. Various bio-mechanical factors affecting heat generation during osteotomy preparation: a sys- tematic review. Indian J Dent Res 2018;29(01):81–92 2 Hein C, Inceoglu S, Juma D, Zuckerman L. Heat generation during bone drilling: a comparison between industrial and orthopaedic drill bits. J Orthop Trauma 2017;31(02):e55–e59 2 Hein C, Inceoglu S, Juma D, Zuckerman L. Heat generation during bone drilling: a comparison between industrial and orthopaedic drill bits. J Orthop Trauma 2017;31(02):e55–e59 3 Mishra SK, Chowdhary R. Heat generated by dental implant drills during osteotomy-a review: heat generated by dental implant drills. J Indian Prosthodont Soc 2014;14(02):131–143 3 Mishra SK, Chowdhary R. Heat generated by dental implant drills during osteotomy-a review: heat generated by dental implant drills. J Indian Prosthodont Soc 2014;14(02):131–143 There are many confounding factors in studies evaluating the heat generation during the osteotomy preparation. There- fore, standardization and uniformity in the study design is required to comparetheresultsand to make avalidconclusion. The present review attempted to standardize the processes of identifyingandappraising therelevantarticlesintheliterature despite the lack of standardized appraisal tools for in vitro studies. Finally, a comprehensive evaluation of all the relevant confounding factors within all the studies was conducted, which helped identify the missing gap of knowledge in the literature. The results of this study can be generalized to clinical practice with caution as the included articles were in vitro studies, with a significant dissimilarity between live human and dead animal bone. This, besides the remarkable heterogeneity and potential bias in the study methods. 4 Trisi P, Berardini M, Falco A, Vulpiani MP. Effect of temperature on the dental implant osseointegration development in low-density bone: an in vivo histological evaluation. Implant Dent 2015;24(01):96–100 4 Trisi P, Berardini M, Falco A, Vulpiani MP. Effect of temperature on the dental implant osseointegration development in low-density bone: an in vivo histological evaluation. Implant Dent 2015;24(01):96–100 5 Augustin G, Zigman T, Davila S, et al. Cortical bone drilling and thermal osteonecrosis. Clin Biomech (Bristol, Avon) 2012;27(04): 313–325 5 Augustin G, Zigman T, Davila S, et al. Cortical bone drilling and thermal osteonecrosis. Clin Biomech (Bristol, Avon) 2012;27(04): 313–325 6 Möhlhenrich SC, Modabber A, Steiner T, Mitchell DA, Hölzle F. Heat generation and drill wear during dental implant site prepa- ration: systematic review. Br J Oral Maxillofac Surg 2015;53(08): 679–689 7 Bayerlein T, Proff P, Richter G, Dietze S, Fanghänel J, Gedrange T. Temperature Changes Measurement Temperature Changes Measurement Thermocouples can detect only spot temperatures and not the overall thermal profile. Therefore, factors like depth of recording, distance of the sensor from the osteotomy site, and material of sensor element can affect the overall results. Nine of the included articles have used thermocouples for measuring the changes in the temperature during implant bed site preparation. But their use is not uniform, especially in respect to distance of the sensor from the osteotomy, Heat Generation and Implant Drill Materials Chakraborty et al. 71 which ranges from 0.2 to 1.5 mm. Sumer et al9 and Moshiri et al19 have not even mentioned the distance. However, Koopaie et al22 have used infrared thermometer for heat measurement, which is considered a better option as it provides the overall thermal profile with less degree of error. On the other hand, Scarano et al15 used infrared thermogra- phy for evaluating the change in temperature during implant bed preparation. There is no study in the literature that compares the accuracy of heat generation using both tech- niques. Hence, determining a preference is still difficult. Furthermore, despite the importance of calibrating the in- strument before the experiment to verify the precision of temperature recording (device reliability), only few of the reviewed studies have reported undertaking this using a proper method for calibration. osteotomy. It is important for clinicians to be aware of these findings, as they underscore the need to carefully inspect wear patterns and consider the appropriate drill material when performing osteotomy procedures. By taking these factors into account, clinicians can help minimize the risk of excessive heat generation, which can have a negative impact on implant success rates and treatment outcomes. Study Bias Blinding the operator and assessor is difficult in these studies as the operator needs to see the drills while performing the experiment and the assessor has to measure the heat instan- taneously. Having said that, these types of bias can be reduced by assigning different operators for each group along with an independent assessor. None of the studies have performed this, which makes them all proneto considerablebias. Another important source of bias observed in these studies is sponsor- ship bias. Some articles failed to acknowledge the source of funding andtoprovide a clear statementonconflictof interest. There are many confounding factors in studies evaluating the heat generation during the osteotomy preparation. There- fore, standardization and uniformity in the study design is required to comparetheresultsand to make avalidconclusion. The present review attempted to standardize the processes of identifyingandappraising therelevantarticlesintheliterature despite the lack of standardized appraisal tools for in vitro studies. Finally, a comprehensive evaluation of all the relevant confounding factors within all the studies was conducted, which helped identify the missing gap of knowledge in the literature. The results of this study can be generalized to clinical practice with caution as the included articles were in vitro studies, with a significant dissimilarity between live human and dead animal bone. This, besides the remarkable heterogeneity and potential bias in the study methods. Blinding the operator and assessor is difficult in these studies as the operator needs to see the drills while performing the experiment and the assessor has to measure the heat instan- taneously. Having said that, these types of bias can be reduced by assigning different operators for each group along with an independent assessor. None of the studies have performed this, which makes them all proneto considerablebias. Another important source of bias observed in these studies is sponsor- ship bias. Some articles failed to acknowledge the source of funding andtoprovide a clear statementonconflictof interest. Conflict of Interest None declared. Conflict of Interest None declared. Conclusion 10 Batista Mendes GC, Padovan LEM, Ribeiro-Júnior PD, Sartori EM, Valgas L, Claudino M. Influence of implant drill materials on wear, deformation, and roughness after repeated drilling and steriliza- tion. Implant Dent 2014;23(02):188–194 It is possible to conclude that the literature about the effect of drill materials on heat generation during osteotomy is not conclusive. The lack of standardization and uniformity in the study design, along with potential bias in the study meth- odology, makes it challenging to draw concrete conclusions based on the findings. However, the findings of this study can still serve as a helpful guide for clinicians seeking to optimize their approach to implant procedures. 11 Oliveira N, Alaejos-Algarra F, Mareque-Bueno J, Ferrés-Padró E, Hernández-Alfaro F. Thermal changes and drill wear in bovine bone during implant site preparation. A comparative in vitro study: twisted stainless steel and ceramic drills. Clin Oral Implants Res 2012;23(08):963–969 12 Harder S, Egert C, Wenz HJ, Jochens A, Kern M. Influence of the drill material and method of cooling on the development of intrabony temperature during preparation of the site of an implant. Br J Oral Maxillofac Surg 2013;51(01):74–78 References The use of ceramic drills on a zirconium oxide basis in bone preparation. Folia Morphol (Warsz) 2006;65(01):72–74 8 Scarano A, Carinci F, Quaranta A, Di Iorio D, Assenza B, Piattelli A. Effects of bur wear during implant site preparation: an in vitro study. Int J Immunopathol Pharmacol 2007;20(1, Suppl 1):23–26 9 Sumer M, Misir AF, Telcioglu NT, Guler AU, Yenisey M. Comparison of heat generation during implant drilling using stainless steel and ceramic drills. J Oral Maxillofac Surg 2011;69(05):1350–1354 Limitations of the Study To minimize the effect of clinical heterogeneity and con- founding factors on the study outcomes, this review focused solely on benchtop research related to the subject. Therefore, the generalizability of the findings to clinical practice may be limited. European Journal of Dentistry Vol. 18 No. 1/2024 © 2023. The Author(s). Clinical Implications 13 Allsobrook OFL, Leichter J, Holborrow D, Swain M. Descriptive study of the longevity of dental implant surgery drills. Clin Implant Dent Relat Res 2011;13(03):244–254 13 Allsobrook OFL, Leichter J, Holborrow D, Swain M. Descriptive study of the longevity of dental implant surgery drills. Clin Implant Dent Relat Res 2011;13(03):244–254 The findings of this review emphasize the significant impact of drill material and wear behavior on heat generation during 72 Heat Generation and Implant Drill Materials Chakraborty et al. 14 Koo KT, Kim MH, Kim HY, Wikesjö UME, Yang JH, Yeo IS. Effects of implant drill wear, irrigation, and drill materials on heat genera- tion in osteotomy sites. J Oral Implantol 2015;41(02):e19–e23 19 Moshiri Z, Roshanaei G, Vafaei F, Kadkhodazadeh M. Evaluation the effect of drill type on heat generation in implant drilling site. Res J Med Sci 2013;7(5–6):118–122 15 Scarano A, Lorusso F, Noumbissi S. Infrared thermographic evalu- ation of temperature modifications induced during implant site preparation with steel vs. zirconia implant drill. J Clin Med 2020;9 (01):148 20 Er N, Alkan A, Ilday S, Bengu E. Improved dental implant drill durability and performance using heat and wear resistant protec- tive coatings. J Oral Implantol 2018;44(03):168–175 21 Hochscheidt CJ, Shimizu RH, Andrighetto AR, Moura LM, Golin AL, Hochscheidt RC. Thermal variation during osteotomy with differ- ent dental implant drills: a standardized study in bovine ribs. Implant Dent 2017;26(01):73–79 16 Tur D, Giannis K, Unger E, Mittlböck M, Rausch-Fan X, Strbac GD. Thermal effects of various drill materials during implant site preparation—ceramic vs. stainless steel drills: a comparative in vitro study in a standardised bovine bone model. Clin Oral Implants Res 2021;32(02):154–166 22 Koopaie M, Kolahdouz S, Kolahdouz EM. Comparison of wear and temperature of zirconia and tungsten carbide tools in drilling bone: in vitro and finite element analysis. Br J Oral Maxillofac Surg 2019;57(06):557–565 17 Yoshida K, Uoshima K, Oda K, Maeda T. Influence of heat stress to matrix on bone formation. Clin Oral Implants Res 2009;20(08): 782–790 23 Tehemar SH. Factors affecting heat generation during implant site preparation: a review of biologic observations and future considerations. Int J Oral Maxillofac Implants 1999;14(01): 127–136 23 Tehemar SH. Factors affecting heat generation during implant site preparation: a review of biologic observations and future considerations. Int J Oral Maxillofac Implants 1999;14(01): 127–136 18 Eriksson RA, Adell R. Temperatures during drilling for the place- ment of implants using the osseointegration technique. European Journal of Dentistry Vol. 18 No. 1/2024 © 2023. The Author(s). Clinical Implications J Oral Maxillofac Surg 1986;44(01):4–7
https://openalex.org/W3190795812
https://www.biorxiv.org/content/biorxiv/early/2021/08/04/2021.08.04.455019.full.pdf
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Probing the stability of the SpCas9-DNA complex after cleavage
bioRxiv (Cold Spring Harbor Laboratory)
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Probing the stability of the SpCas9-DNA complex after cleavage Pierre Aldag1, Fabian Welzel1, Leonhard Jakob2, Andreas Schmidbauer2, Marius Rutkauskas1, Fergus Fettes1, Dina Grohmann2,3, and Ralf Seidel1,* 1 Peter Debye Institute for Soft Matter Physics, University of Leipzig, Leipzig, 04103, Germany 2 Institute of Microbiology & Archaea Centre, Single-Molecule Biochemistry Lab, University of Regensburg, Regensburg, 93053, Germany 3 Regensburg Center of Biochemistry (RCB), University of Regensburg, 93053 Regensburg, Germany * To whom correspondence should be addressed. Tel: +49 341 9732501; Email: ralf.seidel@physik.uni- leipzig.de Present Address: Leonhard Jakob, Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Regensburg, Regensburg, 03053, Germany Pierre Aldag1, Fabian Welzel1, Leonhard Jakob2, Andreas Schmidbauer2, Marius Rutkauskas1, Fergus Fettes1, Dina Grohmann2,3, and Ralf Seidel1,* 1 Peter Debye Institute for Soft Matter Physics, University of Leipzig, Leipzig, 04103, Germany 2 Institute of Microbiology & Archaea Centre, Single-Molecule Biochemistry Lab, University of Regensburg, Regensburg, 93053, Germany g g y ( ) y g g g g y * To whom correspondence should be addressed. Tel: +49 341 9732501; Email: ralf.seidel@physik.uni- leipzig.de * To whom correspondence should be addressed. Tel: +49 341 9732501; Email: ralf.seidel@physik.uni- leipzig.de * To whom correspondence should be addressed. Tel: +49 341 9732501; Email: ralf.seidel@physik.uni- leipzig.de Present Address: Leonhard Jakob, Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Regensburg, Regensburg, 03053, Germany Present Address: Leonhard Jakob, Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Regensburg, Regensburg, 03053, Germany Present Address: Leonhard Jakob, Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Regensburg, Regensburg, 03053, Germany Present Address: Leonhard Jakob, Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Regensburg, Regensburg, 03053, Germany . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprin this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455019 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprin this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455019 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455019 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455019 doi: bioRxiv preprint ABSTRACT CRISPR-Cas9 is a ribonucleoprotein complex that sequence-specifically binds and cleaves double- stranded DNA. Wildtype Cas9 as well as its nickase and cleavage-incompetent mutants have been used in various biological techniques due to their versatility and programmable specificity. Cas9 has been shown to bind very stably to DNA even after cleavage of the individual DNA strands, inhibiting further turnovers and considerably slowing down in-vivo repair processes. This poses an obstacle in genome editing applications. Here, we employed single-molecule magnetic tweezers to investigate the binding stability of different S. pyogenes Cas9 variants after cleavage by challenging them with supercoiling. We find that different release mechanisms occur depending on which DNA strand is cleaved. After non-target strand cleavage, supercoils are immediately but slowly released by swiveling of the non-target strand around the DNA with friction. Consequently, Cas9 and its non-target strand nicking mutant stay stably bound to the DNA for many hours even at elevated torsional stress. After target-strand cleavage, supercoils are only removed after the collapse of the R-loop. We identified several states with different stabilities of the R-loop. Most importantly, we find that the post-cleavage state of Cas9 exhibits a higher stability compared to the pre-cleavage state. This suggests that Cas9 has evolved to remain tightly bound to its cut target. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455019 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455019 doi: bioRxiv preprint INTRODUCTION The TS and NTS are cleaved by the HNH and the RuvC domains, respectively, followed by a further conformational change into a post-cleavage product state (16,12). In this state, the HNH domain undocks from the cleavage site and is thought to become completely disordered. After inducing a double-strand break, an extremely slow release of the DNA products essentially limits SpCas9 to undergo further turnovers (17). This results in an inhibited and slow in-vivo DNA repair process (18,19). However, even for target bound SpCas9, the 3’ end of the cleaved NTS can be released and targeted by complementary ssDNA or 3’ to 5’ ssDNA exonucleases (16,19). CRISPR (Clustered Regularly Interspaced Palindromic Repeats)-Cas (CRISPR associated) systems constitute adaptive immune systems against nucleic acid-containing invaders in prokaryotic cells (1). CRISPR-Cas9 from the bacterial species Streptococcus pyogenes (SpCas9) is a 160 kD protein that forms a ribonucleoprotein (RNP) complex with CRISPR-RNA (crRNA) and trans-activating RNA (tracrRNA). This complex can site-specifically target double-stranded DNA (dsDNA) and induces a double-strand break using its RuvC and HNH nuclease domains (2–4). Since its discovery, SpCas9 and other Cas9 variants have been widely used in rapidly emerging genome engineering applications in prokaryotic, plant and animal cells (5–7). The target specificity is mainly encoded by the 20 nt spacer region at the 5’ end of the crRNA of the SpCas9 RNP complex. The tracrRNA and crRNA can be replaced by a single-guide RNA chimera (sgRNA) (4) which simplifies RNP formation. A prerequisite for site-specific DNA targeting is a three-nucleotide PAM sequence (protospacer-adjacent motif, NGG for SpSpas9) next to the target site. SpCas9 weakly interacts with suitable PAM sites via facilitated diffusion (8). PAM recognition triggers an initial melting of the DNA double strand and primes base pairing between the crRNA spacer and the DNA target strand (TS) while displacing the non-target DNA strand (NTS) such that a so-called R-loop structure is formed (9). R-loop expansion is initiated from the PAM proximal nucleotides over the entire spacer region and constitutes the actual recognition of matching targets. In case of strongly mismatched targets, R-loop expansion is impeded and stops or the R-loop collapses (10). Successful base pairing between TS and crRNA spacer is verified by two conformational changes of the SpCas9 RNP. Formation of an R-loop intermediate of 9 bp length triggers the transition into a “checkpoint” state, opening up a channel to further accommodate the TS-crRNA hybrid. INTRODUCTION CRISPR (Clustered Regularly Interspaced Palindromic Repeats)-Cas (CRISPR associated) systems constitute adaptive immune systems against nucleic acid-containing invaders in prokaryotic cells (1). CRISPR-Cas9 from the bacterial species Streptococcus pyogenes (SpCas9) is a 160 kD protein that forms a ribonucleoprotein (RNP) complex with CRISPR-RNA (crRNA) and trans-activating RNA (tracrRNA). This complex can site-specifically target double-stranded DNA (dsDNA) and induces a double-strand break using its RuvC and HNH nuclease domains (2–4). Since its discovery, SpCas9 and other Cas9 variants have been widely used in rapidly emerging genome engineering applications in prokaryotic, plant and animal cells (5–7). The target specificity is mainly encoded by the 20 nt spacer region at the 5’ end of the crRNA of the SpCas9 RNP complex. The tracrRNA and crRNA can be replaced by a single-guide RNA chimera (sgRNA) (4) which simplifies RNP formation. A prerequisite for site-specific DNA targeting is a three-nucleotide PAM sequence (protospacer-adjacent motif, NGG for SpSpas9) next to the target site. SpCas9 weakly interacts with suitable PAM sites via facilitated diffusion (8). PAM recognition triggers an initial melting of the DNA double strand and primes base pairing between the crRNA spacer and the DNA target strand (TS) while displacing the non-target DNA strand (NTS) such that a so-called R-loop structure is formed (9). R-loop expansion is initiated from the PAM proximal nucleotides over the entire spacer region and constitutes the actual recognition of matching targets. In case of strongly mismatched targets, R-loop expansion is impeded and stops or the R-loop collapses (10). Successful base pairing between TS and crRNA spacer is verified by two conformational changes of the SpCas9 RNP. Formation of an R-loop intermediate of 9 bp length triggers the transition into a “checkpoint” state, opening up a channel to further accommodate the TS-crRNA hybrid. This channel is formed by a conformational change of the REC2 and REC3 domains (11,12). In this state, the HNH domain is still positioned more than 30 Å away from the cleavage site in the TS. The second transition into the “docked” state is induced once the R-loop reaches the PAM-distal end, causing a large movement of the HNH domain to reach its site of catalytic activity (13–15). Mg2+ ions as cofactor and a maximum of three terminal mismatches are prerequisites for the transition into the docked state that allows DNA cleavage. INTRODUCTION CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455019 doi: bioRxiv preprint Cas9-mediated functionalities (22,23,21). While a large body of research has investigated the structure of the conformational states of SpCas9 during R-loop formation and after DNA cleavage, the stability of the SpCas9-DNA complex in these states and their dependence on supercoiling remains elusive. Developing a thorough understanding of the post-cleavage stability of SpCas9 and the respective mutants can be helpful to improve the efficiencies of genome editing techniques. Here, we investigated the post-cleavage state stability of SpCas9 and its respective nickase mutants by challenging it with supercoiling using single-molecule magnetic tweezers experiments (9,24,25). We show that SpCas9 bound to a target with a dsDNA break or a nicked NTS can easily relax superhelical tension. It can thus not be dissociated in processes that generate this kind of mechanical stress, such as transcription and replication (26–28). In contrast, SpCas9 bound to an intact target or a target with a nicked TS cannot release superhelical tension and can thus be dissociated from the DNA when elevated tension is induced. In both cases, protein states with different stabilities are found indicating dynamic structural transitions in the post-celavage state. We show that dissociation from a target DNA with a nicked TS requires considerably higher levels of supercoiling compared to an intact target, suggesting an increased stability of the post-cleavage conformational state. DNA substrates for magnetic tweezers measurements Double-stranded DNA constructs for magnetic tweezers experiments with lengths of 1500 bp and 2700 bp were prepared as previously described (25). A single copy of a given protospacer matching the spacer of the SpCas9 sgRNA and including a 5’-AGG-3’PAM on the non-target strand (see Table S1) was cloned into the SmaI site of a pUC19 plasmid. From the plasmid, a 1.5 kbp or 2.7 kbp fragment including the SpCas9 target site was amplified by PCR, using primers in which either a NotI or a SpeI restriction enzyme site was introduced (see Table S1). After digestion with NotI and SpeI, the fragment was ligated at either end to ~600-bp-PCR fragments containing multiple biotin (SpeI site) or digoxigenin (NotI site) modifications (29). INTRODUCTION This channel is formed by a conformational change of the REC2 and REC3 domains (11,12). In this state, the HNH domain is still positioned more than 30 Å away from the cleavage site in the TS. The second transition into the “docked” state is induced once the R-loop reaches the PAM-distal end, causing a large movement of the HNH domain to reach its site of catalytic activity (13–15). Mg2+ ions as cofactor and a maximum of three terminal mismatches are prerequisites for the transition into the docked state that allows DNA cleavage. The TS and NTS are cleaved by the HNH and the RuvC domains, respectively, followed by a further conformational change into a post-cleavage product state (16,12). In this state, the HNH domain undocks from the cleavage site and is thought to become completely disordered. After inducing a double-strand break, an extremely slow release of the DNA products essentially limits SpCas9 to undergo further turnovers (17). This results in an inhibited and slow in-vivo DNA repair process (18,19). However, even for target bound SpCas9, the 3’ end of the cleaved NTS can be released and targeted by complementary ssDNA or 3’ to 5’ ssDNA exonucleases (16,19). Introducing inactivating point mutations at positions D10A (RuvC) or H840A (HNH) turns SpCas9 into TS or NTS nickases (4). Cas9 nickase mutants and cleavage-incompetent Cas9, which carries both mutations, have been used in various genome editing applications to increase specificity or to avoid double-strand breaks (20,21). Fusion of Cas9 variants with other enzymes, such as the repair enzyme Rad51, the endonuclease FokI, or an engineered reverse transcriptase, expanded the repertoire of Introducing inactivating point mutations at positions D10A (RuvC) or H840A (HNH) turns SpCas9 into TS or NTS nickases (4). Cas9 nickase mutants and cleavage-incompetent Cas9, which carries both mutations, have been used in various genome editing applications to increase specificity or to avoid double-strand breaks (20,21). Fusion of Cas9 variants with other enzymes, such as the repair enzyme Rad51, the endonuclease FokI, or an engineered reverse transcriptase, expanded the repertoire of . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455019 doi: bioRxiv preprint . Preparation of SpCas9 Plasmids for the expression of wildtype SpCas9 and the catalytically inactive SpCas9 variants carrying the mutations D10A/H840A were obtained from Addgene (plasmid ID: 39312 and 39318). SpCas9 variants that carried a single mutation of the catalytic site (D10A or H840A) were generated by site- directed mutagenesis using the wildtype SpCas9 expression plasmid as template. Expression was performed in E. coli BL21 (DE3) cells. Expression and cell lysis conditions were chosen as described by Jinek et al. (4). The cleared cell lysate was applied to a HisTrap FF Ni-NTA affinity column (GE healthcare) equilibrated with HisA buffer (20 mM HEPES pH 7.5, 100 mM NaCl, 5 mM MgCl2, 5% (v/v) glycerol, 20 mM imidazole). The column was washed with 2 column volumes (cv) of HisA buffer and 10 cv of HisA buffer containing 1 M NaCl to liberate unspecifically bound nucleic acids from SpCas9. Subsequently, the column was equilibrated with 2 cv of HisA buffer and pre-eluted with 2 cv buffer HisA with increased imidazole concentration of 40 mM. In the final elution step, the column was washed with 5 cv elution buffer (20 mM HEPES pH 7.5, 100 mM NaCl, 5 mM MgCl2, 5% (v/v) glycerol, 200 mM imidazole). Elution fractions were pooled and digested with 50 U AcTEV protease (Thermo Fisher Scientific) over night at 4 °C. The cleaved His6-MBP-tag was removed by gel filtration using a HiLoad 16/60 Superdex 200 column (GE healthcare) equilibrated in size exclusion buffer (20 mM Tris-HCl, 150 mM NaCl, 5 mM MgCl2, 5% (v/v) glycerol, 1 mM DTT). Elution fractions contained nucleic acid free SpCas9 or SpCas9 variants (A260/280 < 0.6). Fractions were flash frozen in liquid nitrogen and stored at -80 °C. sgRNA synthesis for magnetic tweezers experiments The template for T7 transcription of the SpCas9 single sgRNA was generated by PCR, using the High Fidelity Phusion Polymerase (Thermo Fisher Scientific) and overlapping oligonucleotides. RNAs were produced by in vitro transcription using the TranscriptAid T7 High Yield Transcription Kit (Thermo Fisher Scientific) and purified using the GeneJet RNA Cleanup and Concentration Kit (Thermo Fisher Scientific). The sequences of the oligonucleotides and sgRNA used in this study are available in Table S1. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455019 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455019 doi: bioRxiv preprint Reconstitution of guideRNA-SpCas9 complexes For characterizing DNA cleavage, SpCas9 was pre-loaded with either a sgRNA or a pre-annealed crRNA:tracrRNA complex (Table S1 and S2). To reconstitute SpCas9-RNA complexes, a stock solution containing 1 µM SpCas9 and 1 µM guide RNA in 1x Cas Buffer (20 mM Tris–HCl pH 7.5, 100 mM NaCl, 10 mM MgCl2, 2% (v/v) glycerol, 1 mM DTT, 0.05% (v/v) Tween20) was incubated for 10 minutes at room temperature followed by a centrifugation step (20.000 g, 5 min, room temperature). Plasmid-DNA cleavage assays As substrate for plasmid-DNA cleavage assays, we cloned target plasmid (pM53.1; 6290 bp) containing the sequence of the S. cerevisiae PHO5 gene with its native genomic context 500 bp upstream and downstream of the gene in a pBluescript vector backbone. The PHO5 locus was amplified from yeast genomic DNA using primers 5’-GGCCGCAGATCAAGTCAGAG-3’ and 5’- GCAAGTCACGAGAAATACCA-3’. The gene is flanked by a Z. rouxii sequence-specific recombinase R recognition site (RS) upstream, and a triple E. coli LexA binding site downstream of the gene followed by another RS element. The plasmid was generated making use of pM49.2 containing the upstream RS element and the downstream triple LexA binding site and RS element (for details of the cloning procedure see (30)). Reconstituted SpCas9-sgRNA complexes were added in 10-fold excess to 5 nM pM53.1 plasmid DNA containing the target sequence and a 5’-AGG-3’ PAM motif in 1x Cas buffer. For kinetic analysis, 15 µl samples were taken at different time points and the reactions were stopped by addition of EDTA (83 mM final concentration). Subsequently, SpCas9 was digested by the addition of . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455019 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455019 doi: bioRxiv preprint 0.36 U Proteinase K (Thermo Fisher Scientific) followed by an incubation at 55 °C for one hour. After Proteinase K digestion, 6x DNA loading dye (10 mM Tris-HCl pH 7.5, 0.03% bromophenol blue (w/v), 60% glycerol (v/v), 6 mM EDTA) was added to the samples and fragments were resolved on a 0.9% agarose gel. Cleavage products were visualized using a ChemiDoc Imaging System (Bio-Rad) after ethidium bromide staining of the agarose gel. Short target DNA cleavage assay for determination of SpCas9 strand-specific cleavage Fluorescently labeled double-stranded target DNA was formed annealing two single-stranded oligonucleotides. Single strands were purchased from Microsynth Seqlab (Göttingen, Germany) with a fluorescent label coupled to the 5‘-end. Plasmid-DNA cleavage assays The target strand carried a Cy3 label and the non-target strand a Cy5 label (Table S2). Annealing was performed by incubation of equimolar amounts of TS and NTS in annealing buffer (final concentration: 3 mM HEPES pH 7.4, 10 mM CH3COOK, 0.2 mM Mg(CH3COO)2 at 95 °C for 3 min and passive cool-down to room temperature. The reconstituted SpCas9-crRNA:tracrRNA complex (final concentration of 250 nM) was added to 5 nM of the double- stranded target DNA in a total volume of 10 µl in 1x Cas buffer. Cleavage reactions were incubated at 37 °C for 1 h and the reaction was stopped by the addition of 0.36 U Proteinase K (Thermo Fisher Scientific), followed by incubation at 55 °C for 45 min. The samples were mixed with loading dye (final concentration: 47.5% (v/v) formamide, 0.01% (w/v) SDS, 0.01% (w/v) bromophenol blue, 0.005% (w/v) xylene cyanol, 0.5 mM EDTA). Immediately prior to loading of the samples, the samples were denatured at 95 °C for 5 min. Denatured samples were separated for 30 min on a pre-heated 15% PAA, 6 M urea, 1× TBE gel (300 V, 45 min). Cleavage products were visualized using a ChemiDoc Imaging System (Bio-Rad). Magnetic Tweezers experiments The measurements were performed in a custom-built magnetic tweezers setup allowing the GPU- assisted real-time tracking of the DNA length for up to 100 DNA molecules in parallel (31). DNA constructs for the tweezers experiments were bound to magnetic beads of 1 µm-diameter (MyOne; Invitrogen) and flushed into the glass flowcell of the setup allowing the anchoring of the digoxigen- modified end to the anti-digoxigenin coated surface of the cell (29,32). After removing unbound beads by flushing, force was applied by lowering the magnets towards the flow cell and DNA tethered beads were selected. Using bead tracking with a camera (Mikrotron EoSens), the length of the individual DNA molecules was determined and the applied forces were calibrated (33). During experiments, desired forces on the DNA construct could be set by placing the magnets at a particular distance from the flow cell according to the calibration results. Supercoiling of DNA was achieved by turning the magnets. The resulting torque that depended mainly on the stretching force was calculated based on previous theoretical work (34,35). Time trajectories of the DNA length were recorded at 120 Hz and smoothed with a sliding average to 6 Hz for analysis. For measurements with SpCas9, reconstituted SpCas9- sgRNA was added in tweezers buffer (20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10 mM MgCl2, and 1 mg/ml BSA) at a concentration of 0.25 nM or 0.5 nM. After adding SpCas9, DNA length changes were . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455019 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455019 doi: bioRxiv preprint monitored in real-time. For experiments where we exclusively challenged the DNA-bound SpCas9 complex, SpCas9 was allowed to prebind to negatively supercoiled DNA in the flowcell for 20 minutes to ensure efficient R-loop formation on most molecules before the experiment was started. The successful formation of an R-loop was confirmed by the occurrence of a pronounced change in DNA length. Data Analysis Data analysis for the tweezers experiments was performed with Origin (OriginLab) and custom-built Matlab (The Mathworks Inc.) scripts. Supercoil release velocities Supercoil release velocities For Cas9dHNH experiments, a slow supercoil release was observed. The velocities of the release were determined in Matlab by performing linear fits to the individual release events (see Fig. 2). The velocities at each particular torque were then plotted in a histogram and fitted with a Gamma distribution. The Gamma distribution describes the total waiting time 𝑡 of 𝑛 successive events with exponentially distributed dwell times with mean value 𝜆. The Gamma distribution is given by: 𝑓(𝑡) = 𝑒−𝜆𝑡⋅𝜆𝑛⋅𝑡𝑛−1 (𝑛−1)! . 𝑓(𝑡) = 𝑒−𝜆𝑡⋅𝜆𝑛⋅𝑡𝑛−1 (𝑛−1)! . 𝑓(𝑡) = 𝑒−𝜆𝑡⋅𝜆𝑛⋅𝑡𝑛−1 (𝑛−1)! . By using this distribution, we assume that SpCas9 releases the non-target strand one turn at a time, with each turn released after an exponentially distributed time with the same mean waiting time 1/𝜆. R-loop collapse times To determine transition times, R-loop transitions into the intermediate state or full collapse were fitted by step functions in Matlab. Survival plots of the collapse times were then fitted with bi- or tri-exponential functions using Origin. Investigating the post-cleavage behavior of SpCas9 in single-molecule multiplex magnetic tweezers measurements To investigate the post-cleavage behavior of different SpCas9 variants, we employed a single-molecule DNA twisting assay on a high-throughput magnetic tweezers platform (31). Numerous 2.7 kbp dsDNA molecules containing a single SpCas9 target site matching the crRNA spacer and multiple biotin or digoxigenin modifications at either end were attached on the respective DNA end to 1 µm streptavidin- coated superparamagnetic beads and to the antidigoxigenin-coated surface of the flowcell of the setup. The three-dimensional positions of multiple DNA-tethered beads were tracked in parallel in a home- built microscope (36) allowing the real-time observation of the DNA length of the molecules (Fig. 1A). The field gradient from a pair of magnets enabled the application of force onto the DNA molecule. The . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455019 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455019 doi: oRxiv preprint . CC-BY 4.0 International license made available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455019 doi: oRxiv preprint force could be controlled by adjusting the distance between the magnets and the flow cell. Turning the magnets allowed supercoiling of the DNA molecules in parallel. To monitor R-loop formation by SpCas9, we applied negative supercoiling which reduces the DNA length due to writhe formation (Fig 1B, top). R-loop formation results in untwisting of the DNA by about two turns, which absorbs part of the applied supercoiling such that it becomes visible as a sudden increase in the DNA length (Fig. 1B bottom). Figure 1. (A) Schematic representation of the magnetic tweezers assay used to twist single double- stranded DNA molecules that are tethered at either end to the surface of the fluidic cell and to a magnetic microbead. Investigating the post-cleavage behavior of SpCas9 in single-molecule multiplex magnetic tweezers measurements CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455019 doi: bioRxiv preprint As was previously shown for cleavage-incompetent dCas9 and other CRISPR-Cas effector complexes, the application of positive twist on DNA can promote the collapse of the R-loop and dissociation of the complex (9,25,10). In a simplified view, the R-loop is wrung out with the opposing superhelicity. At positive supercoiling, R-loop collapse on intact target DNA is also observable as a sudden DNA length change that, however, occurs in two steps (Fig. 1D, first trajectory). The first step corresponds to the transition into the checkpoint state, corresponding to the formation of the partial R-loop, while the second step corresponds to the total collapse of the R-loop and typically the dissociation of the protein. We started to test the stability of the post-cleavage product state by challenging R-loops formed by wildtype SpCas9 (Cas9wt thereafter) (Fig. 1C), SpCas9 with a mutated HNH domain (Cas9dHNH thereafter), and SpCas9 with a mutated RuvC domain (Cas9dRuvC thereafter) with positive supercoiling. Even though Cas9wt induced double-strand breaks (DSBs) within ~2 min after enzyme addition (Fig. S1), the DNA tethers in the tweezers stayed in place (Fig. S2) for several hours, indicating that Cas9wt remained stably bound to both DNA ends as previously reported (13,16). We observed different behaviors of post-cleavage SpCas9-DNA complexes depending on which strand was cleaved (Fig. 1D). For Cas9wt we did not observe the characteristic R-loop collapse with a 2-step supercoil release limited to ~2 turns. Instead, an immediately starting but slow release of all applied supercoiling was observed (Fig. 1D, second trajectory). This behavior resembled that of eukaryotic topoisomerase IB, which nicks DNA and releases twist by a free swivel mechanism in which the release velocity is reduced by protein friction (37). Cas9dHNH, which solely nicks the NTS of the DNA, showed a similar behavior as Cas9wt indicating that also in this case a swivel mechanism causes the slow release of supercoiling (Fig. 1D, third trajectory). In contrast, for Cas9dRuvC, which induces a nick on the TS, no immediately starting supercoil release was observed. Instead, the supercoiling level remained constant for prolonged times and was completely released in a fast, sudden step (Fig. Investigating the post-cleavage behavior of SpCas9 in single-molecule multiplex magnetic tweezers measurements 1D, fourth trajectory). Such fast supercoil release is indicative of a DNA nick in absence of bound protein (38) suggesting that Cas9dRuvC suddenly dissociated leaving a free nick behind. This idea was supported by the observation that the fast release of all supercoils was frequently preceeded by a short substep indicative of the first step of R-loop collapse as seen for dCas9 (see small step in Fig. 1D, fourth trajectory). Investigating the post-cleavage behavior of SpCas9 in single-molecule multiplex magnetic tweezers measurements A pair of permanent magnets allows the application of a stretching force and molecule twisting. Up to one hundred beads were tracked in parallel (right: full microscope image with enlarged view of a single bead in the inset). (B) Top: Representative DNA supercoiling curve taken at 0.3 pN showing the characteristic DNA length reduction due to writhe formation upon twisting. Bottom: Representative time trajectory of the DNA length of a negatively supercoiled molecule (- 6 turns, 0.3 pN) exhibiting a DNA length jump due to R-loop formation by Cas9wt. (C) Schematic representation of the SpCas9-sgRNA ribonucleoprotein complex forming an R-loop on double-stranded DNA. The scissors indicate the active sites of the RuvC and HNH nuclease domains. The orange section on the NTS represents the PAM. (D) Representative time trajectories when challenging R-loops formed by dCas9, Cas9wt, Cas9dHNH, and Cas9dRuvC with positive supercoiling. Figure 1. (A) Schematic representation of the magnetic tweezers assay used to twist single double- stranded DNA molecules that are tethered at either end to the surface of the fluidic cell and to a magnetic microbead. A pair of permanent magnets allows the application of a stretching force and molecule twisting. Up to one hundred beads were tracked in parallel (right: full microscope image with enlarged view of a single bead in the inset). (B) Top: Representative DNA supercoiling curve taken at 0.3 pN showing the characteristic DNA length reduction due to writhe formation upon twisting. Bottom: Representative time trajectory of the DNA length of a negatively supercoiled molecule (- 6 turns, 0.3 pN) exhibiting a DNA length jump due to R-loop formation by Cas9wt. (C) Schematic representation of the SpCas9-sgRNA ribonucleoprotein complex forming an R-loop on double-stranded DNA. The scissors indicate the active sites of the RuvC and HNH nuclease domains. The orange section on the NTS represents the PAM. (D) Representative time trajectories when challenging R-loops formed by dCas9, Cas9wt, Cas9dHNH, and Cas9dRuvC with positive supercoiling. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455019 doi: bioRxiv preprint . Bound Cas9dHNH releases supercoiling through detachment of the NTS in a swivel-like manner governed by torque and friction Next, we investigated the different responses of the SpCas9 variants to supercoiling after DNA cleavage in more detail. Both, Cas9wt and Cas9dHNH released supercoiling slowly by a swivel mechanism (Fig. 2A). The slow supercoil release was observed repeatably, indicating that the protein remained stably bound to the target. Otherwise, supercoils would be very rapidly released by a protein-free nick (38) and the introduction of additional supercoils in the DNA would be inhibited (as observed for Cas9dRuvC, see below). Hence, we observed a similar behavior for the Cas9wt and Cas9dHNH variants, which both nick the NTS, suggesting that occasional detachment of the NTS from the protein enables the swiveling/supercoil release (Fig. 2B). Despite the presence of a TS nick for Cas9wt, the DNA tethers did not rupture during supercoil release, indicating that the TS is tightly bound. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455019 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455019 doi: bioRxiv preprint For Cas9dHNH, we carefully characterized the supercoil release velocity as function of the applied positive and negative supercoiling levels. For both supercoiling directions, a similar release behavior was recorded. Application of different forces allowed varying the acting torque during supercoil release. We measured the velocity of individual release events by applying a linear fit through each event. At each respective torque, we determined the linear slopes of the supercoiling curve which allowed us to convert the velocity from nanometers per second to turns per second. The distributions of the release velocities had a pronounced maximum and were well described by a Gamma distribution (Fig. 2C). The Gamma distribution describes a process containing 𝑛 substeps with exponentially distributed dwell times (see Methods). Figure 2. (A) Representative time trajectories of repeated supercoil release measured with magnetic tweezers (smoothed to 6Hz). Upon repeated step-wise DNA supercoiling, the DNA length is shortened. Bound Cas9dHNH releases supercoiling through detachment of the NTS in a swivel-like manner governed by torque and friction It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455019 doi: bioRxiv preprint For the parameter which describes the number of substeps in the process, we obtained on average 𝑛 = 6.6 ± 1.3 and 𝑛 = 4.3 ± 0.3 for positive and negative supercoiling, respectively. These numbers agree well with the number of turns within the linear regime of the supercoiling curve, indicating the release of supercoils one turn at a time. The mean release velocities approximately showed an exponential increase with the applied torque for positive and negative supercoiling (Fig. 2D). Following Arrhenius transition-state rate theory, we modeled the supercoil release as a step-wise process in which supercoils can be released (or introduced) as single full turns. For this process, a periodic energy landscape can be drawn with energy minima at full turns (corresponding to the NTS docked onto Cas9dHNH) and transition state maxima in between (corresponding to a detached NTS and partially turned DNA) (Fig. 2E). Applied torque tilts the energy landscape to favor supercoil release. The torque dependence can be described using an exponential Arrhenius-term in which the activation energy required to reach the transition state is changed by the work to reach the transition state in presence of the torque. To consider supercoil release in positive and negative direction and to ensure a zero net release at zero torque, two torque-dependent Arrhenius-terms were used: 𝑣(𝛤) = 𝑣0 ⋅(exp (𝛤⋅ 2𝜋⋅Δ𝑁+ 𝑘𝑏𝑇 ) −exp (−𝛤⋅2𝜋⋅Δ𝑁− 𝑘𝑏𝑇 )), (1) (1) where 𝑣0 is the spontaneous swiveling rate in either direction at zero torque, 𝛤 is the applied torque, and Δ𝑁+/− are the angular distances of the transition state from the energy minima for positive and negative supercoil release, respectively. Fitting this model to the data in Figure 2C resulted in transition state distances of Δ𝑁+= 0.4 ± 0.1 turns and Δ𝑁−= 0.3 ± 0.1, indicating that the transition state is located approximately half a turn from the energy minima. The swiveling rate at zero torque was 𝑣0= 0.04 ± 0.01 turns/s, i.e. Cas9dHNH would spontaneously introduce or deplete a single supercoil every ~25s even for relaxed DNA. Overall, this data supports a model in which Cas9dHNH releases the NTS by a swiveling mechanism governed by torque and friction. Due to the slight difference in the transition state distance, the release occurred slightly faster at positive than negative torque. Bound Cas9dHNH releases supercoiling through detachment of the NTS in a swivel-like manner governed by torque and friction Supercoil release can be detected as a slow increase in DNA length, which is limited by friction due to the presence of Cas9dHNH. (B) Cartoon illustrating the supercoil release for DNA nicked by Cas9dHNH. When the NTS of a supercoiled dsDNA is cleaved, the applied torque is released by swivelling of the NTS around the TS. The swivelling is sterically obstructed by the protein. (C) Histogram of the measured supercoil release velocities given in turns per second (blue bars). The red line shows a fit with a Gamma distribution. (D) Torque-dependence of the supercoil release velocity (blue). The red line shows a fit of the data with Eq. (1) (E) Hypothetic scheme of the energy landscape for NTS release. The energy landscape is tilted downhill if positive or negative torque is applied (red line) in contrast to the absence of torque (blue line). Figure 2. (A) Representative time trajectories of repeated supercoil release measured with magnetic tweezers (smoothed to 6Hz). Upon repeated step-wise DNA supercoiling, the DNA length is shortened. Supercoil release can be detected as a slow increase in DNA length, which is limited by friction due to the presence of Cas9dHNH. (B) Cartoon illustrating the supercoil release for DNA nicked by Cas9dHNH. When the NTS of a supercoiled dsDNA is cleaved, the applied torque is released by swivelling of the NTS around the TS. The swivelling is sterically obstructed by the protein. (C) Histogram of the measured supercoil release velocities given in turns per second (blue bars). The red line shows a fit with a Gamma distribution. (D) Torque-dependence of the supercoil release velocity (blue). The red line shows a fit of the data with Eq. (1) (E) Hypothetic scheme of the energy landscape for NTS release. The energy landscape is tilted downhill if positive or negative torque is applied (red line) in contrast to the absence of torque (blue line). . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455019 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. TS cleavage causes two differently stable SpCas9 states In a next step, we explored the stability of Cas9dRuvC in the post-cleavage state on DNA when subjected to supercoiling. Cas9dRuvC only cleaves the TS, such that supercoils cannot be released by NTS detachment and swiveling around the protein (Fig. 3A). Experimentally, this is seen as a constant supercoiling level over extended time periods (Fig. 3B). Full supercoil release can be observed as a sudden rapid step similar to the free nick generated by a nicking enzyme (38). This suggests that the twist is only released after R-loop collapse. Notably, the sudden supercoil release due to R-loop collapse is preceeded by a substep corresponding to a partial R-loop collapse i.e. the transition into the intermediate R-loop state. Subsequent repeated introduction of additional supercoiling failed, indicating that the enzyme dissociated upon R-loop collapse. After a waiting time of several minutes, the introduction of supercoils became possible again, indicating that a new Cas9dRuvC molecule had bound to the nicked target site (Fig S3). . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455019 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455019 doi: bioRxiv preprint We carefully investigated the stability of Cas9dRuvC bound to the nicked target site. To this end, we incubated the protein and DNA in the flowcell at negative supercoiling to allow R-loop formation. We then induced positive twist on the DNA to enforce the collapse of the R-loop and characterized the time between introduction of positive supercoiling and the collapse of the full R-loop. The collapse times were best described by a bi-exponential distribution (Fig. 3C). We, therefore, propose the existence of two distinct states distinguishable by their collapse times which were on the order of minutes for the unstable state and hours for the stable state. Repeating the experiment at different applied torques, we observed an exponential dependence of the R-loop collapse time on the torque for both states (Fig. 3D). The post-cleavage product state stabilizes the R-loop in a torque-dependent manner To reveal the relative stability of the post-cleavage state, we finally investigated the stability of SpCas9 in the pre-cleavage state. To this end, we challenged intact R-loops formed by a catalytically fully inactive dCas9 variant with positive supercoiling (Fig. 4A). The measurements were performed in presence of magnesium, allowing dCas9 to adopt the “docked” conformation of the HNH domain. As described before, R-loops formed by dCas9 collapsed in two steps at positive torque (Fig. 4B). The first step can be attributed to the transition from the docked to the intermediate state as observed for Cas9dRuvC . The second step, which was limited only to partial supercoiling release due to the lack of a DNA nick, can be attributed to the collapse of the intermediate state and dissociation of dCas9. Allowing a new R-loop formation at negative turns, the collapse process could be repeated multiple times (Fig. S5). The R-loop collapse times were best described by an exponential distribution with three distinct components, therafter called stable, semi-stable, and unstable state (Fig. 4C). As for Cas9dRuvC, R-loop collapse occurred in two distinct steps for all three components. The respective collapse times fell into the range of hours, minutes, and seconds with each component showing a slight torque dependence (Fig. 4D). Compared to Cas9dRuvC, the collapse time for the stable state was slightly decreased, while the collapse time of the unstable state did not significantly change. The occurrence of a torque- dependent semi-stable state was not observed for Cas9dRuvC. This may indicate flexibility of the docked state of dCas9. Similar to Cas9dRuvC, the fraction of molecules in the unstable state did not change with torque (Fig. 4E). However, the fraction of molecules in the semi-stable state increased, while the fraction of molecules in the stable state decreased with increasing torque. Due to the torque dependence of the fraction of molecules found in the different populations, the mean time of the R-loop collapse was strongly dependent on the torque applied (Fig. 4F). When comparing these results to Cas9dRuvC (Fig. 4F), we found that the mean time of the R-loop collapse was generally higher for Cas9dRuvC compared to dCas9. This indicates a further stabilization of SpCas9 after TS cleavage. The stabilization was found to be torque-dependent with pronounced stabilization at high torques. TS cleavage causes two differently stable SpCas9 states CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455019 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455019 doi: bioRxiv preprint This corresponds to a DNA rewinding (most likely at the PAM-distal end) of about 2 and 1 bp. Furthermore, the fitting allowed to extrapolate the collapse times to zero torque for which we obtained 408 ± 58 min and 3.2 ± 1.5 min for the stable and the unstable state, respectively. On average, 72 ± 3% of the R-loops were in the stable state and 28% ± 3% in the unstable state with no obvious torque- dependence (Fig. 3E). We did not observe any dsDNA release by Cas9wt until many hours after cleavage. This suggests that the occurrence of the unstable state after TS cleavage is unique to Cas9dRuvC and does not occur in case of Cas9wt, since stable TS capturing would only be possible for a formed R-loop. Overall, the results support that Cas9dRuvC stays tightly bound to the target after TS cleavage until the full R-loop collapses. During this time, the Cas9dRuvC-sgRNA-DNA complex can adopt different conformational states, possibly in a dynamic manner. TS cleavage causes two differently stable SpCas9 states This indicated the presence of a transition state for R-loop collapse (similar to the supercoil release above) as observed previously for various CRISPR-Cas proteins (25,10,39,9). Fitting the torque dependent data with a single Arrhenius function provided angular distances to the transition states of 0.18 ± 0.03 turns and 0.07 ± 0.01 turns for the unstable and stable state, respectively. Figure 3. (A) Cartoon illustrating the nicking of the TS by Cas9dRuvC. The TS is nicked but remains tightly bound to Cas9dRuvC such that supercoils are not released in presence of the protein. (B) Representative magnetic tweezers trajectory (smoothed to 6 Hz) showing data for an experiment in which Cas9dRuvC- nicked the DNA and the complex is challenged by strong positive supercoiling (high positive torque). No supercoils are released until part of the R-loop collapses (see orange arrow). This is followed by a rapid release of all supercoiling as observed for nicked DNA in absence of protein (red arrow). (C) Survival plot of the introduced R-loops (black squares) fitted with a bi-exponential decay function (orange/blue line). (D) Characteristic time constants for the R-loop collapse as function of torque obtained from the bi-exponential fits revealing an unstable (orange) and a stable (blue) population of R- loops. (E) Torque-dependence of the fractions of unstable (orange) and stable (blue) R-loops. Figure 3. (A) Cartoon illustrating the nicking of the TS by Cas9dRuvC. The TS is nicked but remains tightly bound to Cas9dRuvC such that supercoils are not released in presence of the protein. (B) Representative magnetic tweezers trajectory (smoothed to 6 Hz) showing data for an experiment in which Cas9dRuvC- nicked the DNA and the complex is challenged by strong positive supercoiling (high positive torque). No supercoils are released until part of the R-loop collapses (see orange arrow). This is followed by a rapid release of all supercoiling as observed for nicked DNA in absence of protein (red arrow). (C) Survival plot of the introduced R-loops (black squares) fitted with a bi-exponential decay function (orange/blue line). (D) Characteristic time constants for the R-loop collapse as function of torque obtained from the bi-exponential fits revealing an unstable (orange) and a stable (blue) population of R- loops. (E) Torque-dependence of the fractions of unstable (orange) and stable (blue) R-loops. . The post-cleavage product state stabilizes the R-loop in a torque-dependent manner To correlate the two states with different stabilities to known conformations of SpCas9, we carried out experiments under Mg2+-free conditions at which the docked state of SpCas9 is suppressed (14). Even though the docked state is . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455019 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455019 doi: bioRxiv preprint suppressed, a full R-loop is formed (Fig. S6A). We obtained an average collapse time that matches the collapse time of the previously observed unstable state (Fig. S6B). This suggests that the stable and semi-stable states represent the docked conformation of SpCas9. suppressed, a full R-loop is formed (Fig. S6A). We obtained an average collapse time that matches the collapse time of the previously observed unstable state (Fig. S6B). This suggests that the stable and semi-stable states represent the docked conformation of SpCas9. In conclusion, we showed that dCas9 adopts three different conformations when a full R-loop has been formed. Additionally, we observed a torque-dependent stabilization of the R-loop after cleavage of the TS. Figure 4. (A) Cartoon illustrating the pre-cleavage state of SpCas9, a state produced by the cleavage- incompetent dCas9 variant. (B) Representative magnetic tweezers trajectory (smoothed to 6 Hz) when challenging an R-loop formed by dCas9 with positive supercoiling. The R-loop spontaneously collapses in two characteristic steps. Absence of full supercoil release indicates that the DNA was not nicked by dCas9. (C) Survival plot of R-loops formed by dCas9 (black triangles) fitted with a tri-exponential decay function (orange/purple/blue line). (D) Characteristic time constants for the R-loop collapse as function of torque obtained from the tri-exponential fits revealing an unstable (orange), a semi-stable (purple), and a stable (blue) population of R-loops. (E) Torque-dependence of the fractions of unstable (orange), semi-stable (purple), and stable (blue) R-loops. (F) Mean R-loop collapse time averaged over all populations for dCas9 (green) and Cas9dRuvC (red). Cleavage of the TS appears to stabilize SpCas9 on the DNA. Figure 4. DISCUSSION Several studies showed that SpCas9 stays tightly bound to its target DNA after cleavage of double- stranded DNA is completed. In this study, we elucidate the mechanisms by which SpCas9 achieves this high stability even under unfavorable conditions of positive supercoiling, a situation encountered during transcription and replication of genomic DNA. Using single-molecule magnetic tweezers measurements, we show that the post-cleavage behavior of SpCas9 strongly depends on which strand of the target site is cleaved. NTS cleavage allows the NTS to detach from the protein and to swivel around the TS under friction. By this mechanism, even high supercoiling levels are quickly relaxed such that R-loop collapse and SpCas9 dissociation are circumvented. We determined a spontaneous swivel rate of 0.04 ± 0.01 turns/s at zero torque. This provides evidence for a spontaneous release of the NTS even in absence of supercoiling such that it becomes accessible for complementary ssDNA or exonucleases (16). Characterizing the torque dependence of this supercoil release allowed us to determine the transition state distances, indicating that the transition state lies at approximately half a turn. A different behavior was observed for a SpCas9 variant that can bind the DNA target site but can only cleave the TS. Here, a facilitated supercoil release was impeded. As observed in the cryo-EM structure of the post-cleavage state of SpCas9 (12), the cleaved TS stays hybridized to the sgRNA in the post- cleavage state which can explain the absence of an immediate release of supercoiling. Applying elevated positive torque resulted in the collapse of the R-loop and dissociation of Cas9dRuvC. By carefully analyzing the collapse times, we observed two states of different stabilities with collapse times in the order of a few minutes and several hours, respectively. After TS cleavage, the HNH domain has been reported to be highly flexible (16,12). The different stability states observed in this work might stem from this flexibility. Additionally, there has been some debate on the dynamics of the pre-cleavage conformational states. While some studies have shown that SpCas9 reaches a stable, cleavage- competent ‘docking’ conformation in the presence of Mg2+ ions (14,40), other studies have observed only brief visits of the docked state with the majority of time spent in the ‘checkpoint’ conformation (15,41,42). The post-cleavage product state stabilizes the R-loop in a torque-dependent manner (A) Cartoon illustrating the pre-cleavage state of SpCas9, a state produced by the cleavage- incompetent dCas9 variant. (B) Representative magnetic tweezers trajectory (smoothed to 6 Hz) when challenging an R-loop formed by dCas9 with positive supercoiling. The R-loop spontaneously collapses in two characteristic steps. Absence of full supercoil release indicates that the DNA was not nicked by dCas9. (C) Survival plot of R-loops formed by dCas9 (black triangles) fitted with a tri-exponential decay function (orange/purple/blue line). (D) Characteristic time constants for the R-loop collapse as function of torque obtained from the tri-exponential fits revealing an unstable (orange), a semi-stable (purple), and a stable (blue) population of R-loops. (E) Torque-dependence of the fractions of unstable (orange), semi-stable (purple), and stable (blue) R-loops. (F) Mean R-loop collapse time averaged over all populations for dCas9 (green) and Cas9dRuvC (red). Cleavage of the TS appears to stabilize SpCas9 on the DNA. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455019 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455019 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455019 doi: bioRxiv preprint DISCUSSION Our results suggest that even though a complete R-loop is formed and cleavage is carried out, Cas9dRuvC exhibits conformational flexibility, transitioning between an undocked state and a stabilized post-cleavage state. It is possible that the undocked state corresponds to the previously reported ‘checkpoint’ state. However, we note that even in the undocked state a full R-loop is formed, as confirmed by the extent of the length jump and the presence of the intermediate step during R-loop collapse (Figure S7). We show that the lifetime of the undocked state resembles that of a Mg2+-free control for which the “docked” state is not formed. For wildtype SpCas9, we did not observe DNA release after a cleavage of both DNA strands even after several hours of measurement indicating that it does not exhibit the undocked state after cleavage which was observed for Cas9dRuvC. Cleavage-incompetent dCas9 exhibits even three distinct states and, potentially, a higher conformational flexibility. Combining the results of Cas9dRuvC and dCas9 we suggest that the stable state that is observed for both variants corresponds to the post-cleavage product state. This state would appear to be transiently visited even for the uncleaved target, albeit only at low levels of supercoiling. The semi-stable state is observed when dCas9 is bound to the target DNA without DNA cleavage . This state dominates under conditions . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455019 doi: bioRxiv preprint . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455019 doi: bioRxiv preprint of high supercoiling and most likely corresponds to the pre-cleavage state with a docked HNH domain. The unstable state is found for both dCas9 and Cas9dRuvC and most likely represents the undocked state in agreement with the previously observed dynamic sampling between the different states (15,41,42) (Fig. 5). Overall, the dominance of the post-cleavage product state accompanying TS cleavage stabilizes the R-loop compared to the uncut target, especially at higher torques. DISCUSSION This suggests that, under in-vivo conditions, SpCas9 has evolved to remain bound to its cut target. Possibly, this not only inactivates the invader DNA but also blocks transcription and/or replication of the foreign DNA, thereby fully blocking replication of a bacteriophage. However, this represents a challenge for efficient genome engineering applications. Overall, the presented results expand the knowledge about the post-cleavage state(s) of SpCas9 and the mechanisms by which SpCas9 avoids R-loop collapse and dissociation even under high twist that can be generated in vivo by the genome processing machineries. Nickase mutants of Cas9 have been used in various approaches for genome editing with strong differences in results depending on which nickase was used (22,43,44). Our findings may help to understand the described differences and might facilitate future genome editing applications with SpCas9 nickases. Figure 5. Schematic overview of the different states identified for SpCas9 bound to intact DNA and DNA with a nicked TS. We assigned the unstable state to the intermediate state with undocked HNH domain, the semi-stable state to the precleavage state with the HNH domain docked to the active site, and the stable state to the post-cleavage product state with a disordered (or flexible) HNH domain (12,16). Catalytically inactive dCas9 can occupy all states and most likely samples these states in a dynamic manner. Cas9dRuvC was predominantly found in the stable (post-cleavage) state and rarely occupied the unstable state. Figure 5. Schematic overview of the different states identified for SpCas9 bound to intact DNA and DNA with a nicked TS. We assigned the unstable state to the intermediate state with undocked HNH domain, the semi-stable state to the precleavage state with the HNH domain docked to the active site, and the stable state to the post-cleavage product state with a disordered (or flexible) HNH domain (12,16). Catalytically inactive dCas9 can occupy all states and most likely samples these states in a dynamic manner. Cas9dRuvC was predominantly found in the stable (post-cleavage) state and rarely occupied the unstable state. We thank Joachim Griesenbeck for providing the pM53.1 plasmid. Furthermore, we thank Virginijus Siksnys, Tautvydas Karvelis, and Greta Bigelytė for providing the sgRNA. REFERENCES 1. Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., Romero, D.A. and Horvath, P. (2007) CRISPR provides acquired resistance against viruses in prokaryotes, Science (New York, N.Y.), 315, 1709–1712. 2. 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It is The copyright holder for this preprint this version posted August 4, 2021. ; https://doi.org/10.1101/2021.08.04.455019 doi: bioRxiv preprint
https://openalex.org/W3209619552
https://zenodo.org/record/5594474/files/UKavak_PhDThesis_Title_and_contents.pdf
English
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Interaction of Massive Stars with Gas Clouds in the Milky Way
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cc-by
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Citation for published version (APA): Kavak, U. (2021). Interaction of Massive Stars with Gas Clouds in the Milky Way: from shooting stars to breaking bubbles. University of Groningen. https://doi.org/10.33612/diss.187660161 University of Groningen University of Groningen Kavak, Umit DOI: 10.33612/diss.187660161 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Kavak, U. (2021). Interaction of Massive Stars with Gas Clouds in the Milky Way: from shooting stars to breaking bubbles. University of Groningen. https://doi.org/10.33612/diss.187660161 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne- amendment. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Take-down policy If b li h Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 23-10-2021 Download date: 23-10-2021 Download date: 23-10-2021 Cover design by: Yakup Kurt/Adgency Koeln. The background image on the front cover is a star-forming region known as LH-95 in the Large Magellanic Cloud taken from hubblesite.org. The interpretation of Starry Night on the back-cover and figures at the end of each chapter were provided by Garip Ay. The blue tulip created by Evren Kaan at page 220 was taken from kulturpor- tali.gov.tr with ID of #8942. Printed by: Gildeprint Contents 1 Introduction 1 1.1 Interstellar Medium . . . . . . . . . . . . . . . . . . . . . 2 1.2 Low and Massive Star Formation . . . . . . . . . . . . . . 3 1.3 Massive Stars . . . . . . . . . . . . . . . . . . . . . . . . . 7 1.3.1 Jets and Outflows . . . . . . . . . . . . . . . . . . 9 1.3.2 Photodissociation Regions . . . . . . . . . . . . . . 10 1.3.3 Interstellar Bubbles . . . . . . . . . . . . . . . . . . 13 1.4 Feedback from Massive Stars . . . . . . . . . . . . . . . . 15 1.4.1 Mechanical Feedback . . . . . . . . . . . . . . . . . 16 1.4.2 Radiative Feedback . . . . . . . . . . . . . . . . . . 16 1.5 Aims and Methods . . . . . . . . . . . . . . . . . . . . . . 18 1.5.1 Herschel Space Observatory . . . . . . . . . . . . . 18 1.5.2 Very Large Array (VLA) . . . . . . . . . . . . . . . 20 1.5.3 Stratospheric Observatory for Infrared Astronomy (SOFIA) . . . . . . . . . . . . . . . . . . . . . . . . 21 1.5.4 Meudon PDR code . . . . . . . . . . . . . . . . . . 22 1.5.5 RADEX . . . . . . . . . . . . . . . . . . . . . . . . 23 1.5.6 GILDAS . . . . . . . . . . . . . . . . . . . . . . . . 23 1.5.7 CASA . . . . . . . . . . . . . . . . . Ümit Kavak Supervisors Prof. F. F. S. van der Tak Prof. A. G. G. M. Tielens Assessment committee Prof. J. Tan Prof. G. J. Stacy Prof. I. E. E. Kamp Assessment committee Prof. J. Tan Prof. G. J. Stacy Prof. I. E. E. Kamp To my family... Cover design by: Yakup Kurt/Adgency Koeln. The background image on the front cover is a star-forming region known as LH-95 in the Large Magellanic Cloud taken from hubblesite.org. The interpretation of Starry Night on the back-cover and figures at the end of each chapter were provided by Garip Ay. The blue tulip created by Evren Kaan at page 220 was taken from kulturpor- tali.gov.tr with ID of #8942. Printed by: Gildeprint Contents . . . . . . . . 23 1.5.8 HIPE . . . . . . . . . . . . . . . . . . . . . . . . . 24 1.5.9 Python Libraries . . . . . . . . . . . . . . . . . . . 24 1.6 Goals of this thesis . . . . . . . . . . . . . . . . . . . . . . 24 1.7 Outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 2 Search for radio jets from massive young stellar objects. Association of radio jets with H2O and CH3OH masers 29 2.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 i 1 Introduction 1 1.1 Interstellar Medium . . . . . . . . . . . . . . . . . . . . . 2 1.2 Low and Massive Star Formation . . . . . . . . . . . . . . 3 1.3 Massive Stars . . . . . . . . . . . . . . . . . . . . . . . . . 7 1.3.1 Jets and Outflows . . . . . . . . . . . . . . . . . . 9 1.3.2 Photodissociation Regions . . . . . . . . . . . . . . 10 1.3.3 Interstellar Bubbles . . . . . . . . . . . . . . . . . . 13 1.4 Feedback from Massive Stars . . . . . . . . . . . . . . . . 15 1.4.1 Mechanical Feedback . . . . . . . . . . . . . . . . . 16 1.4.2 Radiative Feedback . . . . . . . . . . . . . . . . . . 16 1.5 Aims and Methods . . . . . . . . . . . . . . . . . . . Contents . . . 18 1.5.1 Herschel Space Observatory . . . . . . . . . . . . . 18 1.5.2 Very Large Array (VLA) . . . . . . . . . . . . . . . 20 1.5.3 Stratospheric Observatory for Infrared Astronomy (SOFIA) . . . . . . . . . . . . . . . . . . . . . . . . 21 1.5.4 Meudon PDR code . . . . . . . . . . . . . . . . . . 22 1.5.5 RADEX . . . . . . . . . . . . . . . . . . . . . . . . 23 1.5.6 GILDAS . . . . . . . . . . . . . . . . . . . . . . . . 23 1.5.7 CASA . . . . . . . . . . . . . . . . . . . . . . . . . 23 1.5.8 HIPE . . . . . . . . . . . . . . . . . . . . . . . . . 24 1.5.9 Python Libraries . . . . . . . . . . . . . . . . . . . 24 1.6 Goals of this thesis . . . . . . . . . . . . . . . . . . . . . . 24 1.7 Outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 2 Search for radio jets from massive young stellar objects. Association of radio jets with H2O and CH3OH masers 29 2.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 1 Introduction 2.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 31 2.3 Observations . . . . . . . . . . . . . . . . . . . . . . . . . Contents 33 2.3.1 Selected sample . . . . . . . . . . . . . . . . . . . . 33 2.3.2 VLA observations . . . . . . . . . . . . . . . . . . . 35 2.4 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 2.4.1 Continuum emission . . . . . . . . . . . . . . . . . 36 2.4.2 Spectral index analysis . . . . . . . . . . . . . . . . 37 2.4.3 Maser emission . . . . . . . . . . . . . . . . . . . . 41 2.5 Analysis and discussion . . . . . . . . . . . . . . . . . . . 41 2.5.1 Nature of the radio continuum emission . . . . . . 42 2.5.2 Association with molecular outflows . . . . . . . . 44 2.5.3 Association with EGOs . . . . . . . . . . . . . . . 45 2.5.4 Association with masers . . . . . . . . . . . . . . . 48 2.5.5 Radio-jet candidates . . . . . . . . . . . . . . . . . 48 2.6 Implications for high-mass star formation . . . . . . . . . 57 2.7 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 2.8 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . 60 2.9 Comments on individual sources . . . . . . . . . . . . . . 60 2.10 Catalog of the continuum sources . . . . . . . . . . . . . . 72 3 Origin of hydrogen fluoride emission in the Orion Bar. An excellent tracer for CO-dark H2 gas clouds 109 3.1 Abstract . . . . . . . . Contents . . . . . . . . . . . . . . . . . . . . 109 3.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 111 3.3 Observation and data reduction . . . . . . . . . . . . . . . 113 3.4 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 3.5 Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 3.5.1 Column density . . . . . . . . . . . . . . . . . . . . 119 3.5.2 Spatial distribution of HF . . . . . . . . . . . . . . 122 3.6 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 3.6.1 Collisional excitation . . . . . . . . . . . . . . . . . 124 3.6.2 Infrared pumping . . . . . . . . . . . . . . . . . . . 129 3.6.3 Chemical Pumping . . . . . . . . . . . . . . . . . . 130 3.7 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . 130 3.8 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . 131 3.9 Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 3.9.1 SEDs of Three Positions in the HF map . . . . . . 133 ii 4 Breaking Orion’s Veil bubble with fossil outflows 139 4.1 Abstract . . . . . . . . . . . . . Contents . . . . . . . . . . . . . . . 139 4.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 141 4.3 Observations . . . . . . . . . . . . . . . . . . . . . . . . . 145 4.3.1 [C ii] Observations . . . . . . . . . . . . . . . . . . 145 4.3.2 Molecular Gas Observations . . . . . . . . . . . . . 147 4.3.3 Ionized Gas Observations . . . . . . . . . . . . . . 148 4.3.4 Far-IR photometric observations . . . . . . . . . . 148 4.3.5 Mid-IR Observations . . . . . . . . . . . . . . . . . 150 4.4 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150 4.5 Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152 4.5.1 Expansion Velocity . . . . . . . . . . . . . . . . . . 152 4.5.2 Morphology of the protrusion . . . . . . . . . . . . 152 4.5.3 Expansion Timescale . . . . . . . . . . . . . . . . . 155 4.5.4 Line Profile Analysis . . . . . . . . . . . . . . . . . 155 4.5.5 Kinetic Energy and Momentum . . . . . . . . . . . 157 4.6 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . 160 4.6.1 Persistence of fossil outflow cavity . . . . . . . . . 165 4.6.2 Ionizing source . . . . . . . . . . . . . . . . . . . Contents . 166 4.6.3 Correlation of Intensities . . . . . . . . . . . . . . . 167 4.7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 4.8 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . 170 4.9 Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . 170 4.9.1 Geometric correction Factor . . . . . . . . . . . . . 170 4.10 Additional Maps . . . . . . . . . . . . . . . . . . . . . . . 172 5 Unveiling the Veil: Protostellar feedback in Orion 183 5.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 5.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 185 5.3 Observations . . . . . . . . . . . . . . . . . . . . . . . . . 187 5.3.1 [C ii] observations . . . . . . . . . . . . . . . . . . 187 5.3.2 Molecular Gas observations . . . . . . . . . . . . . 189 5.3.3 Mid-IR observations . . . . . . . . . . . . . . . . . 189 5.3.4 Far-IR photometric observations . . . . . . . . . . 189 5.4 Identification of Dents . . . . . . . . . . . . . . . . . . . . 190 5.4.1 Position-velocity (PV) Diagrams . . . . . . . . . . 190 5.4.2 High-velocity [C ii] emission . . . . . . . . . . . . . 193 iii 5.4.3 Line profiles . . . . . . . . . . . Contents . . . . . . . . . . . 193 5.5 Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194 5.5.1 Characteristics of the dents . . . . . . . . . . . . . 194 5.5.2 Origin of the dents . . . . . . . . . . . . . . . . . . 196 5.5.3 Momentum of the dents . . . . . . . . . . . . . . . 196 5.5.4 Potential shock signature of the dents . . . . . . . 199 5.5.5 Collimation factor and opening angle . . . . . . . . 201 5.6 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . 202 5.7 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . 203 5.8 Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . 204 5.8.1 Gaussian Fitting Results . . . . . . . . . . . . . . . 204 5.8.2 Massive Stars and Geometry . . . . . . . . . . . . 204 5.8.3 PV diagram of the dents . . . . . . . . . . . . . . . 205 6 Conclusions and Outlook 215 6.1 Summary and conclusions . . . . . . . . . . . . . . . . . . 215 6.2 Future Outlook . . . . . . . . . . . . . . . . . . . . . . . . 218 7 Additional Sections 223 7.1 Contributed Publications . . . . . . . . . . . . . . . . . . 223 7.2 Talks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224 7.3 Posters . Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224 7.4 Türkçe Özet . . . . . . . . . . . . . . . . . . . . . . . . . . 226 7.5 Nederlandse samenvatting . . . . . . . . . . . . . . . . . . 231 7.6 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . 236 iv iv
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Digital Recruitment and Acceptance of a Stepwise Model to Prevent Chronic Disease in the Danish Primary Care Sector: Cross-Sectional Study
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Digital Recruitment and Acceptance of a Stepwise Model to Prevent Chronic Disease in the Danish Primary Care Sector: Cross-Sectional Study Lars Bruun Larsen1, MPH; Jens Sondergaard1, PhD; Janus Laust Thomsen2, PhD; Anders Halling3, PhD; Anders Larrabee Sønderlund1, PhD; Jeanette Reffstrup Christensen1, PhD; Trine Thilsing1, PhD 1Research Unit of General Practice, Institute of Public Health, University of Southern Denmark, Odense, Denmark 2Research Unit for General Practice, Department of Clinical Medicine, Aalborg University, Aalborg, Denmark 3Department of Clinical Sciences in Malmö, Centre for Primary Health Care Research, Lund University, Lund, Sweden Corresponding Author: Lars Bruun Larsen, MPH Research Unit of General Practice Institute of Public Health University of Southern Denmark JB Winsløws Vej 9A Odense, 5000 Denmark Phone: 45 65503830 Email: lblarsen@health.sdu.dk Abstract Background: During recent years, stepwise approaches to health checks have been advanced as an alternative to general health checks. In 2013, we set up the Early Detection and Prevention project (Tidlig Opsporing og Forebyggelse, TOF) to develop a stepwise approach aimed at patients at high or moderate risk of a chronic disease. A novel feature was the use of a personal digital mailbox for recruiting participants. A personal digital mailbox is a secure digital mailbox provided by the Danish public authorities. Apart from being both safe and secure, it is a low-cost, quick, and easy way to reach Danish residents. Objective: In this study we analyze the association between the rates of acceptance of 2 digital invitations sent to a personal digital mailbox and the sociodemographic determinants, medical treatment, and health care usage in a stepwise primary care model for the prevention of chronic diseases. Methods: We conducted a cross-sectional analysis of the rates of acceptance of 2 digital invitations sent to randomly selected residents born between 1957 and 1986 and residing in 2 Danish municipalities. The outcome was acceptance of the 2 digital invitations. Statistical associations were determined by Poisson regression. Data-driven chi-square automatic interaction detection method was used to generate a decision tree analysis, predicting acceptance of the digital invitations. Results: A total of 8814 patients received an invitation in their digital mailbox from 47 general practitioners. A total of 40.22% (3545/8814) accepted the first digital invitation, and 30.19 % (2661/8814) accepted both digital invitations. The rates of acceptance of both digital invitations were higher among women, older patients, patients of higher socioeconomic status, and patients not diagnosed with or being treated for diabetes mellitus, chronic obstructive pulmonary disease, or cardiovascular disease. Conclusions: To our knowledge, this is the first study to report on the rates of acceptance of digital invitations to participate in a stepwise model for prevention of chronic diseases. More studies of digital invitations are needed to determine if the acceptance rates seen in this study should be expected from future studies as well. Similarly, more research is needed to determine whether a multimodal recruitment approach, including digital invitations to personal digital mailboxes will reach hard-to-reach subpopulations more effectively than digital invitations only. Original Paper Digital Recruitment and Acceptance of a Stepwise Model to Prevent Chronic Disease in the Danish Primary Care Sector: Cross-Sectional Study Lars Bruun Larsen1, MPH; Jens Sondergaard1, PhD; Janus Laust Thomsen2, PhD; Anders Halling3, PhD; Anders Larrabee Sønderlund1, PhD; Jeanette Reffstrup Christensen1, PhD; Trine Thilsing1, PhD 1Research Unit of General Practice, Institute of Public Health, University of Southern Denmark, Odense, Denmark 2Research Unit for General Practice, Department of Clinical Medicine, Aalborg University, Aalborg, Denmark 3Department of Clinical Sciences in Malmö, Centre for Primary Health Care Research, Lund University, Lund, Sweden Larsen et al JOURNAL OF MEDICAL INTERNET RESEARCH JOURNAL OF MEDICAL INTERNET RESEARCH Larsen et al Original Paper (J Med Internet Res 2019;21(1):e11658) doi: 10.2196/11658 (J Med Internet Res 2019;21(1):e11658) doi: 10.2196/11658 J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 1 (page number not for citation purposes) Background General health checks are seen as one way to mitigate the rising prevalence of chronic diseases such as cardiovascular disease (CVD), type 2 diabetes mellitus (T2DM), and chronic obstructive pulmonary disease (COPD). Consequently, periodical general health checks are provided to citizens by various national health care systems around the world, including those in the United States, South Korea, Australia, and Germany. However, general health checks have not only failed to show population health effects on CVD and total mortality but may have also widen health inequalities [1-6]. This is probably because of the generally higher uptake of health check initiatives in the populations who are likely to benefit the least—including most notably women and patients of higher age, better health, and higher socioeconomic status (SES). Population-level uptake seems to be determined by an interrelationship between individual and societal facilitators and barriers as well as self-selection [7,8]. A novel feature of the pilot study was the use of a personal digital mailbox for recruiting participants. A personal digital mailbox is a secure digital mailbox provided by the Danish public authorities. It is accessed either via a webpage or an app developed for all major operating systems. The digital mailbox is secured by a national 2-phased log-in system (NemID) and is used by all public authorities as well as an increasing number of private companies such as banks and insurance companies. Beyond being both safe and secure, it is a low-cost, quick, and easy way to reach Danish residents [19]. Permanent residents of Denmark are obliged by law to have a digital mailbox and are expected to check it regularly. Short message service text message and mail reminders are optional. Opting out is only possible in special cases, mainly in the event of low information and technology literacy (usually age-related) or cognitive impairment. A total of 90% of the entire population in Denmark and 95% of the target population have a digital mailbox (May 2016) [20]. During recent years, stepwise approaches to health checks have been advanced as an alternative to general health checks [9,10]. Stepwise approaches usually comprise a risk assessment to identify the at-risk population, followed by health checks tailored to this population. If deemed necessary, behavior change interventions, preventive medical treatment, or a combination of the two may also be included. KEYWORDS promotion of health; clinical decision support systems; cross-sectional studies http://www.jmir.org/2019/1/e11658/ XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Larsen et al targeted intervention would be complemented by further behavior change intervention or preventive medical treatment. Patients already diagnosed with hypertension, hypercholesterolemia, T2DM, CVD, or COPD by the GP, or who displayed no health risk behaviors were only offered the joint intervention. In line with the Medical Research Council’s recommendations for complex interventions, we tested the acceptability, feasibility, and short-term effects of the intervention in a pilot study in 2 municipalities between April and December 2016 [18]. Objective This study reports on the association between the rates of acceptance of 2 digital invitations sent to a personal digital mailbox and sociodemographic determinants, medical treatment, and health care usage in a stepwise primary care model for the prevention of chronic diseases. Design We conducted a cross-sectional analysis of the rates of acceptance of 2 digital invitations sent to residents from 2 Danish municipalities randomly selected to take part in a pilot study of the TOF project (NCT02797392). Population The target population consisted of citizens born between 1957 and 1986 and residing in Haderslev or Varde, 2 rural municipalities located in the southern part of Jutland, Denmark. The population of both municipalities totals 106,081 citizens (2015). Background Various stepwise approaches to health checks have been tested in research studies; however, no long-term effects have been reported [11-15]. In the Danish health care system, health checks are provided to the general population on an opportunistic, nonperiodic, and nontargeted basis. On the basis of a technical feasibility study from 2012 [16], we set up the Tidlig Opsporing og Forebyggelse (TOF; early detection and prevention) project in a partnership with the general practitioners’(GPs) organization and 10 municipalities of the Region of Southern Denmark [17]. Over a period of 2 years, we developed a stepwise model for systematic and targeted prevention of chronic diseases to be used in the Danish primary care sector. The intervention consisted of a joint intervention and a targeted intervention. The joint intervention was applied to the entire study population in the form of a personal digital health profile. The targeted intervention was only applied to patients who were deemed to potentially benefit from either a health check at their GP or lifestyle coaching provided by the municipal health center. Patients at high risk of a chronic disease were identified using validated risk algorithms for COPD, T2DM, and CVD and were offered a health check at their GP in the form of a medical examination and a health dialogue. Patients with health risk behavior included patients who were not at high risk as determined by the risk algorithms but who engaged in one or more health-risk behaviors such as smoking, high-risk alcohol consumption, poor dietary habits, sedentary behavior, and/or a body mass index above 35. This cohort was offered a short 15-min telephone-based health dialogue with a health professional from the municipal health center. For patients with limited health capabilities, the initial telephone-based health dialogue could be followed up by a 1-hour face-to-face health dialogue. If deemed necessary, the J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 2 (page number not for citation purposes) http://www.jmir.org/2019/1/e11658/ Variables In January 2016, all 66 GPs residing in the municipalities of Varde and Haderslev received a written invitation, with an enclosed project agreement form and a prepaid return envelope. Nonresponse was followed up by a telephone call to the GP. Using the Regional Primary Care Administrative System (KMD Sygesikring), the regional health authorities identified a source population of 200 randomly selected patients extracted from each of the participating GPs’ patient lists. From the source population, we excluded patients having either no digital mailbox or residing outside the municipalities of Varde or Haderslev. Registry variables for the entire study population were retrieved from the administrative registry and Statistics Denmark and linked with the patients’Danish Personal Identification numbers. EPR information was retrieved directly from participating GPs’ EPR systems and related purely to consenting patients (Table 1). All participants were pseudonymized when linking project data and national registers from Statistics Denmark. Age was categorized in 10-year age groups. Country of origin was retrieved for the year 2016 and categorized as Danish, Western, or non-Western origin. Cohabitational status was retrieved for the year 2016 and categorized as cohabiting or single. Highest attainable educational level was retrieved for October 2015 and categorized as secondary school, high school, vocational education, or higher education. Occupation was retrieved for November 2014 and categorized according to the Organisation for Economic Co-operation and Development equivalence scales into 5 groups: employed, self-employed, unemployed or on benefits, social welfare recipients, or other [30]. The distinction between unemployment benefits and social welfare is that unemployment benefits are accessible to citizens who have been unemployed for less than 2 years and who are members of a voluntary unemployment benefit fund. Social welfare benefits are for all other unemployed persons who can take up a job. Others represent, for example, nonworking persons from a family that relies on 1 income only. Family income was retrieved for 2013, 2014, and 2015, defined by the mean annual net income of the household, and categorized into quartiles. “Partner in project” describes whether your partner (if cohabiting) participated as well. Partner in project is categorized in a binary yes or no variable. Participants were recruited using 2 digital invitations sent to their personal digital mailbox (Multimedia Appendix 1). a personal digital health profile. The results from the second digital invitation are presented in Multimedia Appendices 2 and 3. a personal digital health profile. The results from the second digital invitation are presented in Multimedia Appendices 2 and 3. secondary prevention, including treatment for hypertension, hypercholesterolemia, and diabetes. secondary prevention, including treatment for hypertension, hypercholesterolemia, and diabetes. Variables Both digital invitations consisted of a 1-page PDF file in Danish and included a highly visible hyperlink to a Web-based digital support system, on which participants would provide both consent and access to the personal digital health profile [22]. The first digital invitation was sent out in April 2016, with the aim of obtaining consent to participate in the study and to access specific information from their GP’s electronic patient record (EPR) system, including International Classification of Primary Care, 2nd edition) codes for diagnoses and anatomical therapeutic chemical (ATC) codes for medical prescriptions. The second digital invitation was aimed at providing participants with a digital health profile. This invitation was sent out in September 2016 to participating patients who were registered with the same GP as when they consented and who still resided in the municipalities of Varde or Haderslev. A nonresponse triggered up to 2 reminders 1 week apart. J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 3 (page number not for citation purposes) Setting The Danish health care system comprises a strong, publicly funded primary care sector, which includes municipal health centers and GP clinics [21]. GPs operate a patient list system, with an average of 1600 patients per GP. On average, 2 GPs work in a given clinic. Municipal health centers serve the entire population with primary prevention such as smoking cessation and dietary advice, whereas GPs manage and coordinate http://www.jmir.org/2019/1/e11658/ XSL•FO RenderX XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Larsen et al JOURNAL OF MEDICAL INTERNET RESEARCH JOURNAL OF MEDICAL INTERNET RESEARCH Table 1. Analyses of associations between patient characteristics and acceptance. Second digital invitation First digital invitation Variable Multimedia Appendix 3 Multimedia Appendix 2 Results section Presentation of results Consent to the first digital invitation Study population Study population Denominator Uptake or nonuptake of the second digital invitation Uptake or nonuptake of the second digital invitation Consent or nonconsent to the first digital invitation Outcome variable Exposures Agea,b Agea,b Agea,b Sociodemographics   Sexa,b Sexa,b Sexa,b     Country of origin Country of origin Country of originb   Highest educational attainmentc Highest educational attainmentc Highest educational attainmentc   Occupational statusd Occupational statusd Occupational statusd   Family incomee Family incomee Family incomee   Cohabitational statusb Cohabitational statusb Cohabitational statusb   Partner consentf Partner consentf Partner consentf   Prescriptions from primary care or hospitals (ATC codes)g Prescriptions from primary care or hospitals (ATC codes)g Prescriptions from primary care or hospitals (anatomical therapeutic chemical [ATC] codes)g Medical treatment   Hospital discharge diagnoses (ICD- 10 codes)h Hospital discharge diagnoses (ICD- 10 codes)h Hospital discharge diagnoses (Interna- tional Classification of Diseases 10th edition [ICD-10] codes)h     Administrative primary care codesi,j; health checksi,j Administrative primary care codesi,j Administrative primary care codesi,j Health care usage   aDanish National Administrative Primary Care System (Praksys). bDanish Civil Registration System [23]. cDanish Education Register [24]. dDanish Registers of Labour Market Affiliation [25]. eDanish Registers of Personal Income and Transfer Payments [26]. fQuestionnaire data. gDanish National Prescription Registry [27]. hDanish National Patient Registry [28]. iGeneral practitioners’ electronic patient record. jDanish National Health Service Register [29]. Table 2. International Classification of Diseases 10th edition codes and anatomical therapeutic chemical classification codes used to define when a medical condition of chronic obstructive pulmonary disease, cardiovascular disease, or diabetes mellitus had been registered. ATCb therapeutic codes for prescribed medicine registered from May 2014 to April 2016 ICD-10a codes registered from January 2013 to April 2016 Medical condition R03AC18, R03AC19, R03AL03, R03AL04, R03AL05, R03BB04, R03BB05, and R03BB06 J44 COPDc C (except: C01CA and C05) I1-I7 and E78 (except: I0, I16, I60, I73, and I78) CVDd A10 E10-E13 Diabetes mellitus aICD-10: International Classification of Diseases 10th edition. bATC: anatomical therapeutic chemical. cCOPD h i b i l di Larsen et al JOURNAL OF MEDICAL INTERNET RESEARCH Table 1. Analyses of associations between patient characteristics and acceptance. Outcomes In this paper, we report on both the consent to take part in the study and the uptake of the personal digital health profile. The main outcome relates to the acceptance of the first digital invitation and is operationalized in terms of consent or nonconsent to take part in the study. Consent is defined as the provision of informed consent to participate in the study; nonconsent includes both nonresponse and active nonconsent. The second outcome relates to the acceptance of the second digital invitation and is operationalized in terms of uptake or nonuptake of the personal digital health profile. Uptake is defined as patients who gave their active consent and received Information on prescriptions and diagnoses was combined as a proxy for medical treatment (Table 2). Prescriptions were retrieved for the period from May 2014 to April 2016 as ATC codes. We chose a 2-year period, as prescriptions may be filed up to 2 years after their date of issue. International Classification of Diseases 10th edition (ICD-10) codes were retrieved for the period from January 2013 to April 2016. Medical treatment was defined as either registered with an ATC code, ICD-10 code, or both during the periods specified above. J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 3 (page number not for citation purposes) http://www.jmir.org/2019/1/e11658/ http://www.jmir.org/2019/1/e11658/ XSL•FO RenderX http://www.jmir.org/2019/1/e11658/ JOURNAL OF MEDICAL INTERNET RESEARCH Second digital invitation First digital invitation Variable Multimedia Appendix 3 Multimedia Appendix 2 Results section Presentation of results Consent to the first digital invitation Study population Study population Denominator Uptake or nonuptake of the second digital invitation Uptake or nonuptake of the second digital invitation Consent or nonconsent to the first digital invitation Outcome variable Exposures Agea,b Agea,b Agea,b Sociodemographics   Sexa,b Sexa,b Sexa,b     Country of origin Country of origin Country of originb   Highest educational attainmentc Highest educational attainmentc Highest educational attainmentc   Occupational statusd Occupational statusd Occupational statusd   Family incomee Family incomee Family incomee   Cohabitational statusb Cohabitational statusb Cohabitational statusb   Partner consentf Partner consentf Partner consentf   Prescriptions from primary care or hospitals (ATC codes)g Prescriptions from primary care or hospitals (ATC codes)g Prescriptions from primary care or hospitals (anatomical therapeutic chemical [ATC] codes)g Medical treatment   Hospital discharge diagnoses (ICD- 10 codes)h Hospital discharge diagnoses (ICD- 10 codes)h Hospital discharge diagnoses (Interna- tional Classification of Diseases 10th edition [ICD-10] codes)h     Administrative primary care codesi,j; health checksi,j Administrative primary care codesi,j Administrative primary care codesi,j Health care usage   aDanish National Administrative Primary Care System (Praksys). Table 1. Analyses of associations between patient characteristics and acceptance. Exposures aDanish National Administrative Primary Care System (Praksys). bDanish Civil Registration System [23]. cDanish Education Register [24]. dDanish Registers of Labour Market Affiliation [25]. eDanish Registers of Personal Income and Transfer Payments [26]. fQuestionnaire data. gDanish National Prescription Registry [27]. hDanish National Patient Registry [28]. iGeneral practitioners’ electronic patient record. jDanish National Health Service Register [29]. Table 2. International Classification of Diseases 10th edition codes and anatomical therapeutic chemical classification codes used to define when a medical condition of chronic obstructive pulmonary disease, cardiovascular disease, or diabetes mellitus had been registered. ATCb therapeutic codes for prescribed medicine registered from May 2014 to April 2016 ICD-10a codes registered from January 2013 to April 2016 Medical condition R03AC18, R03AC19, R03AL03, R03AL04, R03AL05, R03BB04, R03BB05, and R03BB06 J44 COPDc C (except: C01CA and C05) I1-I7 and E78 (except: I0, I16, I60, I73, and I78) CVDd A10 E10-E13 Diabetes mellitus aICD-10: International Classification of Diseases 10th edition. bATC i l h i h i l ATCb therapeutic codes for prescribed medicine registered from May 2014 to April 2016 J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 4 (page number not for citation purposes) J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 4 (page number not for citation purposes) http://www.jmir.org/2019/1/e11658/ JOURNAL OF MEDICAL INTERNET RESEARCH Larsen et al Health care usage was determined from administrative codes registered by the GP and retrieved for the period from May 2013 to April 2016. To this end, we also examined EPR information on laboratory test results. Administrative codes were used to extract frequent GP attenders, GP attenders, and usage of specific administrative codes pertaining to laboratory tests and preventive consultations. Frequent attenders referred to the top 10% of patients, who on average contacted the GP the most—either in person or by phone—during the 3 years from May 2013 to April 2016 [31]. GP attenders were defined as patients having contacted their GP during the 2-year period from May 2014 to April 2016. Laboratory tests comprised blood samples (administrative codes 2101 and 2601), peak flow tests (7183), spirometries (7113), and home blood pressure monitoring (2146) during the period from May 2014 to April 2016. Furthermore, we retrieved information on specific preventive consultations (0120)—that is, special consultations for coaching patients with health risk behaviors and patients diagnosed with a chronic disease. Ethics Approval and Consent to Participate The study was approved by the Danish Data Protection Agency (J.Number 2015-57-0008) and registered at Clinical Trial Gov (Unique Protocol ID: TOFpilot2016). According to Danish regulations (Act on Research Ethics Review of Health Research Projects [section 14,2]), this study does not need approval from a health research ethics committee as no research on human tissue or other biological material is performed. The study complies with the Helsinki Declaration, with informed consent to study participation and to disclosure of data from the GPs’ EPR obtained from all participants. Exposures To determine if a patient had received a preventive health check within a period of 2 years before consenting to the study (May 2014 to May 2016), we retrieved blood pressure (systolic and diastolic blood pressure), lung function (forced expiratory volume [FEV1], forced vital capacity [FVC], and FEV1/FVC), glycated hemoglobin, and lipids (total cholesterol, high-density lipoprotein, and low-density lipoprotein) measurements from the GPs’ EPR systems. A health check was defined as having had 2 or more of the above-mentioned values measured in the same consultation. tests of interdependence, a CHAID analysis clusters categories within each predictor variable to determine what predictor variables are associated with the outcome. Subsequently, the predictor variables associated with the outcome are ordered hierarchically. The specific variable order is determined by the Bonferroni P value of each variable such that the predictor variable with the smallest P value (strongest association) is placed at the top of the hierarchy (parent node). The minimum number of observations for each split in the decision tree was set at 500 (child node) and 200 for each node (terminal node). In the analysis of the acceptance of the second digital invitation, each split in the decision tree was set to 100 (child node) and 20 for each node (terminal node) because of the limited number of observations. Statistical analysis was performed on secure servers at Statistics Denmark using Stata 14 (Statacorp). J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 5 (page number not for citation purposes) Recruitment and Overall Uptake Statistical associations are presented as crude figures, age- and sex-adjusted figures, and as models minimally adjusted for known confounders. Poisson regression with robust variance error was used rather than logistic regression to obtain incidence rate ratios (IRR). The binary outcome variable of consent or nonconsent was interpreted as a continuous variable with the only counts being 0 or 1. The minimally adjusted model was developed from a causal direct acyclic graph (DAG), built on the current evidence of the determinants of attendance at health checks [32]. Attendance at health checks was the outcome variable of the DAG. Significance level was set at P<.05. Of the 68 GPs residing in the 2 municipalities, 47 GPs from 18 clinics agreed to participate in the study (Figure 1). This provided us with a source population of 9400 patients. However, a total of 586 patients did not meet the inclusion criteria, which is why only 8814 received the first invitation. Initially, a total of 3587 patients consented to participate, but among them, 30 patients moved from the municipality to a nonparticipating GP, and 12 withdrew their consent after receiving the second invitation. This resulted in 3545 active consenters from the first round of invitations (Multimedia Appendix 2). Of the patients who accepted the first digital invitation (n=3545), 75.06% (2661/3545) also accepted the second digital invitation (Multimedia Appendix 3; Table 3). A data-driven chi-square automatic interaction detection (CHAID) method was used to generate a decision tree analysis to identify interactions and a hierarchy of variables predicting the chosen outcome variable (root node) [33]. Using chi-square J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 5 (page number not for citation purposes) http://www.jmir.org/2019/1/e11658/ http://www.jmir.org/2019/1/e11658/ JOURNAL OF MEDICAL INTERNET RESEARCH Larsen et al Figure 1. Flow diagram from source population to study population. GP: general practitioner. Larsen et JOURNAL OF MEDICAL INTERNET RESEARCH J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 6 (page number not for citation purposes) J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 6 (page number not for citation purposes) http://www.jmir.org/2019/1/e11658/ JOURNAL OF MEDICAL INTERNET RESEARCH Larsen et al Table 3. Descriptive analysis of determinants of the acceptance of the first digital invitation. J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 7 (page number not for citation purposes) J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 7 ( b f i i ) J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 7 http://www.jmir.org/2019/1/e11658/ (page number not for citation purposes) FO http://www.jmir.org/2019/1/e11658/ Recruitment and Overall Uptake Missing, n (% Total (N=8814), n (%) Nonconsenters (N=5269), n (%) Consenters (N=3545), n (%) Determinants Demographya Age (years)   0 (0.00) 2653 (30.09) 1921 (36.45) 732 (20.64) 29-39     0 (0.00) 3026 (34.33) 1875 (35.58) 1151 (32.46) 40-49     0 (0.00) 3135 (35.56) 1473 (27.95) 1662 (46.88) 50-60     Sex   0 (0.00) 4379 (49.68) 2845 (53.99) 1590 (44.85) Male     0 (0.00) 4435 (50.31) 2424 (46.00) 1955 (55.14) Female     Country of origin   18 (0.20) 7831 (88.44) 4446 (84.38) 3385 (95.48) Denmark     18 (0.20) 549 (6.22) 458 (8.69) 91 (2.56) Western     18 (0.20) 416 (4.71) 347 (6.58) 69 (1.94) Non-Western     Cohabitation   18 (0.20) 2242 (25.43) 1516 (28.77) 726 (20.47) Single     18 (0.20) 6554 (74.35) 3735 (70.88) 2819 (79.52) Cohabiting     Partner in project   18 (0.20) 4752 (53.91) 2715 (51.52) 2037 (57.46) Yes     18 (0.20) 4044 (45.88) 2536 (48.13) 1508 (42.53) No     Socioeconomy Educational attainment     583 (6.61) 1707 (19.36) 1194 (22.66) 513 (14.47) Secondary school     583 (6.61) 356 (4.03) 213 (4.04) 143 (4.103) High school     583 (6.61) 3803 (46.14) 2199 (41.73) 1604 (45.24) Vocational education     583 (6.61) 2365 (26.83) 1149 (21.80) 1216 (34.30) Higher education     Employment status   105 (1.19) 6610 (74.99) 3719 (70.58) 2891 (81.55) Employed     105 (1.19) 430 (4.87) 260 (4.93) 170 (4.79) Self-employed     105 (1.19) 272 (3.08) 184 (3.49) 88 (2.48) Benefits     105 (1.19) 1146 (13.00) 806 (15.29) 340 (9.59) Social welfare     105 (1.19) 251 (2.84) 200 (3.79) 51 (1.43) Other     Family income   130 (1.47) 2047 (23.22) 1488 (28.24) 559 (15.76) Low     130 (1.47) 2147 (24.35) 1341 (25.45) 806 (22.73) Middle-low     130 (1.47) 2235 (25.35) 1246 (24.64) 989 (27.89) Middle-high     130 (1.47) 2255 (25.58) 1072 (20.34) 1183 (33.4) High     Medical treatmentb Prescriptions and diagnoses     0 (0.00) 1736 (19.69) 973 (18.46) 763 (21.52) Treatment     0 (0.00) 7078 (80.30) 4296 (81.53) 2782 (78.47) No treatment     Health care usagec Att d t l titi (GP) Table 3. Descriptive analysis of determinants of the acceptance of the first digital invitation. Recruitment and Overall Uptake Missing, n (%) Total (N=8814), n (%) Nonconsenters (N=5269), n (%) Consenters (N=3545), n (%) Determinants http://www.jmir.org/2019/1/e11658/ XSL•FO RenderX XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Missing, n (%) Total (N=8814), n (%) Nonconsenters (N=5269), n (%) Consenters (N=3545), n (%) Determinants 0 (0.00) 7545 (85.60) 4372 (82.97) 3173 (89.50) Yes     0 (0.00) 1269 (14.39) 897 (17.02) 372 (10.49) No     Frequent attender   0 (0.00) 952 (10.80) 584 (11.08) 368 (10.38) Yes     0 (0.00) 7862 (89.19) 4685 (88.91) 3177 (89.61) No     Laboratory tests at GP   0 (0.00) 4524 (51.32) 2471 (46.89) 2053 (57.91) Yes     0 (0.00) 4290 (48.67) 2798 (53.10) 1492 (42.08) No     Preventive consultation at GP   0 (0.00) 947 (10.74) 516 (9.79) 431 (12.15) Yes     0 (0.00) 7867 (89.25) 4753 (90.20) 3114 (87.84) No     aSocial registries. bAnatomical therapeutic chemical codes and International Classification of Diseases 10th edition codes related to diabetes, cardiovascular disease, and chronic obstructive pulmonary disease. Social registries. bAnatomical therapeutic chemical codes and International Classification of Diseases 10th edition codes related to diabetes, cardiovascular disease, and chronic obstructive pulmonary disease. bAnatomical therapeutic chemical codes and International Classification of Diseases 10th edition codes related to diabetes, cardiovascular disease, and chronic obstructive pulmonary disease. cAdministrative codes from the general practitioner. invitation and the frequency of GP appointments, being registered with a preventive consultation, or having a partner that also consented to the study. J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 8 (page number not for citation purposes) Acceptance of the First Digital Invitation The Poisson regressions showed that a higher rate of acceptance of the first digital invitation was associated with sociodemographic factors, including higher age, income, and educational attainment (Table 4). A higher rate of acceptance was also associated with being female, employed, born in Denmark, and cohabiting. Patients not diagnosed with or in treatment for T2DM, CVD, or COPD were more likely to accept the first digital invitation than patients in treatment. Similarly, the acceptance rate was higher among patients who had seen their GP or who had registered 1 or more laboratory tests at their GP within 2 years of giving consent. We found no association between the likelihood of accepting the first digital The CHAID analysis showed that age was the strongest predictor of accepting the first digital invitation followed by the educational attainment in patients below the age of 50 years and income in patients above the age of 50 years (Figure 2). The CHAID showed large subgroup differences in acceptance rates. Of patients below the age of 40 years, with secondary school as the highest educational attainment, 15.92% accepted the first digital invitation. By contrast, the acceptance rate among patients above the age of 50 years, with high income and with at least a bachelor-level education was 68.58 %. http://www.jmir.org/2019/1/e11658/ XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Larsen et al Table 4. Analysis of associations between acceptance of the first digital invitation and sociodemographic determinants, medical treatment, and heal care usage. Acceptance of the First Digital Invitation Model 3 (minimally adjusted) Model 2 (adjusted for age and sex) Model 1 (crude) Sample size (N) Determinants P value IRR (95% CI) P value IRR (95% CI) P value IRRa (95% CI)     Ageb (years) — 1 (0) — 1 (0) —c 1 (0) 2653 29-39   .001 1.08 (1.06-1.10) .001 1.08 (1.06-1.10) .001 1.08 (1.06-1.10) 3026 40-49   .001 1.20 (1.18-1.22) .001 1.20 (1.18-1.22) .001 1.20 (1.18-1.22) 3135 50-60   Sexb — 1 (0) — 1 (0) — 1 (0) 4435 Female   .001 0.94 (0.93-0.95) .001 0.94 (0.93-0.95) .001 0.94 (0.93-0.95) 4379 Male   Country of originb — 1 (0) — 1 (0) — 1 (0) 7831 Denmark   .001 0.81 (0.79-0.84) .001 0.84 (0.81-0.86) .001 0.81 (0.79-0.84) 549 Western   .001 0.81 (0.79-0.84) .001 0.84 (0.81-0.86) .001 0.81 (0.79-0.84) 416 Non-Western   Cohabitationd — 1 (0) — 1 (0) — 1 (0) 2242 Single   .001 1.05 (1.03-01.07) .001 1.07 (1.05-01.08) .001 1.08 (1.06-01.10) 6554 Cohabiting   Partner in projecte — 1 (0) — 1 (0) — 1 (0) 4752 Yes   .70 1.00 (0.99-1.02) .001 0.96 (0.95-0.98) .001 0.96 (0.95-0.98) 4044 No   Educational attainmentf — 1 (0) — 1 (0) — 1 (0) 1707 Secondary school   .001 1.09 (1.05-1.14) .001 1.09 (1.05-1.13) .001 1.08 (1.04-1.12) 356 High school   .001 1.10 (1.08-1.12) .001 1.10 (1.08-1.12) .001 1.09 (1.07-1.12) 3803 Vocational education   .001 1.16 (1.14-1.19) .001 1.17 (1.14-1.19) .001 1.16 (1.14-1.19) 2365 Higher education   Employment statusd — 1 (0) — 1 (0) — 1 (0) 6610 Employed   .05 0.97 (0.94-1.00) .03 0.96 (0.93-1.00) .089 0.97 (0.94-1.00) 430 Self-employed   .03 0.95 (0.91-1.00) .001 0.93 (0.89-0.97) .001 0.92 (0.88-0.96) 272 Benefits   .001 0.94 (0.92-0.96) .001 0.90 (0.88-0.92) .001 0.90 (0.88-0.92) 1146 Social welfare   .005 0.92 (0.87-0.97) .001 0.85 (0.82-0.89) .001 0.84 (0.80-0.87) 251 Other   Family incomed — 1 (0) — 1 (0) — 1 (0) 2047 Low   .001 1.05 (1.02-1.07) .001 1.07 (1.05-1.10) .001 1.08 (1.06-1.10) 2147 Middle-low   .001 1.06 (1.04-1.09) .001 1.11 (1.09-1.14) .001 1.13 (1.11-1.16) 2235 Middle-high   .001 1.08 (1.05-1.10) .001 1.14 (1.12-1.17) .001 1.20 (1.17-1.22) 2255 High   Prescriptions and diagnosesd — 1 (0) — 1 (0) — 1 (0) 1736 Treatment   .04 1.02 (1.00-1.04) .02 1.02 (1.00-1.04) .001 0.97 (0.95-0.99) 7078 No treatment   Attendance at general practitioner (GP)d Table 4. Analysis of associations between acceptance of the first digital invitation and sociodemographic determinants, medical treatment, and health care usage. care usage. J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 9 (page number not for citation purposes) | | | | p (page number not for citation purposes) Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 9 (page number not for citation purposes) J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 10 (page number not for citation purposes) Acceptance of the First Digital Invitation Sample s Determinants     Ageb (years) 2653 29-39   3026 40-49   3135 50-60   Sexb 4435 Female   4379 Male   Country of originb 7831 Denmark   549 Western   416 Non-Western   Cohabitationd 2242 Single   6554 Cohabiting   Partner in projecte 4752 Yes   4044 No   Educational attainmentf 1707 Secondary school   356 High school   3803 Vocational education   2365 Higher education   Employment statusd 6610 Employed   430 Self-employed   272 Benefits   1146 Social welfare   251 Other   Family incomed 2047 Low   2147 Middle-low   2235 Middle-high   2255 High   Prescriptions and diagnosesd 1736 Treatment   7078 No treatment   Attendance at general practitioner (G 7545 Yes http://www.jmir.org/2019/1/e11658/ XSL•FO RenderX XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Model 3 (minimally adjusted) Model 2 (adjusted for age and sex) Model 1 (crude) Sample size (N) Determinants P value IRR (95% CI) P value IRR (95% CI) P value IRRa (95% CI)     .001 0.95 (0.93-0.98) .001 0.93 (0.91-0.95) .001 0.91 (0.89-0.93) 1269 No   Frequent attender to GPd — 1 (0) — 1 (0) — 1 (0) 952 Yes   .07 1.02 (1.00-1.05) .007 1.03 (1.01-1.06) .30 1.01 (0.99-1.04) 7862 No   Laboratory tests at GPd — 1 (0) — 1 (0) — 1 (0) 4524 Yes   .001 0.95 (0.94-0.97) .001 0.95 (0.94-0.97) .001 0.93 (0.91-0.94) 4290 No   Preventive consultation at GPd — 1 (0) — 1 (0) — 1 (0) 947 Yes   .66 0.99 (0.97-1.02) .94 1.00 (0.98-1.02) .001 0.96 (0.94-0.98) 7867 No   aIncidence rate ratio. bModel 3 adjustments: no adjustments. cReference category. dModel 3 adjustments: age, sex, country of origin, and education. eModel 3 adjustments: cohabitation. fModel 3 adjustments: age, sex, and country of origin. fModel 3 adjustments: age, sex, and country of origin. http://www.jmir.org/2019/1/e11658/ XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Larsen et al Figure 2. Chi-square automatic interaction detection analysis of the acceptance of the first digital invitation. GP: general practitioner. Acceptance of the Second Digital Invitation Acceptance of the Second Digital Invitation Among Acceptance of the Second Digital Invitation Among Consenters (N=3545) J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 11 (page number not for citation purposes) http://www.jmir.org/2019/1/e11658/ Main Findings In this study, 40.22% of our sample accepted the first digital invitation and 30.19% accepted both digital invitations. That is, among those who accepted the first digital invitation, 75.06% also accepted the second. The rates of acceptance of both digital invitations were higher among women, elderly patients, patients of higher SES, and patients not diagnosed with or in treatment for T2DM, COPD, or CVD. Patients who had seen their GP within the past 2 years were also more likely to accept the digital invitations. The frequency of GP appointments, registering for a preventive consultation, or having a partner who had accepted the first digital invitation showed no association with the acceptance of either invitation. In the subpopulation of patients (N=2661) who accepted the first digital invitation, women, patients of relatively high age and SES, and patients who had not seen their GP for a period of 2 years before giving their consent showed a higher rate of acceptance of the second invitation. No other health care usage, including having had a health check within the previous 2 years or being diagnosed with or in treatment for T2DM, COPD, or CVD showed any association with the rate of acceptance. Low patient uptake of stepwise models for preventing chronic diseases seems to be the current norm. This trend may be attributed to a combination of a recent overall increase in the use of preventive health checks in primary care and a decrease in response rates to research studies in general [34,35]. A recent Dutch study of a stepwise prevention model showed an uptake rate of 29% in patients aged 45 to 70 years, whereas an Australian study had an initial uptake rate of 31% in patients aged 40 to 64 years [11,36]. A feasibility study to this study showed an uptake rate of 63% using paper-based invitations, with a link to a Web-based questionnaire and an enclosed hard copy questionnaire and return envelope [37]. Two other Danish studies of stepwise models showed uptake rates of 55% in a general population aged 30 to 49 years and 30% in a population of social housing residents aged 45 to 70 years [38,39]. Both Danish studies used a proactive approach by which paper-based invitations indicated a prebooked time and date—a method which has been shown to garner increased response rates in a previous study [40]. Acceptance of the Second Digital Invitation Acceptance of the Second Digital Invitation In the entire study population, the rate of acceptance of the second digital invitation showed associations similar to the ones in the analysis of the first digital invitation. The only differences were higher rates of acceptance among frequent GP attenders and no association with attendance or nonattendance to the GP. The similarity also applied to the CHAID analysis, in which age was shown to be the strongest predictor, followed by educational attainment in the age below 50-years bracket and income in the age above 50-years bracket. The CHAID analysis showed a rate of acceptance of 8.37% in patients below the age of 40, with secondary school as their highest educational attainment and income below 50% of the median. By contrast, among patients aged more than 50 years, with an income of more than 50% above the median and at least a bachelor-level education, 60.40% accepted the second digital invitation. Among the patients who accepted the first digital invitation, the Poisson regressions indicated associations between the rate of acceptance of the second digital invitation and most sociodemographic variables. The rate of acceptance increased with age, educational attainment, and income. Being female, employed, and born in Denmark also correlated positively with rates of acceptance. We saw higher rates of acceptance among patients who had not seen their GP within 2 years before consenting to the study. No other variables describing health status or health care usage showed an association with rates of acceptance—that includes having received a health check during a period of 2 years before consenting to the study. The CHAID analysis showed that age was the strongest predictor of acceptance of the second digital invitation among those who http://www.jmir.org/2019/1/e11658/ J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 11 (page number not for citation purposes) XSL•FO RenderX XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Larsen et al evidence of the association between uptake of stepwise models and medical treatment or health care usage is scarce and largely inconclusive. This is presumably because of a general lack of health and health care information on nonresponders. Nonetheless, the results of medical treatment and health care usage in this study differ somewhat from previous studies. We show slightly higher rates of acceptance of the digital invitations among patients not diagnosed with or in treatment for T2DM, COPD, or CVD. Acceptance of the Second Digital Invitation This is in line with a comparable Danish study [38], whereas other studies report either higher uptake among patients with chronic diseases [42,43] or no association [44,45]. The inconsistency in results could be explained by different definitions of medical treatment. Similarly, different definitions of preventive services could explain why we see no association between acceptance of the digital invitations and use of preventive consultations at the GP, whereas other studies suggest an association [38]. We saw no association between acceptance of either invitation and having had a health check during a period of 2 years leading up to consent; however, other studies have consistently found that prior use of health checks seems to increase the likelihood of getting another health check [46-48]. Interestingly, we found an association between not having had a GP appointment during the previous 2 years and a higher rate of acceptance of the second digital invitation among those who also accepted the first digital invitation. This may suggest that true compliers to the study (ie, patients who would not have taken up the offer had they not been invited) are more likely to also accept the second digital invitation than always-users (ie, patients who always respond to invitations to participate in preventive services) [49]. As we saw no association between the rates of acceptance of the second digital invitation and other variables of health care utilization, this result should be interpreted with great care and examined further in future studies. accepted the first digital invitation and the only predictor in patients younger than 50 years. In patients older than 50 years, income followed age as the second strongest predictor. Specifically, when it came to the patients older than 50 years, with a middle-low or middle-high income, the CHAID analysis revealed that nonattendance at the GP and not receiving medical treatment during a period of 2 years before consenting were both strong predictors of whether or not participants accepted the second digital invitation. J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 12 (page number not for citation purposes) http://www.jmir.org/2019/1/e11658/ Main Findings Furthermore, the associations between acceptance of digital invitations and socioeconomic determinants are in line with the evidence from other Danish and European studies of health checks [6,38,41]. However, the CHAID analyses showed that the differences in rates of acceptance among the SES groups in our study are larger than those observed in the 2 Danish studies mentioned above [38,39]. The Efficacy of Digital Recruitment To our knowledge, this is the first study to report on the rates of acceptance of digital invitations to participate in a stepwise model for prevention of chronic diseases. Moreover, it is most likely the first to report on digital recruitment to a health intervention using digital invitations. We cannot establish whether rates of acceptance would have been different if recruitment had been paper-based, as we did not include a random subpopulation, which received paper-based invitations. To our knowledge, comparisons of digital and paper-based invitations sent by regular mail have only been reported once in a randomized study by Ebert et al [19]. This study showed that 50- to 59-year-old responders to the digital invitation were more likely to be of higher SES than their counterparts who responded to paper invitations. However, no differences were seen in those aged 30 to 39 years. The overall rate of acceptance of digital invitations and paper-based invitations was comparable. The rate of acceptance of digital invitations in this study and the results reported by Ebert et al may suggest slightly lower overall acceptance rates and slightly larger SES differences when using digital invitations. However, the rate of acceptance of digital invitations combined with Web-based data collection may resemble the emergence of the combined paper-based invitation and Web-based data-collection approach, http://www.jmir.org/2019/1/e11658/ XSL•FO RenderX XSL•FO RenderX JOURNAL OF MEDICAL INTERNET RESEARCH Larsen et al Larsen et al data from the primary care sector on specific tasks performed as part of a consultation, such as taking a blood sample, a spirometry, or having a preventive consultation, may be incomplete and more prone to human error and should as such be interpreted with care. Another strength is the combination of DAG and CHAID analyses to establish a both theory-driven and data-driven analytical approach. The DAG established adjustments to the Poisson regression models based on a theoretical and evidence-based causal model. The CHAID used the data from this study and identified the strongest predictors of attendance. However, residual confounding and collider bias cannot be eliminated in the regressions because of both a rather complex causal model, conditional independence between exposures, and the unavailability of a number of exposures—especially health-risk behaviors as well as cognitive and psychological parameters [6]. Acknowledgments The project received funding from the Region of Southern Denmark and the Danish Ministry of Health. The funders had no role in the analysis or in the interpretation of the data, writing of the manuscript, or the decision to submit it for publication. Authors' Contributions All authors participated in the design of the study. LBL analyzed and interpreted the patient data and was the main contributor to the manuscript. All authors contributed to the interpretation of patient data and the manuscript. All authors read and approved the final manuscript. Conclusions To our knowledge, this is the first study to report on the rates of acceptance of digital invitations to participate in a stepwise model for prevention of chronic diseases. We show acceptance rates of 40% for a first digital invitation and 30% for a second digital invitation, the rates being higher among women, elderly patients, and patients of higher SES; patients not diagnosed with, or in treatment for T2DM, COPD, and/or CVD; and patients having attended the GP within a period of 2 years before consent. A total of 75% of those who accepted the first digital invitation also accepted the second. On one hand, the 2 digital invitations seem to deepen the sociodemographic differences in acceptance compared with a single digital invitation; on the other hand, patients who had not consulted a GP during a period of 2 years before the study, and who were not receiving medical treatment, showed a significantly higher rate of acceptance of the second invitation when it came to those who had accepted the first digital invitation. This suggests that compliers are more prone to accepting the second digital invitation than always-users are. In future studies, multimodal recruitment approaches, which complement digital invitations, are warranted to increase the rates of acceptance among harder-to-reach subpopulations. Nonetheless, more studies of digital invitations are needed to determine if the acceptance rates seen in this study and in the study by Ebert et al could be expected from future studies as well. Similarly, more research is needed to determine whether a multimodal recruitment approach, including digital invitations to personal digital mailboxes will reach other subpopulations more effectively than digital invitations only. Strengths and Limitations The main strengths of this study relate to the high validity of the registries of Statistics Denmark and the random sampling of patients from a large number of GP clinics. Especially, the health and social registries are of high quality, with few missing cases and up-to-date information that has been registered either immediately before study commencement or during another specified period before study commencement [54]. Thus, the impact is most likely negligent because of the strong association between acceptance of the digital invitations and SES. All direct contacts with the GP are most likely both valid and complete as this type of information is automatically registered onto patients’ personal health insurance cards. Other administrative Efficacy of Digital Recruitment Finally, the invitations sent to the digital mailbox and the digital support system were in Danish language only, which may have had a negative impact on the rate of acceptance among people originating from outside of Denmark. where the advent of Web-based data collection methods precipitated an initial drop in the rate of acceptance [34]. In addition, digital invitations sent to personal digital mailboxes seem to be an especially suitable and low-cost recruitment method for patients of high SES. It is well known that risk factors of chronic lifestyle-related diseases are clustered in low SES populations [50]. To generate population health effects, stepwise models for the prevention of chronic diseases may have to employ other low-tech recruitment approaches, which complement digital invitations. Results from the health check program of the British National Health Service indicate that uptake may increase over time, with a clear focus on the hardest-to-reach populations [51]. Thus, it would appear that the lower uptake among patients of lower SES found in this as well as in many other studies can be eliminated, or even inverted, by a focused recruitment effort aimed at deprived communities and outreach services [52,53]. However, at present, digital recruitment is only applicable in a few countries, among others the Nordic countries of Denmark, Norway, and Sweden. When digital mail gets more widespread, the results from the described recruitment procedure and intervention may be well applicable in other settings as well. Multimedia Appendix 2 Data on the acceptance of the second digital invitation in the entire study population. [PDF File (Adobe PDF File), 290KB-Multimedia Appendix 2] Data on the acceptance of the second digital invitation in the entire study population. 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J Epidemiol Community Health 1993 Jun;47(3):224-228 [FREE Full text] [Medline: 8350036] g , , , , , g p health check. J Epidemiol Community Health 1993 Jun;47(3):224-228 [FREE Full text] [Medline: 8350036] 47. Labeit A, Peinemann F, Baker R. http://www.jmir.org/2019/1/e11658/ J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 16 (page number not for citation purposes) J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 17 (page number not for citation purposes) Abbreviations ATC: anatomical therapeutic chemical CHAID: chi-square automatic interaction detection COPD: chronic obstructive pulmonary disease CVD: cardiovascular disease DAG: direct acyclic graph EPR: electronic patient record FEV1: forced expiratory volume in one second FVC: forced vital capacity GP: general practitioner ICD-10: International Classification of Diseases 10th Edition IRR: incidence rate ratio SES: socioeconomic status J Med Internet Res 2019 | vol. 21 | iss. 1 | e11658 | p. 16 (page number not for citation purposes) http://www.jmir.org/2019/1/e11658/ JOURNAL OF MEDICAL INTERNET RESEARCH JOURNAL OF MEDICAL INTERNET RESEARCH Larsen et al T2DM: type 2 diabetes mellitus T2DM: type 2 diabetes mellitus Edited by G Eysenbach; submitted 22.07.18; peer-reviewed by J Ebert, J Apolinário-Hagen; comments to author 19.09.18; revised version received 27.09.18; accepted 29.09.18; published 21.01.19 Please cite as: Please cite as: Larsen LB, Sondergaard J, Thomsen JL, Halling A, Sønderlund AL, Christensen JR, Thilsing T Digital Recruitment and Acceptance of a Stepwise Model to Prevent Chronic Disease in the Danish Primary Care Sector: Cross-Sectional Study J Med Internet Res 2019;21(1):e11658 URL: http://www.jmir.org/2019/1/e11658/ doi: 10.2196/11658 PMID: 30664466 ©Lars Bruun Larsen, Jens Sondergaard, Janus Laust Thomsen, Anders Halling, Anders Larrabee Sønderlund, Jeanette Reffstrup Christensen, Trine Thilsing. 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Unraveling the Complexity of Cardiac Distress: A Study of Prevalence and Severity
Frontiers in psychiatry
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This is an open access article published under a Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. g Copyright 2022 the authors. py g This is an open access article published under a Creative Commons Attribution License (https://creativecommons.org/ which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited Take down policy Th R h P Take down policy The Research Portal is Queen's institutional repository that provides access to Queen's research output. Every effort has been made to ensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in the Research Portal that you believe breaches copyright or violates any law, please contact openaccess@qub.ac.uk. Open Access This research has been made openly available by Queen's academics and its Open Research team. We would love to hear how access to this research benefits you. – Share your feedback with us: http://go.qub.ac.uk/oa-feedback Published in: Frontiers in psychiatry Published in: Frontiers in psychiatry Published in: Frontiers in psychiatry Document Version: Publisher's PDF, also known as Version of record General rights g Copyright for the publications made accessible via the Queen's University Belfast Research Portal is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Take down policy The Research Portal is Queen's institutional repository that provides access to Queen's research output. Every effort has been made to ensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in the Research Portal that you believe breaches copyright or violates any law, please contact openaccess@qub.ac.uk. Unraveling the Complexity of Cardiac Distress: A Study of Prevalence and Severity Jackson, A. C., Rogerson, M. C., Amerena, J., Smith, J., Hoover, V., Alvarenga, M. E., Higgins, R. O., Grande, M. R. L., Ski, C. F., Thompson, D. R., & Murphy, B. M. (2022). Unraveling the Complexity of Cardiac Distress: A Study of Prevalence and Severity. Frontiers in psychiatry, 13, Article 808904. https://doi.org/10.3389/fpsyt.2022.808904 Unraveling the Complexity of Cardiac Distress: A Study of Prevalence and Severity Jackson, A. C., Rogerson, M. C., Amerena, J., Smith, J., Hoover, V., Alvarenga, M. E., Higgins, R. O., Grande, M. R. L., Ski, C. F., Thompson, D. R., & Murphy, B. M. (2022). Unraveling the Complexity of Cardiac Distress: A Study of Prevalence and Severity. Frontiers in psychiatry, 13, Article 808904. https://doi.org/10.3389/fpsyt.2022.808904 Unraveling the Complexity of Cardiac Distress: A Study of Prevalence and Severity Jackson, A. C., Rogerson, M. C., Amerena, J., Smith, J., Hoover, V., Alvarenga, M. E., Higgins, R. O., Grande, M. R. L., Ski, C. F., Thompson, D. R., & Murphy, B. M. (2022). Unraveling the Complexity of Cardiac Distress: A Study of Prevalence and Severity. Frontiers in psychiatry, 13, Article 808904. https://doi.org/10.3389/fpsyt.2022.808904 Jackson, A. C., Rogerson, M. C., Amerena, J., Smith, J., Hoover, V., Alvarenga, M. E., Higgins, R. O., Grande, M. R. L., Ski, C. F., Thompson, D. R., & Murphy, B. M. (2022). Unraveling the Complexity of Cardiac Distress: A Study of Prevalence and Severity. Frontiers in psychiatry, 13, Article 808904. https://doi.org/10.3389/fpsyt.2022.808904 Unraveling the Complexity of Cardiac Distress: A Study of Prevalence and Severity Alun C. Jackson 1,2,3*, Michelle C. Rogerson 1, John Amerena 4,5, Julian Smith 6,7, Valerie Hoover 8, Marlies E. Alvarenga 1,9,10,11, Rosemary O. Higgins 1,12,13, Michael R. Le Grande 1,14, Chantal F. Ski 1,15,16, David R. Thompson 1,16,17 and Barbara M. Murphy 1,2,18 Alun C. Jackson 1,2,3*, Michelle C. Rogerson 1, John Amerena 4,5, Julian Smith 6,7, Valerie Hoover 8, Marlies E. Alvarenga 1,9,10,11, Rosemary O. Higgins 1,12,13, Michael R. Le Grande 1,14, Chantal F. Ski 1,15,16, David R. Thompson 1,16,17 and Barbara M. Murphy 1,2,18 1 Australian Centre for Heart Health, Melbourne, VIC, Australia, 2 Center on Behavioral Health, Faculty of Health, Deakin University, Geelong, VIC, Australia, 3 Behavioral Health, University of Hong Kong, Pokfulam, Hong Kong SAR, China, 4 Barwon Health, Geelong, VIC, Australia, 5 Deakin School of Medicine, University Hospital Geelong, Geelong, VIC, Australia, 6 Department of Cardiothoracic Surgery, Monash Health, Melbourne, VIC, Australia, 7 Department of Surgery, School of Clinical Sciences, Monash Health, Melbourne, VIC, Australia, 8 Psychiatry and Behavioral Sciences, Stanford Women’s Heart Health Clinic, Stanford, CA, United States, 9 School of Health and Life Sciences, Federation University Australia, Berwick, VIC, Australia, 10 Victorian Heart Institute, Melbourne, VIC, Australia, 11 Monash Health & Department of Medicine, Monash University, Melbourne, VIC, Australia, 12 Department of Psychology, Deakin University, Geelong, VIC, Australia, 13 Department of Physiotherapy, University of Melbourne, Melbourne, VIC, Australia, 14 Centre for Behaviour Change, Melbourne School of Psychological Sciences, University of Melbourne, Melbourne, VIC, Australia, 15 Integrated Care Academy, University of Suffolk, Ipswich, United Kingdom, 16 Department of Psychiatry, University of Melbourne, Melbourne, VIC, Australia, 17 School of Nursing and Midwifery, Queen’s University Belfast, Belfast, United Kingdom, 18 Department of Psychology, University of Melbourne, Melbourne, VIC, Australia Edited by: Melissa Thong, German Cancer Research Center (DKFZ), Germany Reviewed by: Gianluca Serafini, San Martino Hospital (IRCCS), Italy Daniela Doege, German Cancer Research Center (DKFZ), Germany Introduction: While much research attention has been paid to anxiety and depression in people who have had a recent cardiac event, relatively little has focused on the broader concept of cardiac distress. Cardiac distress is a multidimensional construct that incorporates but extends beyond common mood disorders such as anxiety and depression. In the present study we assessed the prevalence, severity and predictors of a broad range of physical, affective, cognitive, behavioral and social symptoms of cardiac distress. This is the first study to investigate cardiac distress in this comprehensive way. *Correspondence: Alun C. Jackson alun.jackson@ australianhearthealth.org.au Specialty section: This article was submitted to Psychological Therapy and Psychosomatics, a section of the journal Frontiers in Psychiatry Method: A sample of 194 patients was recruited from two hospitals in Australia. Eligible participants were those who had recently been hospitalized for an acute cardiac event. Data were collected at patients’ outpatient clinic appointment ∼8 weeks after their hospital discharge. Using a questionnaire developed through a protocol-driven 3-step process, participants reported on whether they had experienced each of 74 issues and concerns in the past 4 weeks, and the associated level of distress. They also provided sociodemographic and medical information. Regression analyses were used to identify risk factors for elevated distress. Received: 04 November 2021 Accepted: 23 February 2022 Published: 31 March 2022 Accepted: 23 February 2022 Published: 31 March 2022 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 ORIGINAL RESEARCH published: 31 March 2022 doi: 10.3389/fpsyt.2022.808904 Unraveling the Complexity of Cardiac Distress: A Study of Prevalence and Severity Alun C. Jackson 1,2,3*, Michelle C. Rogerson 1, John Amerena 4,5, Julian Smith 6,7, Valerie Hoover 8, Marlies E. Alvarenga 1,9,10,11, Rosemary O. Higgins 1,12,13, Michael R. Le Grande 1,14, Chantal F. Ski 1,15,16, David R. Thompson 1,16,17 and Barbara M. Murphy 1,2,18 INTRODUCTION and self-concept have been reported (12), as have worries about getting back to one’s previous sense of self (13). These are often tied to changes in roles and role function (8, 12), including loss of the ability to provide (11) or be the “breadwinner” or “homemaker” (7). Impacts on intimate relationships are also a concern for some survivors (13), including concerns about resuming sexual activity due to fear of causing another heart attack (7, 14). Challenges navigating the health system are also evident, including difficulties in obtaining information and advice (13), and concerns about having to rely on help from health professionals (9). The psychosocial impacts of an acute cardiac event have gained increasing recognition in the past two decades. It is now well- accepted that both anxiety and depression are common after heart attack and heart surgery (1–3). In a recent Australian study involving over 900 patients admitted to hospital after acute myocardial infarction (AMI) or to undergo coronary artery bypass graft surgery (CABGS), over 40% had elevated anxiety and over 20% had symptoms of depression in the period shortly after hospital discharge, while 31% had anxiety or depression at 6–12 months post-event (3). These rates are up to four times higher than in the general population. The cardiac event also triggers difficulties in coping with change (9), including difficulties adjusting to limitations in everyday life (6) and living with pain (7), resistance to being on medications (9), hypervigilance regarding bodily sensations (7), concerns about making lifestyle changes (7, 15), and difficulties associated with resuming work or being unable to work (7). Some survivors express difficulty in accepting the diagnosis and the disease itself (6). For some, the trauma associated with the cardiac event can trigger the resurfacing of past traumas or unresolved grief, with concomitant intrusive thoughts and nightmares (7). Fear about the future (8, 11, 13) and concerns about having to reprioritise goals and life plans in a foreshortened future (8, 11) have also been reported. Concerns about having another heart attack and fear of dying can also emerge (7, 10), as survivors are forced to confront their mortality, possibly for the first time in their life (7, 8). There is also growing evidence that acute cardiac events are often experienced as traumatic, thereby predisposing survivors to experiencing post-traumatic stress symptoms (PTSS) or disorder (PTSD). Citation: Jackson AC, Rogerson MC, Amerena J, Smith J, Hoover V, Alvarenga ME, Higgins RO, Grande MRL, Ski CF, Thompson DR and Murphy BM (2022) Unraveling the Complexity of Cardiac Distress: A Study of Prevalence and Severity. Front. Psychiatry 13:808904. doi: 10.3389/fpsyt.2022.808904 Results: Across the 74 issues and concerns, prevalence ratings ranged from a high of 66% to a low of 6%. The most commonly endorsed items were within the domains of dealing with symptoms, fear of the future, negative affect, and social isolation. Common experiences were “being physically restricted” (66%), “lacking energy” (60%), “being short of breath” (60%), “thinking I will never be the same again” (57%), and “not sleeping well” (51%). While less prevalent, “not having access to the health care I need,” “being March 2022 | Volume 13 | Article 808904 Frontiers in Psychiatry | www.frontiersin.org Jackson et al. Unraveling the Complexity of Cardiac Distress concerned about my capacity for sexual activity,” and “being unsupported by family and friends” were reported as highly distressing (74, 64, and 62%) for those experiencing these issues. Having a mental health history and current financial strain were key risk factors for elevated distress. Conclusion and Implications: Specific experiences of distress appear to be highly prevalent in people who have had a recent cardiac event. Understanding these specific fears, worries and stressors has important implications for the identification and management of post-event mental health and, in turn, for supporting patients in their post-event cardiac recovery. Keywords: psychocardiology, cardiac distress, anxiety, stress, depression, fear of progression, secondary prevention Frontiers in Psychiatry | www.frontiersin.org INTRODUCTION The traumatic components of a cardiac event include its abruptness, the risk of death, and a sense of helplessness and loss of control during and after the event (4). It has been suggested that around 12% of patients experience acute coronary syndrome (ACS)-induced PTSD (5). But is there more to a cardiac event than just anxiety, depression and PTSD? Qualitative studies involving cardiac event survivors and clinicians who support them provide greater insight into the breadth of experiences, concerns and worries expressed during convalescence after an acute cardiac event and therefore provide a nuanced understanding of cardiac distress. One qualitative study of survivors concluded that they experience “being forced into a demanding life-shaking journey” (6), while another study of clinicians described it as “a lonely journey, an existential crisis” (7). It appears that the acute cardiac event can trigger the beginning of a completely new life chapter, involving new and difficult emotions, changes in self-concept and identity, and other unexpected challenges, fears and concerns. These challenging emotions, changes and experiences that follow an acute cardiac event can all be conceptualized as “cardiac distress.” We have defined this multi-dimensional construct in previous publications: Cardiac distress is a persistent negative emotional state rather than a transient state; involving multiple psychosocial domains; that challenges a patient’s capacity to cope with living with their heart condition, the treatment of the condition, and the resultant changes to daily living; and challenges the person’s sense of self and future orientation (16, 17). Reported experiences gleaned from qualitative studies include a range of emotions, such as feelings of uncertainty (8– 10), vulnerability (6), loneliness and fear of being alone (7), hopelessness and helplessness (6, 7), anger and resentment (7), sadness, grief and loss (7, 8). Specific losses include loss of independence (7, 11), loss of health and physical strength (8, 11), and loss of control (7). Similarly, various changes in self-identify Importantly cardiac distress spans multiple psychosocial domains, thereby incorporating patients’ responses to physical, affective, cognitive, behavioral and social symptoms and experiences related to their cardiac event and their recovery (16). March 2022 | Volume 13 | Article 808904 Frontiers in Psychiatry | www.frontiersin.org 2 Unraveling the Complexity of Cardiac Distress Jackson et al. factors (high blood pressure, high cholesterol, obesity, and positive family history), and significant comorbidity (diabetes, musculoskeletal conditions, chronic obstructive pulmonary disorder, cancer, stroke, and dementia). Participant Recruitment A sample of 194 patients was recruited from two hospitals in Australia, one in metropolitan Melbourne (Monash Health) and one in regional Victoria (Barwon Health, Geelong). The procedure at each of the two hospitals differed slightly. At Barwon Health, the majority of participants were recruited while they were inpatients at the University Hospital Geelong following their hospital admission for either AMI, CABGS or PCI. At this contact, the Research Nurse provided eligible patients with a brief explanation of the study and asked if they would be willing to participate. Interested patients were then provided with the consent form. Consent was obtained to re- contact participants via telephone ∼6–8 weeks later to complete the questionnaire. A smaller number of participants were recruited during their attendance at cardiac rehabilitation (CR). At Monash Health, participants were recruited at the time of their appointment at the Cardiothoracic Preadmission Clinic, prior to their hospital admission for CABGS, or in the Cardiac Care Unit (CCU) for those with AMI, PCI, and other cardiac conditions. At this contact, the Research Nurse provided eligible patients with a brief explanation of the study and asked if they would be willing to participate. Interested patients were then provided with the consent form. Consent was obtained to re-contact participants at their routine 6–8-week follow- up appointment. INTRODUCTION Psychosocial data included living alone (Y/N), partner status (partnered/unpartnered), presence of a close confidante (Y/N), loss of a close relative or friend in the past 12 months (Y/N; defined as “recent bereavement”), having been diagnosed with a mental health disorder prior to the cardiac event (Y/N, defined as “mental health history”), and financial strain reported on a 5-point Likert scale from 0 “no financial strain” to 4 “extreme financial strain.” The questionnaire was prepared in both hardcopy form for mailout and return, and via the REDCap (Research Electronic Data Capture) system for online completion. The definition also highlights both present and future concerns, thereby taking into account impacts on current self-perception as well as fears about the future (16). The definition also highlights both present and future concerns, thereby taking into account impacts on current self-perception as well as fears about the future (16). Current understanding of the prevalence and correlates of cardiac distress has been limited by a lack of quantitative studies on this topic. While several qualitative studies have been undertaken to explore survivors’ post-event experiences, as outlined earlier, no quantitative studies have investigated the broad range of cardiac distress experiences across multiple psychosocial domains, and incorporating both current and future-orientations. Demographic, Medical, and Psychosocial Questions Questions regarding demographic, medical and psychosocial characteristics were also included. Demographic information included age, sex, country of birth, education, and employment status. Medical information included event type, cardiac rehabilitation (CR) attendance (Y/N), cardiovascular risk Inclusion and Exclusion Criteria Eligible participants were those who have had an acute coronary event, namely acute myocardial infarction (AMI), percutaneous coronary intervention (PCI), coronary artery bypass graft surgery (CABGS), valve issues, heart rhythm disturbance, spontaneous coronary artery dissection (SCAD), or cardiac arrest in the previous 12 months and who attended an outpatient clinic at participating hospitals. Patients who did not have adequate English language proficiency to read and understand the PICF and questionnaire were excluded. Item Generation and Distress Questionnaire Preparation Q p A broad pool of items was generated following a strict protocol which has been described in full elsewhere (17). In brief, the process involved three key steps. First, items included in instruments to measure cardiac anxiety, depression, quality of life and other cardiac-related constructs were reviewed, as were measures of distress used in the oncology and diabetes settings, with a view to creating a pool of cardiac distress-related items. Second, qualitative studies from the cardiac literature were reviewed to identify relevant constructs and generate further items. Third, the item pool was reviewed by an expert multidisciplinary committee of cardiac researchers and clinicians to identify missing constructs and fine-tune item wording. Following these steps, a set of 74 items addressing various issues and concerns was generated. These assessed issues and concerns across seven key conceptual domains, determined a priori by the project team, namely symptoms, self-perception, concerns about the future, negative affect, self-management, social functioning and role functioning. Participants reported on whether or not they had experienced each of 74 issues or concerns in the past 4 weeks by responding Yes or No for each item. For endorsed items, participants then reported on the level of distress associated with the issue, using a response scale where 0 = “no distress at all,” 1 = “slight distress,” 2 = “moderate distress,” and 3 = “severe distress.” Aims of the Study In the present study we assessed the prevalence and severity of a broad range of physical, affective, cognitive, behavioral and social symptoms of cardiac distress. We also investigated the correlates of distress by identifying the patient characteristics that predict these distressing experiences. This is the first study to assess the prevalence, severity and predictors of cardiac distress in this comprehensive way. Questionnaire Completion Due to the COVID-19 pandemic and imposed lockdowns, participant recruitment varied between face-to-face and via telehealth. Questionnaires were either completed in hard copy March 2022 | Volume 13 | Article 808904 Frontiers in Psychiatry | www.frontiersin.org 3 Unraveling the Complexity of Cardiac Distress Jackson et al. TABLE 1 | Characteristics of participants. TABLE 1 | Characteristics of participants. N n % Sociodemographic characteristics Sex Male 194 140 72.2 Female 53 27.3 Prefer not to say 1 0.5 Country of birth Australia 194 138 71.1 Outside Australia 56 28.9 Marital status Married/living with partner 194 137 70.6 Divorced/separated 26 13.4 Widowed 15 7.7 Never married 16 8.2 Education Primary 193 3 1.6 Secondary 83 43.0 Trade or certificate 55 28.5 University degree/post-graduate 52 26.9 Employment status In workforce 194 98 50.5 Not in workforce 96 49.5 Psychosocial characteristics Lives alone 189 43 22.8 Recent bereavement 179 43 24.0 Financial strain 179 73 40.8 Mental health history 194 64 33.0 Medical characteristics Event type Acute myocardial infarction 194 102 52.6 Coronary artery bypass graft surgery 74 38.1 Percutaneous coronary intervention 57 29.4 Other 41 21.1 Attended cardiac rehabilitation 180 80 44.4 Significant co-morbidity 194 113 58.2 N = 179–194 with variations due to incompletion of some questionnaires. Recent bereavement = having lost of relative or friend in the past 12 months. Financial strain = reports of moderate, considerable, or extreme financial strain. “Other” event type includes valve issues (n = 21), heart rhythm disturbance (n = 15), SCAD (n = 6), and cardiac arrest (n = 6). TABLE 1 | Characteristics of participants. ion of some questionnaires. Recent bereavement = having lost of relative or friend in the past 12 months. Financial strain = reports of train. “Other” event type includes valve issues (n = 21), heart rhythm disturbance (n = 15), SCAD (n = 6), and cardiac arrest (n = 6). Social functioning (16 items), Role functioning (five items), and Self-management (13 items). For prevalence, positively endorsed items within each domain were added together to provide domain prevalence scores. Within the “Symptoms” domain, two sub-domains of “physical symptoms” (seven items) and “cognitive symptoms” (five items) were also created. Higher domain and sub-domain prevalence scores indicated endorsement of more items (issues and concerns) within that domain. For distress severity ratings, a distress severity score for each domain was calculated by taking the mean severity ratings of endorsed items within that domain. Ethics Approval This study had ethics approval from the Monash Health Human Research Ethics Committee (approval number: RES-19- 0000631A – 55979, which covered data collection at both the Monash Health and the Barwon University Hospital sites. Questionnaire Completion For each domain and sub-domain, for both prevalence scores and distress severity scores, bivariate analyses (t-tests) were undertaken to identify variations in terms of demographic (age, sex, and employment status), medical (event type, CVD risk factors, comorbidities, and CR attendance), and psychosocial (mental health history, social isolation, and financial strain) characteristics. These particular psychosocial characteristics were selected for examination as they have been previously demonstrated to be strongly predictive of persistent or worsening post-event anxiety and depression (18). Using significant variables from the bivariate analyses, a series of multivariable linear regression analyses were then or online. Thus, instead of completing the questionnaire while waiting for their clinic appointments as originally intended, participants were either directed to the website of the Australian Center for Heart Health (ACHH) to use an online link to the REDCap questionnaire or were mailed a hard copy of the questionnaire for completion at home and return in a reply- paid envelope to the ACHH. The questionnaire took ∼25 min to complete. No identifying information was collected as no participant follow-up was involved. Data Analysis Frequencies were calculated for the prevalence and the distress severity ratings of each of the 74 issues and concerns. Items were clustered into the seven pre-determined domains of Symptoms (15 items), Self-perception (nine items), Concerns about the future (eight items), Negative Affect (eight items), March 2022 | Volume 13 | Article 808904 Frontiers in Psychiatry | www.frontiersin.org 4 Unraveling the Complexity of Cardiac Distress Jackson et al. TABLE 2 | Prevalence of issues and concerns in rank order. Issue/item n % Being physically restricted 126 65.6 Lacking energy 115 60.3 Being short of breath 115 59.6 Thinking I will never be the same again 110 57.0 Not sleeping well 98 50.5 Thinking about having another heart event 93 48.4 Being irritated by little things 93 48.4 Thinking my condition might get worse 91 46.9 Having chest discomfort 87 45.1 Avoiding situations and activities 86 45.0 Being unable to do things that I know will improve my health 87 44.8 Having difficulty concentrating 86 44.6 Being overly aware of my heart in my chest 85 44.0 Thinking that I am not the person that I used to be 84 43.8 Not knowing how my family will cope if something should happen to me 82 42.9 Having difficulty remembering things 78 40.4 Not knowing what the future holds for me 77 40.3 Avoiding activities that make my heart beat faster 76 39.4 Being unsure about how much exercise or physical activity I should be doing 75 39.3 Having changes in my usual roles 72 37.9 Being tearful more easily than before 72 37.1 Forgetting things more than before 70 36.6 Not knowing what will happen to other people if I die 68 35.4 Having to make difficult lifestyle changes because of my heart condition 65 33.9 Being emotionally exhausted 63 33.3 Being afraid of dying 64 33.0 N = 189–194 with variations due to incompletions in some questionnaires. Only items endorsed by >33% of participants shown. As shown in Table 1, participants were mostly male (72%) and married or partnered (71%). Most (71%) were born in Australia and for those who were not (n = 56), the majority were born in the United Kingdom (n = 18), and New Zealand (n = 7). Nearly half of the participants were no longer in the paid workforce, and 40% were experiencing financial strain. Most Common Issues and Concerns The most commonly endorsed issues and concerns are shown in Table 2 and Figure 1, with those items endorsed by over a third of participants depicted. The data indicate the proportion of participants who had experienced each of these in the previous 4 weeks. Table 2 shows the items in rank order from highest to lowest, while Figure 1 shows the same items grouped according to the seven pre-determined domains. N = 189–194 with variations due to incompletions in some questionnaires. Only items endorsed by >33% of participants shown. N = 189–194 with variations due to incompletions in some questionnaires. Only items endorsed by >33% of participants shown. undertaken to identify the key predictors of prevalence and severity for each domain and sub-domain. For each model the variables entered included: age (years), sex, event type (CABGS vs. non-CABGS), living alone status, financial stress, history of mental illness, bereavement, and employment status. For the financial strain variable, responses of 3 “moderate,” 4 “considerable,” and 5 “extreme financial strain” were combined to indicate presence of financial strain. CVD risk factors (high blood pressure, high cholesterol, obesity, positive family history), various event types (AMI, PCI, and “other”), CR attendance, and comorbidities (Diabetes Mellitus, musculoskeletal, and “other” comorbid conditions) were not significant at the bivariate level and were therefore not entered into the multivariate analyses. As shown in Table 2, the most commonly endorsed items were being physically restricted, lacking energy, being short of breath, thinking I will never be the same again, and not sleeping well. Each of these five items was endorsed by ≥50% of participants. A further 10 items were endorsed by between 40 and 50% of participants, including thinking about having another heart event, being irritated by little things, thinking my condition might get worse, having chest discomfort, avoiding situations and activities, being unable to do things that I know will improve my health, having difficulty concentrating, being overly aware of my heart in my chest, thinking that I’m not the person I used to be, and not knowing how my family will cope if something should happen to me. A further 11 items were endorsed by between 33 and 40% of participants. The remaining 48 items (from the full pool of 74 items) were each endorsed by fewer than 33% of participants (not shown in table). Data Analysis Approximately one quarter lived alone and for those who lived with others (n = 146), the majority lived with their partner (n = 83), or their partner and children (n = 40). All participants had experienced their event in the previous 12 months, with the majority (94%) occurring within the past 3 months (mean = 2.2, SD = 1.9 months). Most participants had an AMI, CABGS, or PCI, while a smaller number (21%) reported having experienced other heart issues (valve issues, n = 21; heart rhythm disturbance, n = 15; SCAD, n = 6; cardiac arrest, n = 6). More than half (n = 100, 56%) of the participants did not attend CR, with the most common reasons being people waiting for CR (42%), declining attendance (21%), and concerns associated with COVID-19 (14%), including barriers associated with using telehealth. Just over half the participants (58%) had at least one significant co-morbidity, most often diabetes (n = 58, 30%) or musculoskeletal conditions (n = 40, 32%). In addition, cardiovascular risk factors such as hypertension (37%), high cholesterol (31%), obesity (13%), and obstructive sleep apnoea (12%) were present. One third of participants had a history of mental health problems (depression or anxiety), with one in four having experienced depression and one in five having experienced anxiety. Participants Three of the four next most distressing items belonged to the “Social” domain. For both the “Social” and “Symptoms” domains, three items were rated as moderately or severely distressing by over 50% of those who experienced these issues, whereas this was the case for only one item within each of the “Future,” “Affect,” and “Role functioning” domains. Overall though, the pattern evidenced in Figure 2 shows that a range of items across all six domains were identified as highly distressing. Most Distressing Issues and Concerns While some items were highly prevalent, others were highly distressing. The items which elicited the highest ratings in terms of the severity of distress they caused are shown in Table 3. Not having access to needed health care was identified as the most distressing issue, being rated as either moderately or severely distressing by 74% of those who experienced this issue. Notably though, only 14% of participants actually reported experiencing this issue. Being concerned about capacity for sexual activity was the next most distressing issue, being rated as moderately or severely distressing by 64% of those who experienced it. However, only around one in five participants reported experiencing this issue. Being unsupported by family and friends, being isolated from family and friends, and being unavailable to family and friends were each reported as highly distressing albeit not commonly experienced issues. Notably, “not knowing how my family will cope if something happens Participants As shown in Figure 1, four of the five most commonly endorsed items belonged to the “Symptoms” domain and more specifically, the “physical symptoms” sub-domain. Indeed, endorsement of items within the Symptoms domain was Participants ranged in age from 22 to 90 years, with a mean (SD) age of 63.7 (11.2) years. Participant sociodemographic, psychosocial and medical characteristics are shown in Table 1. March 2022 | Volume 13 | Article 808904 Frontiers in Psychiatry | www.frontiersin.org 5 Unraveling the Complexity of Cardiac Distress Jackson et al. FIGURE 1 | Prevalence of issues and concerns in domains. N = 189–194 with variations due to incompletions in some questionnaires. Only items endorsed by >33% of participants shown. FIGURE 1 | Prevalence of issues and concerns in domains. N = 189–194 with variations due to incompletions in some questionnaires. Only items endorsed by >33% of participants shown FIGURE 1 | Prevalence of issues and concerns in domains. N = 189–194 with variations due to incompletions in some questionnaires. Only items endorsed by >33% of participants shown. s and concerns in domains. N = 189–194 with variations due to incompletions in some questionnaires. Only items endorsed by to me” and “not sleeping well” were rated as both distressing and common. common, with nine of the symptom-related items each being endorsed by over a third of participants. Items within the “Future” domain were the next most commonly endorsed, with three items from this domain being in the top 10, and six being endorsed by over a third of participants. Items in the “Affect” domain were the next most prevalent, with four being endorsed by over a third of participants. In contrast, items within the domains of “Social functioning,” “Self- management,” “Self-perception,” and “Role functioning” were the least commonly endorsed. In terms of the experience of “severe distress,” having bad dreams and nightmares was identified as the most severely distressing for those who experienced it; 28% of those who experienced bad dreams and nightmares reported that this was “severely distressing.” Being unsupported by family and friends and being unable to work were also commonly identified as causing severe distress amongst those who experienced these issues. As shown in Figure 2, the two most distressing issues belonged to the “Self-management” and “Self-perception” domains, although other items within these two domains were rated as less distressing. Frontiers in Psychiatry | www.frontiersin.org Predictors of Prevalence and Severity Ratings Results of the multivariate regression analyses are shown in Table 4. In terms of prevalence ratings, financial strain was a significant predictor for all domains, and history of mental illness was a significant predictor for all but two domains. Living alone was March 2022 | Volume 13 | Article 808904 Frontiers in Psychiatry | www.frontiersin.org 6 Unraveling the Complexity of Cardiac Distress Jackson et al. TABLE 3 | Most distressing issues and concerns in rank order. TABLE 3 | Most distressing issues and concerns in rank order. Prevalence Level of distress caused Moderate Severe Moderate or severe Not having access to the health care I need 14.0 66.7 7.4 74.1 Being concerned about my capacity for sexual activity 22.0 47.6 16.7 64.3 Being unsupported by my friends or family 6.8 38.5 23.1 61.6 Being isolated from family and friends 15.3 44.8 13.8 58.6 Not knowing how my family will cope if something happens to me 42.9 39.0 18.3 57.3 Being unavailable to my family and friends 17.3 45.5 9.1 54.6 Not sleeping well 50.5 43.3 10.3 53.6 Having more pain than I expected to have 22.8 36.4 15.9 52.3 Having bad dreams and nightmares 24.0 23.9 28.3 52.2 Not being able to cope effectively with my heart condition 27.1 40.4 11.5 51.9 Lacking energy 60.3 36.0 15.8 51.8 Becoming a burden to my family 31.4 38.3 13.3 51.6 Not knowing what the future holds for me 40.3 42.9 7.8 50.7 Being unsure about how much exercise I should be doing 39.3 45.3 5.3 50.6 Being emotionally exhausted 33.3 42.9 6.3 49.2 Being unable to deal with stress 32.8 38.7 9.7 48.4 Being unable to do things that will improve my health 44.8 41.4 6.9 48.3 Being unable to plan for the future 24.9 39.6 8.3 47.9 Not being able to return to work or continue working 23.7 25.6 22.2 47.8 Thinking that I’m not the person I used to be 43.8 40.5 7.1 47.6 Not having anyone to talk to who understands my difficulties 17.9 35.3 11.8 47.1 Being afraid of dying 33.0 35.9 10.9 46.8 Feeling lonely 24.5 38.3 8.5 46.8 Lacking purpose and meaning in life 27.1 28.8 17.3 46.1 Having difficulty making decisions 25.0 4.2 4.2 45.9 N = 189–194 with variations due to incompletions in some questionnaires. Only 25 most distressing items shown. Predictors of Prevalence and Severity Ratings relating to cardiac symptoms, self-perception, concerns about the future, negative affect, social functioning, role functioning, and self-management. The most commonly endorsed items were within the domains of dealing with symptoms, fear of the future, negative affect, and social functioning, and included “being physically restricted,” “lacking energy,” “being short of breath,” “thinking I will never be the same again,” and “not sleeping well.” While less prevalent, “not having access to the health care I need,” “being concerned about my capacity for sexual activity,” and “being unsupported by family and friends” were reported as highly distressing for those experiencing these issues. The study has also identified key demographic and psychosocial predictors of distress. In doing so, the study has expanded our understanding of the multi-faceted nature of distress caused by a cardiac event. predictive of prevalence ratings for the “Cognitive symptoms” sub-domain and the “Social” domain. Females had significantly higher prevalence for the “Symptoms,” “Future,” “Affect,” and “Social” domains, and the “Physical symptoms” sub-domain, while those who had undergone CABGS had higher prevalence for the “Symptoms” domain and the “Physical symptoms” sub- domain. Bereavement was associated with higher prevalence for the Symptoms domain [aOR = 3.82 (95% CI 1.19–12.33), p = 0.025], while employment status was not significantly associated with prevalence ratings for any of the domains (not shown in table). In terms of severity ratings, history of mental illness was a significant predictor across all but one domain, while financial strain was a significant predictor for the “Affect” and “Self- management” domains. Younger age was associated with higher severity ratings for the “Social” domain [aOR = 0.99 (95% CI 0.97–1.00), p = 0.016; not shown in table]. Female sex, event type, living alone, and employment status were not significantly associated with severity ratings for any of the domains. Many of the highly prevalent issues and concerns identified by these cardiac event survivors related to physical symptoms and the fear that they would not ever be the same again. Concerns about loss of health have been identified previously in qualitative studies (6, 8, 11), as have concerns about loss of one’s previous sense of self (13). Many of these concerns about symptoms and physical adjustment are addressed and normalized in CR, thereby reinforcing the need to increase the rate of referral to and attendance at CR as a crucial step in recovery. DISCUSSION The findings of the present study document the prevalence and severity of cardiac distress across 74 items within seven domains March 2022 | Volume 13 | Article 808904 Frontiers in Psychiatry | www.frontiersin.org 7 Unraveling the Complexity of Cardiac Distress Jackson et al. FIGURE 2 | Issues and concerns rated as severely or moderately distressing. N = 189–194 with variations due to incompletions in some questionnaires. Dark color indicates rating of “severe” distress, light color indicates rating of “moderate distress.” Only 25 most distressing items shown. FIGURE 2 | Issues and concerns rated as severely or moderately distressing. N = 189–194 with variations due to incompletions in some questionnaires. Dark color indicates rating of “severe” distress, light color indicates rating of “moderate distress.” Only 25 most distressing items shown. Concerns about the future were also highly prevalent. These included fears about worsening symptoms, having a recurrent event and, ultimately, fears about death. Again, these issues have been identified in previous qualitative studies (7, 8, 10, 11, 13). Additionally, future-related items were also identified as highly distressing, particularly those centering on the issue of an uncertain future. Previous qualitative studies have similarly highlighted the distress caused by uncertainty and the inherent inability to plan for the future post-cardiac event (8–11). disturbance, including poor sleep quality and nightmares, and concerns about how patients’ families will cope, were both highly endorsed and very distressing. These issues are very important to address, to enhance patients’ physical and emotional recovery. We have argued elsewhere for the need for screening for sleep disorders in the recovery phase during CR (19) so that survivors are given relevant and timely assistance with this issue. Similarly, health professionals engaging with cardiac patients in recovery need to be alert to survivors’ concerns about family and, indeed, to the importance of knowing about the degree of connectedness survivors have with their family or within their community. Those experiencing difficulties in this area should be considered for referral to a family-oriented service. y p p While recognizing the highly prevalent nature of physical symptoms in particular, this study highlights the importance of assessing not just what people experience, but how distressing these issues are perceived to be. Not having access to needed health care was identified as the most distressing issue, extending previous qualitative findings regarding worries about obtaining professional help and advice (9, 13). Frontiers in Psychiatry | www.frontiersin.org DISCUSSION Given that the present study was conducted during the COVID-19 pandemic, with concomitant restrictions in face-to-face healthcare delivery and pivoting to telehealth support options, it is perhaps not surprising that this issue was identified as highly distressing. Also extending previous qualitative studies (7, 14), ours is the first to highlight concerns about sexual capacity as amongst the most distressing of all issues faced by cardiac event survivors. Concern about future sexual capacity reflects not only a person’s concern with physical functioning but represents an important dimension of personal identity that is under threat, and a concern with acceptance of potential limitations, whether psychological or physical in origin. It is notable that women experienced more of the issues and concerns listed than did men, with female sex a significant predictor of high prevalence across several domains. This is consistent with women’s previously reported higher levels of anxiety and depression (20) and may also be due to women being more expressive than men and highly attuned to identifying and acknowledging their psychosocial concerns. Although female sex was not predictive of distress severity ratings, this may be due to the fact that these analyses were undertaken by domain rather than for individual items. Consistently, it was somewhat surprising that few age differences were identified and, again, this may be due to a lack of fine-tuned comparisons, which was outside the scope of the present study. As may be expected, people with a mental health history experienced more of the issues and concerns, and also rated them as more distressing. Those with a mental health history tend to The present findings highlight a number of issues that can be addressed in cardiac rehabilitation. Difficulties due to sleep March 2022 | Volume 13 | Article 808904 8 Unraveling the Complexity of Cardiac Distress Jackson et al. TABLE 4 | Significant predictors of distress prevalence and severity ratings. DISCUSSION Predictor variables Female sex CABGS Live alone Financial Strain History of mental illness aOR (95%CI) p aOR (95%CI) p aOR (95%CI) p aOR (95%CI) p aOR (95%CI) p Prevalence ratings Symptoms 4.29 (1.33–13.87) 0.015 2.97 (1.33–13.87) 0.042 6.95 (2.46–19.67) <0.001 5.81 (1.95–17.33) 0.002 Sympt-Phys 1.89 (1.02–3.49) 0.042 1.98 (1.14–3.43) 0.015 2.27 (1.31–3.93) 0.004 2.15 (1.21–3.81) 0.009 Sympt-Cog 1.92 (1.08–3.44) 0.027 1.90 (1.17–3.07) 0.009 2.01 (1.21–3.34) 0.007 Future 2.45 (1.01–5.97) 0.049 3.73 (1.69–8.22) 0.001 Affect 2.17 (1.06–4.47) 0.035 3.64 (1.91–6.90) <0.001 5.12 (2.60–10.08) <0.001 Social 3.82 (1.21–12.07) 0.023 6.29 (1.86–21.29) 0.003 6.66 (2.42–18.30) <0.001 5.58 (1.92–16.12) 0.002 Self-perception 3.84 (1.92–7.71) <0.001 2.47 (1.17–5.22) 0.018 Role function 2.44 (1.57–3.80) <0.001 Self-manage 8.38 (3.33–21.10) <0.001 3.11 (1.17–8.30) 0.024 Severity ratings Symptoms 1.42 (1.18–1.72) <0.001 Sympt-Phys 1.42 (1.15–1.76) 0.002 Sympt-Cog 1.45 (1.11–1.89) 0.007 Future 1.35 (1.08–1.69) 0.009 Affect 1.25 (1.02–1.54) 0.032 1.40 (1.13–1.74) 0.002 Social 1.53 (1.25–1.96) <0.001 Self-perception 1.45 (1.12–1.87) <0.001 Role function 1.59 (1.15–2.19) 0.005 Self-manage 1.29 (1.06–1.57) 0.012 Full multivariable linear regression models for each rating included: age (years), sex, CABGS status, live alone status, financial stress, history of mental illness, and bereavement in past 12 months. aOR, adjusted odds ratio; CABGS, coronary artery bypass graft surgery; Symptoms, symptoms domain; Sympt-Phys, Physical symptoms subdomain; Sympt-Cog, Cognitive symptoms subdomain; Future, Concerns about the future domain; Affect, Negative Affect domain; Social, Social functioning domain; Self-Perception, Self-perception domain; Role Function, Role functioning domain; Self-Manage, Self-management domain. TABLE 4 | Significant predictors of distress prevalence and severity ratings. Full multivariable linear regression models for each rating included: age (years), sex, CABGS status, live alone status, financial stress, history of mental illness, and bereavement in past 12 months. aOR, adjusted odds ratio; CABGS, coronary artery bypass graft surgery; Symptoms, symptoms domain; Sympt-Phys, Physical symptoms subdomain; Sympt-Cog, Cognitive symptoms subdomain; Future, Concerns about the future domain; Affect, Negative Affect domain; Social, Social functioning domain; Self-Perception, Self-perception domain; Role Function, Role functioning domain; Self-Manage, Self-management domain. Full multivariable linear regression models for each rating included: age (years), sex, CABGS status, live alone status, financial stress, history of mental illness, and bereavement in past 12 months. aOR, adjusted odds ratio; CABGS, coronary artery bypass graft surgery; Symptoms, symptoms domain; Sympt-Phys, Physical symptoms subdomain; Sympt-Cog, Cognitive symptoms subdomain; Future, Concerns about the future domain; Affect, Negative Affect domain; Social, Social functioning domain; Self-Perception, Self-perception domain; Role Function, Role functioning domain; Self-Manage, Self-management domain. DISCUSSION symptoms, raising concerns for this group in terms of their cardiac recovery. Consistently, a recent study found that death of a spouse or partner during the year prior to a first AMI is associated with an increased risk of recurrent AMI and cardiac- related death (24), highlighting bereavement as a red flag for compromised cardiac recovery. have a lower base of resilience and coping and therefore will experience more cardiac-related issues and find them inherently more distressing. Previous anxiety/depression is a known risk factor for poor post-cardiac event mental health outcomes (3, 18), underscoring the importance of identifying these patients early on and targeting them for mental health support. The present study also identified other known correlates of persistent anxiety and depression such as financial strain (3, 18), and social isolation (3, 18). Indeed, financial strain was a consistent predictor of prevalence ratings across all domains and sub-domains, highlighting this patient characteristic as an important and easily assessed “red flag” for poor post-event mental health recovery, as we have noted previously (3, 18). Feelings of isolation have been exacerbated by the COVID– related mandatory lockdowns and restrictions, and have brought this issue into sharp focus internationally (21). Poignantly, of those who reporting feeling unsupported by family and friends, over 60% found this severely or moderately distressing, with this distress likely heightened by the COVID-19 lockdowns (21). Social isolation as a barrier to good recovery has been identified as a contributor to persistent or worsening mental health post cardiac event (3, 22) as well as conferring a higher risk of premature death (23). Those who had experienced a recent bereavement endorsed more items related to cognitive Frontiers in Psychiatry | www.frontiersin.org DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Limitations Some study limitations should be acknowledged. First, while the present results confirm our definition of cardiac distress as multifactorial and non-transient, a stronger case for the latter point may have been made if the questionnaire had been administered further into convalescence. The 8-week assessment point was chosen to coincide with routine follow-up clinic appointments, in order to optimize participation. However, at this relatively early point of recovery it is possible that symptoms of the cardiac blues, typical during the early post- event adjustment period (25), may not yet have resolved. Future longitudinal studies to investigate the trajectories of the components of distress are needed. Second, due to the large number of distress items assessed, we identified predictors for domains only, rather than for individual items. More fine- tuned analysis to investigate item correlates would provide a more nuanced understanding of sex and age-related differences March 2022 | Volume 13 | Article 808904 9 Unraveling the Complexity of Cardiac Distress Jackson et al. routine screening for cardiac distress in primary care and cardiac rehabilitation settings. The present findings further our understanding of the relative importance of various post-event issues and concerns, thereby providing useful clinical guidance for health professionals working in cardiac rehabilitation and psychocardiology settings. in particular, but was outside the scope of the present study. Again, this could be the focus of more targeted hypothesis testing within specific distress domains and/or for specific patient groups. Third, being undertaken during the COVID- 19 pandemic, the study findings may have been impacted by transient and extraneous stressors that were not measured. Despite these limitations, the study was strengthened by the inclusion of consecutive series of cardiac patients from two large hospitals, representing both metropolitan and regional areas of Australia. FUNDING This study was partially funded by the Angela Anita Reid bequest to the Australian Centre for Heart Health. This study was partially funded by the Angela Anita Reid bequest to the Australian Centre for Heart Health. ETHICS STATEMENT The present study extends the findings of earlier qualitative studies by quantifying common issues and concerns experienced by cardiac event survivors and providing assessment of the level of distress caused by these issues and concerns. It is the first study to explore cardiac distress in this way. The findings highlight the importance of investigating both endorsement of these issues as well as ratings of distress severity in our attempts to understand cardiac distress. The fact that issues such as not being able to access suitable health care and concern about sexual functioning were highly distressing although not highly endorsed, shows the necessity for measuring both, and not simply assuming that high prevalence of an issue corelates with a high level of distress and vice versa. In terms of clinical implications, the findings underscore the importance of providing the opportunity for patients to express the specific nature of their worries and concerns, and to reveal the impacts of these on their psychological wellbeing. Patients expressing high levels of distress should be given the opportunity for psychocardiology-informed counseling to support them in their recovery. Indeed, the findings point to the need for implementing This study had ethics approval from the Monash Health Human Research Ethics Committee (approval number: RES-19- 0000631A – 55979). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. AUTHOR CONTRIBUTIONS BM, MR, ML, and AJ undertook data analysis and data interpretation. AJ, MR, and BM drafted the manuscript. JA, JS, VH, MA, RH, CS, and DT contributed to and approved the manuscript. All authors contributed to study design, questionnaire development, and data collection. All authors contributed to the article and approved the submitted version. BM, MR, ML, and AJ undertook data analysis and data interpretation. AJ, MR, and BM drafted the manuscript. JA, JS, VH, MA, RH, CS, and DT contributed to and approved the manuscript. All authors contributed to study design, questionnaire development, and data collection. 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(2001) 15:71–82. doi: 10.1097/00005082-200104000-00006 March 2022 | Volume 13 | Article 808904 Frontiers in Psychiatry | www.frontiersin.org 10 Unraveling the Complexity of Cardiac Distress Jackson et al. 13. Thompson DR, Ersser SJ, Webster RA. The experiences of patients and their partners 1 month after a heart attack. J Adv Nurs. (1995) 22:707–14. doi: 10.1046/j.1365-2648.1995.22040707.x 22. Jackson A, Murphy B. Loneliness, social isolation and cardiovascular risk. Br J Cardiac Nurs. (2019) 14:1–8. doi: 10.12968/bjca.2019.0093 22. Jackson A, Murphy B. Loneliness, social isolation and cardiovascular risk. Br J Cardiac Nurs. (2019) 14:1–8. doi: 10.12968/bjca.2019.0093 j 23. Holt-Lunstad J, Smith TB, Baker M. Loneliness and social isolation as risk factors for mortality: a meta-analytic review. Perspect Psychol Sci. (2015) 10:227–37. doi: 10.1177/1745691614568352 23. Holt-Lunstad J, Smith TB, Baker M. 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In: D Byrne and M Alvarenga, editors, Handbook of Psychocardiology. Singapore: Springer (2016). p. 57. doi: 10.1007/978-981-287-206-7_57 16. Jackson AC, Murphy BM, Thompson DR, Ski CF, Alvarenga ME, Le Grande MR, et al. What is cardiac distress and how should we measure it? Br J Cardiac Nurs. (2018) 13:286–93. doi: 10.12968/bjca.2018.13.6.286 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. 17. Jackson A, Rogerson M, Le Grande M, Thompson D, Ski C, Alvarenga M, et al. Protocol for the development and validation of a measure of persistent psychological and emotional distress in cardiac patients: the Cardiac Distress Inventory. BMJ Open. (2020) 10:e034946. doi: 10.1136/bmjopen-2019-034946 18. Murphy BM, Elliott PC, Ludeman D, Judd F, Humphreys J, Edington J, et al. ’Red flags’ for anxiety and depression after an acute cardiac event: 6-month longitudinal study in regional and rural Victoria. Eur J Prev Cardiol. (2014) 21:1079–89. doi: 10.1177/2047487313493058 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. 19. Le Grande M, Neubeck L, Murphy BM, McIvor D, Lynch D, McLean H, et al. Screening for obstructive sleep apnoea in cardiac rehabilitation: a position statement from the Australian Centre for Heart Health and the Australian Cardiovascular Rehabilitation Association. Eur J Prev Cardiol. (2017) 23:1466–75. doi: 10.1177/2047487316652975 Copyright © 2022 Jackson, Rogerson, Amerena, Smith, Hoover, Alvarenga, Higgins, Grande, Ski, Thompson and Murphy. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). Copyright © 2022 Jackson, Rogerson, Amerena, Smith, Hoover, Alvarenga, Higgins, Grande, Ski, Thompson and Murphy. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Psychiatry | www.frontiersin.org March 2022 | Volume 13 | Article 808904 REFERENCES 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. 20. O’Neil A, Russell JD, Murphy B. How does mental health impact women’s heart health? Heart, Lung and Circulation. (2020) 30:59–68. doi: 10.1016/j.hlc.2020.05.111 21. Murphy BM, Higgins RO, Jackson AC. The mental health burden of COVID- 19 on cardiac healthcare workers and their patients. Br J Cardiac Nurs. (2020) 15:1–8. doi: 10.12968/bjca.2020.0129 March 2022 | Volume 13 | Article 808904 Frontiers in Psychiatry | www.frontiersin.org 11
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The blood labyrinthine barrier in the human normal and Meniere’s disease macula utricle
Scientific reports
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Gail Ishiyama1, Ivan A. Lopez2, Paul Ishiyama2, Harry V. Vinters3 & Akira Ishiyama2 iyama1, Ivan A. Lopez2, Paul Ishiyama2, Harry V. Vinters3 & Akira Ishiyama2 The ultrastructural organization of the blood labyrinthine barrier (BLB) was investigated in the human vestibular endorgan, the utricular macula, using postmortem specimens from individuals with documented normal auditory and vestibular function and surgical specimens from patients with intractable Meniere’s disease. Transmission electron microscopic analysis of capillaries located in the normal human utricular stroma showed vascular endothelial cells with few pinocytotic vesicles, covered by a smooth and uniform basement membrane surrounded by pericyte processes. Meniere’s disease specimens revealed differential ultrastructural pathological changes in the cellular elements of the microvasculature. With moderate degeneration of the BLB, there were numerous vesicles within the vascular endothelial cells (VECs), with increased numbers at the abluminal face, pericyte process detachment and disruption of the perivascular basement membrane surrounding the VECs. With severe degeneration of the BLB, there was severe vacuolization or frank apparent necrosis of VECs and loss of subcellular organelles. A higher severity of BLB degenerative changes was associated with a higher degree of basement membrane thickening and edematous changes within the vestibular stroma. This study presents the first ultrastructural analysis of the capillaries constituting the BLB in the human vestibular macula utricle from normal and Meniere’s disease. In the inner ear, the term blood labyrinthine barrier (BLB) refers to the barrier between the vasculature and the inner ear fluids, either endolymph or perilymph1, 2. The BLB is critical for the maintenance of the inner ear fluid ionic homeostasis and for the prevention of the entry of deleterious substances into the inner ear3. By studying the penetration of dyes and drugs from the systemic circulation into the fluids of the inner ear, it can be shown that the BLB is selective and that the composition of the inner ear fluids is regulated and markedly different from that of blood or of other fluids such as cerebrospinal fluid2. l pl Understanding the dynamics of the BLB is important to develop therapeutic drug delivery systems to the inner ear to block or enhance the BLB inflammatory response. The BLB in the cochlea is well studied in animal models2, 4, 5. Initial transmission electron microscopy (TEM) studies demonstrated that the BLB in the guinea pig stria vascularis and spiral ligament is composed of vascular endothelial cells (VECs) surrounded by a basement membrane6. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 20 October 2016 Accepted: 20 February 2017 Published: xx xx xxxx The blood labyrinthine barrier in the human normal and Meniere’s disease macula utricle Received: 20 October 2016 Accepted: 20 February 2017 Published: xx xx xxxx Scientific Reports | 7: 253 | DOI:10.1038/s41598-017-00330-5 Gail Ishiyama1, Ivan A. Lopez2, Paul Ishiyama2, Harry V. Vinters3 & Akira Ishiyama2 More recent studies have noted that, in addition, the intrastrial BLB is composed of pericytes and perivascular-resident macrophage-like melanocytes7–11. The VECs line the interior surface of the blood vessels, connected by tight junctions, forming an interface between circulating blood and the rest of the vessel wall. Pericytes are the only cell type to intimately connect with VECs as they lie embedded within the endothelial basement membrane. The perivascular-resident macrophage-like melanocytes have foot processes on the outer surface of capillaries and these cells may play a similar role as astrocytes in the brain to maintain the integrity of the barrier and may play a role in inflammatory responses3, 12. The BLB within the perilymph-filled areas that are part of the blood-perilymph barrier include the cochlear spiral ligament, spiral limbus, modiolus, osseous spiral lamina, vestibular stromal vasculature and the sub-epithelial dark cell area13. The vestibular BLB unit in the rodent is similar to the rodent strial BLB unit at the light microscopic level: composed of vascular endothelial cells, pericytes and perivascular-resident macrophage-like melanocytes10. 1Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, USA. 2Department of Head and Neck Surgery, David Geffen School of Medicine, University of California, Los Angeles, USA. 3Department of Pathology & Laboratory Medicine (Neuropathology), David Geffen School of Medicine, University of California, Los Angeles, USA. Correspondence and requests for materials should be addressed to G.I. (email: gishiyama@mednet.ucla.edu) Scientific Reports | 7: 253 | DOI:10.1038/s41598-017-00330-5 1 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. The BLB in normal capillaries. Capillary located in the stroma of the macula utricle from a normal subject (62-year-old male). (a) Low magnification view. The lumen (lu) of the capillary is smooth and vascular endothelial cells (vec) exhibit normal morphology. The perivascular  basement membrane (pbm) and extracellular matrix (asterisk) are also normal. (b) high magnification view from (a), vec show normal cytoplasm. Tight junctions show a normal organization (tj). Bar (a) = 2 μm, (b) = 300 nm. Figure 1. The BLB in normal capillaries. Capillary located in the stroma of the macula utricle from a normal subject (62-year-old male). (a) Low magnification view. The lumen (lu) of the capillary is smooth and vascular endothelial cells (vec) exhibit normal morphology. The perivascular  basement membrane (pbm) and extracellular matrix (asterisk) are also normal. (b) high magnification view from (a), vec show normal cytoplasm. Tight junctions show a normal organization (tj). Gail Ishiyama1, Ivan A. Lopez2, Paul Ishiyama2, Harry V. Vinters3 & Akira Ishiyama2 Bar (a) = 2 μm, (b) = 300 nm. Recent studies have implicated loss of the integrity of the BLB in several inner ear pathologies including acous- tic trauma, autoimmune inner ear disease, and presbycusis3, 7, 8, 14–16. Meniere’s disease is a disabling syndrome of fluctuating hearing loss, episodic vertigo, and ear fullness, the etiology and pathophysiology of which remain poorly understood. Endolymphatic hydrops, a ballooning of the endolymph fluid, was demonstrated in postmor- tem human temporal bone studies on patients with Meniere’s disease17, 18. Endolymphatic hydrops is character- ized by an excess of endolymph; however, the mechanism whereby vertigo and hearing loss occur is unclear19–21. MRI studies have demonstrated that within patients with Meniere’s disease, the degree of hydrops correlates with the degree of hearing loss22. Hypothesized mechanisms for hearing loss in Meniere’s disease include a decrease in cochlear blood flow to one-third as a direct effect of hydrops23; however, cochlear blood flow measurement in subjects with Meniere’s disease did not differ from control groups24. While pathophysiology of the BLB has been proposed in Meniere’s disease, no prior studies have evaluated the BLB ultrastructure in normal humans or in Meniere’s disease. Of note, Meniere’s disease is a uniquely human disease and there are no animal models which replicate the disease.h p The normal vascular anatomy of the cochlea has been well documented using light, TEM and scanning EM of corrosion cast of the cochlear vasculature25. To our knowledge, there are no previous ultrastructural studies of the human vestibular stromal capillaries. We have previously documented pathological alterations of the vestibular crista ampullaris sub-epithelial basement membrane in patients with intractable Meniere’s disease26. The utricular stroma demonstrated thickening of the basement membrane, fibroblast vacuolization and fibrillary deposition close to the blood vessels. However, the ultrastructural morphology of the human vestibular BLB has not yet been investigated. We report the first ultrastructural study of the normal human BLB of capillaries located underneath the utricular sensory epithelia and compare the results with those from Meniere’s patients. The BLB in subepithelial capillaries of the utricular stroma in Meniere’s disease.  We exam The BLB in subepithelial capillaries of the utricular stroma in Meniere s disease. We examined the ultrastructural changes of the capillaries located within the stroma of the macula utricle from Meniere’s dis- ease patients. There were cases with mild or severe (ischemic) morphological alterations in the VECs. The VECs exhibited abundant vesicular transport and cytoplasmic vacuolization, pseudopodic protrusions into the lumen, microvilli-like projections, and swelling and degeneration of the organelles. In some cases, there were complete degenerative changes and necrosis of the VECs. The perivascular basement membrane, similarly, ranged from normal appearance to severely vacuolated and thickened. VECs with active vesicular transport exhibited hyper- trophic Golgi complexes and markedly increased vesicle formation. Figure 3a shows a relatively intact and smooth lumen of a capillary, which demonstrates mild alterations (39-year-old with delayed endolymphatic hydrops, with history of profound hearing loss as a child with intractable vertigo spells despite medications). The VECs demon- strate some vacuoles and a prominent increase in vesicular transport facing the abluminal side, with some vesicles opening onto the abluminal area (Fig. 3b). The perivascular basement membrane is relatively intact, with uniform thickness. The VEC show mild vacuolar changes. There appears to be detachment of a pericyte process (Fig. 3b), and there are mild edematous changes within the basement membrane. Figure 4 shows the cross section of a capillary located in the stroma of the macula utricle from an 82-year-old male Meniere’s patient with Tumarkin falls and progressive severe loss of caloric function. The VECs exhibit swelling and numerous cytoplasmic vesicles, with narrowed and uneven capillary lumen (Fig. 4a). At higher magnification, vesicles in the endothelial cell cytoplasm are lined up polarized to the abluminal membrane. There are chained or coalesced pinocytotic vesicles forming tubular channel-like formations. Tight junctions joining VECs appear to be intact with no ultrastructural changes apparent. The perivascular basement membrane is mildly disorganized, and the extracellular matrix demonstrates mild edema. The endothelial cell lumen demon- strates multiple areas of microvilli- like projections into the lumen, and protrusion of pseudopodic expansion of the endothelial cell. There is abundant abluminal oriented vesicular transport, facing the basement membrane with some vesicles opening onto the basement membrane. Other areas of the lumen epithelium demonstrate deep invaginations that can be seen ending in micropinocytic vesicles. At higher magnification (Fig. 4b) apparent activation of a Golgi complex within the VEC can be seen. Results Th The normal human BLB in the capillaries of the utricle stroma. The normal human BLB consists of vascular endothelial cells (VECs) surrounded by a perivascular basement membrane with pericyte processes embedded within the basement membrane that surrounds the VECs (Fig. 1a). Figure 1a shows a cross section of a blood vessel (low magnification) located in the stroma underneath the utricular macula sensory epithelia from a 62-year-old individual with normal vestibular or auditory function. The capillaries of the vestibular stroma are continuous, and the endothelial lining is without fenestrations. Higher magnification view of the normal human BLB (Fig. 1b) shows the VECs connected by tight junctions. The capillary endothelial surface facing the lumen is smooth and uniform, and without many pseudopodic extensions; the VECs contain organelles, with numerous mitochondria and a paucity of endocytic vesicles or vacuoles. There are only a few endocytic vesicles and no vacuoles within the cytoplasm of VECs (Fig. 1b). Another component of the BLB, the perivascular base- ment membrane, is a uniform structure with a homogeneous matrix (Fig. 1a and b). The perivascular basement membrane has an even thickness and intermingles uniformly within the abluminal portion of the VECs and per- icytes (Fig. 1b). It completely surrounds the VECs and pericyte dendritic processes, and there is no rarefaction or duplication of the basement membrane. Pericytes have dendritic processes that surround VECs (not seen in this micrograph). There is a paucity of vacuoles or vesicles within the VEC or the pericyte. Scientific Reports | 7: 253 | DOI:10.1038/s41598-017-00330-5 2 www.nature.com/scientificreports/ Figure 2. The BLB in the macula utricle from a normal subject, older age (86-year-old female). Note the normal appearance of the VECs (vec), the basement membrane (bm), extracellular matrix (ecm), tight junctions (tj), and capillary lumen (lu) are also normal. The pericyte (p) cytoplasm appears normal. Bar = 2.5 μm. Figure 2. The BLB in the macula utricle from a normal subject, older age (86-year-old female). Note the normal appearance of the VECs (vec), the basement membrane (bm), extracellular matrix (ecm), tight junctions (tj), and capillary lumen (lu) are also normal. The pericyte (p) cytoplasm appears normal. Bar = 2.5 μm. Figure 2. The BLB in the macula utricle from a normal subject, older age (86-year-old female). Note the normal appearance of the VECs (vec), the basement membrane (bm), extracellular matrix (ecm), tight junctions (tj), and capillary lumen (lu) are also normal. Results Th The pericyte (p) cytoplasm appears normal. Bar = 2.5 μm. The capillary ultrastructure in a normal aging utricle. Figure 2 shows a cross section of a blood vessel (low magnification) located in the stroma underneath the utricular macula sensory epithelia from an 86-year-old individual with normal vestibular function. The VEC demonstrates few vesicles, and the tight junctions remain intact. The basement membrane is smooth, but mildly thickened in comparison with the capillary basement membrane from a younger subject. A pericyte process with normal basement membrane is easily identified. The BLB in subepithelial capillaries of the utricular stroma in Meniere’s disease.  We exam Figure 5 shows the cross section of a capillary located in the stroma of the macula utricle from a 40-year-old individual with congenital deafness, intractable vertigo spells associated with unilateral aural fullness, and caloric paresis. The VEC is degenerated, with loss of organelles, thinning and extensive vacuolization. There is vacuoli- zation of the extracellular space. The basement membrane is thickened, edematous, and irregular. Figure 6 shows the cross section of a capillary located in the stroma of the macula utricle from a 56-year-old with Meniere’s disease with a history of aural fullness, tinnitus, deafness and intractable vertigo spells. The putative endothelial cell demonstrates diffuse vacuolization and loss of organelles. The perivascular basement membrane is thickened and irregular, and the vacuolizations appear to be increased in the boundary. The extracellular perivascular space exhibits vacuolizations and edema. Figure 7 shows the cross section of a capillary located in the stroma of the macula utricle from a 67-year-old individual with Meniere’s disease characterized by intractable vertigo spells, Scientific Reports | 7: 253 | DOI:10.1038/s41598-017-00330-5 3 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 3. The BLB in Meniere’s disease (39-year-old). Capillary located in the utricle stroma underneath the vestibular sensory epithelia. (a) Low magnification view. VECs (vec) show an almost normal appearance, lumen is normal (lu), the extracellular matrix shows edema. (b) High magnification view -insert from (a). Abundant open caveolar flask-like structures that almost touch the abluminal membrane of the vascular  endothelial cell (vec) were observed. The border of the basal membrane (pbm), is almost normal, a pericyte process (pp) is detached. Thick arrowhead points to a large vacuole in the vascular endothelial cell cytoplasm. Bar in (a) = 5 μm, (b) = 2 μm. Figure 3. The BLB in Meniere’s disease (39-year-old). Capillary located in the utricle stroma underneath the vestibular sensory epithelia. (a) Low magnification view. VECs (vec) show an almost normal appearance, lumen is normal (lu), the extracellular matrix shows edema. (b) High magnification view -insert from (a). Abundant open caveolar flask-like structures that almost touch the abluminal membrane of the vascular  endothelial cell (vec) were observed. The border of the basal membrane (pbm), is almost normal, a pericyte process (pp) is detached. Thick arrowhead points to a large vacuole in the vascular endothelial cell cytoplasm. Bar in (a) = 5 μm, (b) = 2 μm. Figure 4. A BLB capillary in Meniere’s disease (82-year-old). The BLB in subepithelial capillaries of the utricular stroma in Meniere’s disease.  We exam Capillary located in the utricle stroma underneath the vestibular sensory epithelia. (a) Low magnification view. The vascular endothelial cell (vec) is swollen with degeneration of organelles and debris within the cytoplasm, the lumen (lu) is uneven and narrowed with pseudopodic and microvilli-like projections, the perivascular basement membrane (bm) exhibits mild thickening. (b) High magnification view (from fig a) of the vascular endothelial cell (vec). The tight junction (tj) appears normal, bm: basement membrane. Bar (a) = 1 μm, (b) = 0.5 μm. Figure 4. A BLB capillary in Meniere’s disease (82-year-old). Capillary located in the utricle stroma underneath the vestibular sensory epithelia. (a) Low magnification view. The vascular endothelial cell (vec) is swollen with degeneration of organelles and debris within the cytoplasm, the lumen (lu) is uneven and narrowed with pseudopodic and microvilli-like projections, the perivascular basement membrane (bm) exhibits mild thickening. (b) High magnification view (from fig a) of the vascular endothelial cell (vec). The tight junction (tj) appears normal, bm: basement membrane. Bar (a) = 1 μm, (b) = 0.5 μm. Tumarkin falls, and unilateral deafness. There is severe perivascular basement membrane disorganization, and diffuse edema in the extracellular matrix. The VECs are severely swollen, with apparent necrosis and no identifi- able subcellular organelles. Tumarkin falls, and unilateral deafness. There is severe perivascular basement membrane disorganization, and diffuse edema in the extracellular matrix. The VECs are severely swollen, with apparent necrosis and no identifi- able subcellular organelles. Discussionh The stroma in the normal utricle is rich in capillaries, with less abundant capillaries in the deeper portion of the sensory endorgan areas, and an abundance of capillaries in the subepithelial region. There is a particularly rich network of fine capillaries beneath the basement membrane of the sensory areas. In the capillaries of the human vestibular stroma, the VECs are thin walled (2–4 μm) with a paucity of pinocytotic vesicles, and pericytes engulf the VECs via dendritic processes. In Iurato et al.27 study of the capillaries of the mammalian crista ampullaris, the flat endothelial cells are noted to be about 0.1 to 1.4 μm, with the outer surface of the VEC in close contact with the basement membrane, and the inner surface facing the capillary lumen. There are no fenestrations in the VECs Scientific Reports | 7: 253 | DOI:10.1038/s41598-017-00330-5 4 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 5. A BLB capillary with moderate degenerative changes in Meniere’s disease (40-year-old). Vacuolization is seen in vascular endothelial cells (vec), and there is apparent degeneration with loss of organelles and thinning. The perivascular basement membrane (bm) shows thickening. Lumen (lu). The stroma shows signs of edema (asterisk). Bar = 2 μm. Figure 5. A BLB capillary with moderate degenerative changes in Meniere’s disease (40-year-old). Vacuolization is seen in vascular endothelial cells (vec), and there is apparent degeneration with loss of organelles and thinning. The perivascular basement membrane (bm) shows thickening. Lumen (lu). The stroma shows signs of edema (asterisk). Bar = 2 μm. Figure 5. A BLB capillary with moderate degenerative changes in Meniere’s disease (40-year-old). Vacuolization is seen in vascular endothelial cells (vec), and there is apparent degeneration with loss of organelles and thinning. The perivascular basement membrane (bm) shows thickening. Lumen (lu). The stroma shows signs of edema (asterisk). Bar = 2 μm. Figure 6. A BLB capillary with moderate degenerative changes in Meniere’s disease (56-year-old). A capillary shows moderate vacuolization of the vascular endothelial cell (vec) and pericytes (pc) and mild edema in the stroma (arrows). The perivascular basement membrane (pbm) is thickened. The stroma shows signs of edema (asterisk). Bar = 2 μm. Figure 6. A BLB capillary with moderate degenerative changes in Meniere’s disease (56-year-old). A capillary shows moderate vacuolization of the vascular endothelial cell (vec) and pericytes (pc) and mild edema in the stroma (arrows). The perivascular basement membrane (pbm) is thickened. The stroma shows signs of edema (asterisk). Discussionh Bar = 2 μm. in the normal rodent cristae ampullaris with cell junctions forming zonulae adherents. Similarly, in this study we found that the capillaries of the human vestibular utricular maculae are of continuous type, and the intercel- lular junctions between VECs are separated by tight junction-like structures. The morphological organization resembles that found in the inner ear of several animal models28. The basal lamina of the capillaries in the human utricular stroma is composed of type IV collagen, laminin and other proteins29, interposed between VECs and pericytes processes.hi in the normal rodent cristae ampullaris with cell junctions forming zonulae adherents. Similarly, in this study we found that the capillaries of the human vestibular utricular maculae are of continuous type, and the intercel- lular junctions between VECs are separated by tight junction-like structures. The morphological organization resembles that found in the inner ear of several animal models28. The basal lamina of the capillaries in the human utricular stroma is composed of type IV collagen, laminin and other proteins29, interposed between VECs and pericytes processes.hi y The site of perilymph production remains controversial, in general it is believed that it is formed as an ultrafil- trate of blood1, 2 with some contribution from filtration from cerebrospinal fluid (CSF). The CSF may also filtrate via the vestibular aqueduct or perivascular channels into the perilymph. The differing protein and amino acid levels of the perilymph, blood serum, and CSF are indicative of the maintenance of ionic and protein composition as part of the function of the BLB. Blood vessels, which exhibit cationic polyethyleneimine distribution, which Scientific Reports | 7: 253 | DOI:10.1038/s41598-017-00330-5 5 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 7. Ultrastructure of a BLB capillary with severe degenerative changes in Meniere’s disease (67-year- old). A capillary located in the macula utricle stroma. The vascular endothelial cell (vec) exhibits degenerative changes with no subcellular organelles and edematous change. The perivascular basement membrane (bm) is disorganized and swollen. There is constriction of the lumen (lu) and edema (asterisk). Bar = 2 μm. Figure 7. Ultrastructure of a BLB capillary with severe degenerative changes in Meniere’s disease (67-year- old). A capillary located in the macula utricle stroma. The vascular endothelial cell (vec) exhibits degenerative changes with no subcellular organelles and edematous change. The perivascular basement membrane (bm) is disorganized and swollen. There is constriction of the lumen (lu) and edema (asterisk). Bar = 2 μm. Discussionh identifies anionic sites, following intravenous administration, are indicative of macromolecular transport systems of the BLB. In the cochlea, anionic sites are present in the basement membrane of Reissner's membrane. In the vestibule, they are localized in the basement membrane of the capillary wall, and the sensory epithelium30. The premature rat does not yet have fully developed anionic sites within the crista ampullaris13. Like the blood brain barrier (BBB), the BLB develops in the early postnatal period of the immature rat. The timing of the development of the BLB has important implications for ototoxicity of agents delivered during prenatal or postnatal periods and to premature infants.i p Intracerebral cortical vessels contain a mean of five caveolae per μm2 in arteriolar endothelium31, much less than that in non-neural vessels, which likely corresponds with limited transcellular traffic of solutes in BBB cap- illaries. Caveolae can undergo budding and fission from the plasma membrane, and translocate to the other side. In the case of Meniere’s disease, there appeared to be increased vesicular formations, with localization of vesicles to the transluminal side. The increased vesicle formation and caveolae-like formations in the VEC, with polariza- tion to the abluminal side, which would lead one to conclude that there is increased transcytosis, possibly vesicles being used to traffic cargo across the VECs. During BBB breakdown, there is also an increase in trans-endothelial channels in VECs32. In the specimens from Meniere’s disease in the present study, the tight junctions appeared to be normal, even at high magnification microscopy and also even in severely edematous BLB cytoarchitecture. In contrast, BBB in vasogenic edema appears to be associated with altered tight junction proteins, and separation noted at tight junctions; however, it is noted that these changes occur late in the course of brain injury32. It is possible that there is altered expression of tight junction proteins in the VEC of Meniere’s disease, without structural changes that can be visualized. Tight junctions are not only important for the regulation of paracellular permeability, but also may be involved in cytoskeletal dynamics and cellular signaling33.h y y y g g The tight junctions in normal human BLB are continuous. No apparent abnormalities of the tight junction were found in the BLB of Meniere’s disease specimens. Discussionh In some cases, the basement membrane appears to be swollen, and in more severe cases, exhibits rarefaction, duplication, and extreme thickening. The subepithelial basement membrane, which lies interposed between the epithelium facing the endolymph and the connective stromal tissue within the peri- lymph, is markedly thickened and under TEM appears to be disorganized fibrils26. Additionally, the composition of the subepithelial basement membrane is altered: collagen IV expression is diminished46, and the sub-epithelial basement membrane thickening correlated with neuroepithelial damage and vestibular hair cell loss26. In Meniere’s disease, both the sub-epithelial basement membrane and the capillary perivascular basement mem- brane forming the blood-perilymph barrier, exhibit thickening, edema, and disorganization in comparison with the normal basement membrane which is homogeneous, thin, with a continuous appearance due to a condensed filamentous pattern and homogeneous matrix.l i p g It has been suggested that Meniere’s disease may be caused by dysfunctional inner ear blood flow47, 48 exacer- bated by the pathological increase in vascular permeability of the blood-labyrinthine barrier3, 49. In severe cases, the capillary lumen is severely compromised due to edematous changes in the endothelial cells, and in some cases, due to apparent debris from necrotic endothelial and pericyte cells. These capillaries are likely to be dysfunctional to deliver nutrients to the neuroepithelium. The endothelial cell appears to be the earliest cell to exhibit damage. ph pp g The finding of increased vesicular transport in the endothelial cell of the capillaries, VEC degenerative changes, and thickening of the basement membrane of capillaries in Meniere’s disease raises the question of a possible inflammatory pathology similar to that proposed as causative in autoimmune inner ear disease. Trune and Nguyen-Huynh50 reviewed the cascade of events that can lead to increased permeability of the BLB mediated by inflammatory cytokines and chemokines that strip off the protective glycocalyx, exposing the endothelial cell, causing a cascade of events leading to loss of BLB integrity and breakdown of stria vascularis blood vessels. While there are no ultrastructural studies of the BLB in human autoimmune inner ear disease, a human temporal bone study of Sjogren's disease, an autoimmune disease associated with hearing loss in 25% of patients, demonstrated thickening of the stria vascularis basement membrane and an associated immunoglobulin deposition in Sjogren's patients with hearing loss51. Discussionh Using in vivo perilymph sampling of extravasated fluorescein from the vascular com- ponent, Hirose et al.43 demonstrated higher fluorescein in the perilymph in LPS treated mice. The region of BLB increased permeability would correspond to capillary beds, which travel through perilymph, filled spaces: spiral ligament, spiral limbus, modiolus, and osseous spiral lamina, and the subepithelial space within the vestibular stroma. In both, the uptake of gadolinium or fluorescein is not significant or nonexistent within the endolymph, indicative that the BLB lining the endolymphatic space is more restrictive than that lining the perilymphatic space. The studies together provide strong animal model evidence that increased gadolinium uptake within the perilymph is indicative of BLB permeability increase.hf p y p p y The role of cochlear macrophages is controversial, as elimination of macrophages did not alter the effect of LPS to induce vascular permeability44. In contrast to the cochlea, the vestibular periphery of the C57BL/6J mouse, exhibited LPS induced activation of perivascular macrophage-like melanocytes, identified using F4/80 stain- ing, and increased vascular permeability as reflected by fluorescein isothiocyanate infiltration into the vestibular periphery11. The role of macrophages or perivascular macrophage-like melanocytes in the vascular permeability seen in the vestibular stromal capillaries from Meniere’s patients is unknown. However, of note, on light micros- copy, there was no evidence for an infiltrate of inflammatory cells. yil y In nearly all Meniere’s cases evaluated, intact VECs of the vestibular BLB exhibited increased vesicular forma- tion, polarized to the abluminal surface, with edema and vacuolization of the perivascular basement membrane and of the extracellular matrix. Similar patterns of endothelial cell activation are noted in cerebral edema, which is also associated with increased permeability of the BBB45. There are two main permeability barriers: the base- ment membrane and the endothelial cell. Increased endothelial cell vesiculation is reported in hypoxic-ischemic stroke, hypertensive encephalopathy, and brain trauma. It is important to note that our histopathological studies are limited, by necessity, to severe intractable stage IV Meniere’s disease associated with profound non-serviceable hearing. Within this group, there were varying degrees of severity of BLB damage. In some cases, the endothelial cell had undergone complete necrosis, and in other cases, active vesicular formation is noted within an endothe- lial cell with relative preservation of subcellular organelles such as mitochondria and endoplasmic reticulum.h p g p The vestibular stromal BLB perivascular basement membrane exhibits thickening, edema, and duplication in Meniere’s disease inner ear. Discussionh All of the capillaries constituting the BLB of the normal human vestibular stroma lacked fenestrations and tight junctions were noted in the intercellular space joining two-endothelial cells, forming a tight barrier which is in alignment with freeze-fracture studies of the guinea pig blood-perilymphatic barrier34. Of note, the stria vascularis exhibits vastly differing permeability as well as degree of pinocytosis. Therefore, the vestibular stromal BLB is similar to that of the cochlear modiolus or spiral limbus, exhibiting a similar morphology to that of the BBB.h Through imaging studies using intravenous gadolinium, there is gathering evidence for blood labyrinthine barrier compromise in Meniere’s disease. Intravenous gadolinium enhanced magnetic resonance imaging (MRI) can be used to visualize and quantify endolymphatic hydrops22, 35. Gadolinium, an agent used in magnetic res- onance imaging (MRI), is taken up into the perilymph, presumably via perfusion through the BLB, specifically through the blood-perilymphatic barrier. Increased gadolinium enhancement in the perilymph on the ipsilater- ally inner ear affected with Meniere’s disease has been noted in multiple studies, indicative of breakdown of the BLB36–39. Of note, in the study of Tagaya et al.37, a high signal intensity ratio correlated with a higher grade (none, mild, or significant) of hydrops in both the cochlea and the vestibule37. Most recently, Pakdaman et al.40 demon- strated increased gadolinium uptake in the perilymph in Meniere’s disease ipsilateral and in the contralateral unaffected ear compared with sudden hearing loss; however, the ratio of affected to unaffected ear in Meniere’s Scientific Reports | 7: 253 | DOI:10.1038/s41598-017-00330-5 6 www.nature.com/scientificreports/ disease was significantly greater than that of sudden hearing loss. This may be indicative that permeability altera- tions of the BLB may be one of the primary causes of endolymphatic hydrops or Meniere’s disease. y p y y p y p Intratympanic injection of lipopolysaccharide (LPS), a substance that breaks down the BLB and also the BBB, is associated with increased gadolinium perilymphatic enhancement41. A similar increase in gadolinium uptake in the perilymph has been noted following high intensity impulse noise acoustic trauma42. The role of infiltration of inflammatory cells is controversial. LeFloc’h et al.41 showed that the reduction of ultrasmall superparamag- netic iron oxide particles, corresponded with cochlear inflammation, which was hypothesized to be secondary to macrophage infiltration. However, conclusive identification of a macrophage infiltrate using CD68 immunohis- tochemistry was negative. Discussionh Specimen Type of Tissue Age (years) Gender Diagnosis Figure 1 Autopsy 62 Male Normal 1a,b 2 Autopsy 86 Female Normal 2 3 Surgical 39 Male Meniere’s 3a,b 4 Surgical 82 Male Meniere’s 4a,b 5 Surgical 40 Female Meniere’s 5 6 Surgical 56 Male Meniere’s 6 7 Surgical 67 Female Meniere’s 7 Table 1. Specimens (utricle) used in the present study. Autopsy: Temporal bones collected between 6 to 8 hrs. postmortem, Normal: indicates normal vestibular and auditory function. Table 1. Specimens (utricle) used in the present study. Autopsy: Temporal bones collected between 6 to 8 hr postmortem, Normal: indicates normal vestibular and auditory function. loss, indicative that this increased permeability likely plays a role in the pathophysiology of Meniere’s disease55. The relation between inner ear autoimmune disease and Meniere’s disease remains to be identified.hh loss, indicative that this increased permeability likely plays a role in the pathophysiology of Meniere’s disease55. The relation between inner ear autoimmune disease and Meniere’s disease remains to be identified.hh hi The diagram in Fig. 8a illustrates schematically the normal BLB. The BLB is maintained by tight junctions between vascular endothelial cells which form a continuous barrier without fenestrations, and are covered by a basement membrane with pericytes intermingled within the basement membrane. Diagram in Fig. 8b, shows the pathological changes noted in Meniere’s disease inner ear BLB: vascular endothelial cells exhibit edema, with an increase in vesicles, with apparent transcytosis of macromolecules. There is vacuolization of endothelial cells and pericytes, and the perivascular basement membrane exhibits thickening and duplication. In some cases, the pericyte processes are detached, and the capillary lumen is compromised by edematous changes of the endothelial cells. In conclusion, the BLB dysfunction in the microvasculature of vestibular endorgans obtained from Meniere’s disease patients likely contributes to edematous changes in the underlying stroma and vacuolization. We hypoth- esize that alterations in BLB could lead to secondary changes in the vestibular sensory epithelia. Discussionh There is only one report on the ultrastructure of the strial cells in the MRL-Fas/lpr mouse model of autoimmune disease52, capillary ultrastructure was found to be normal i.e. revealed no alterations in tight junctions of VECs or alterations in the basement membrane, however, there were signs of hydropic gener- ation in cells surrounding the strial capillaries. In contrast, in the C3H/lpr autoimmune mouse, extensive leakage of ferritin into the perivascular tissues was noted, and a corresponding thickening of the basement membrane of the stria capillaries53. Other studies, however, are indicative that thickening of the basement membrane of the cap- illaries is non-specific, and has been noted in the aging mouse stria vascularis capillary basement membrane as well54. In no previous study in human or mouse, however, there are adequate evaluation of the VECs in the BLB of the peripheral vestibular system to clearly document whether or not there is increase in vesicles or breakdown of tight junctions or other changes. In a recent MRI study, it was noted that the permeability of the capillaries in the inner ear of Meniere’s disease patients was much significantly greater than that in sudden sensorineural hearing Scientific Reports | 7: 253 | DOI:10.1038/s41598-017-00330-5 7 www.nature.com/scientificreports/ Figure 8. Diagram to represent the BLB in (a) normal capillary and (b) Meniere’s disease capillary. In Meniere’s disease, there are excessive vesicular formation (ves), abluminally concentrated, with degenerative changes noted early on in the endothelial cell (vec). The perivascular basement membrane (pbm) is thickened and edematous. Tight junctions (tj) are relatively preserved. Pericytes (p) exhibit vacuolization, and pericyte process detachment. Figure 8. Diagram to represent the BLB in (a) normal capillary and (b) Meniere’s disease capillary. In Meniere’s disease, there are excessive vesicular formation (ves), abluminally concentrated, with degenerative changes noted early on in the endothelial cell (vec). The perivascular basement membrane (pbm) is thickened and edematous. Tight junctions (tj) are relatively preserved. Pericytes (p) exhibit vacuolization, and pericyte process detachment. Specimen Type of Tissue Age (years) Gender Diagnosis Figure 1 Autopsy 62 Male Normal 1a,b 2 Autopsy 86 Female Normal 2 3 Surgical 39 Male Meniere’s 3a,b 4 Surgical 82 Male Meniere’s 4a,b 5 Surgical 40 Female Meniere’s 5 6 Surgical 56 Male Meniere’s 6 7 Surgical 67 Female Meniere’s 7 Table 1. Specimens (utricle) used in the present study. Autopsy: Temporal bones collected between 6 to 8 hrs. postmortem, Normal: indicates normal vestibular and auditory function. Materials and Methodsh Specimens. The Institutional Review Board (IRB) of UCLA approved this study (IRB protocol # 10-001449). All methods used in this study were in accordance with NIH and IRB guidelines and regulations. Appropriate informed consent was obtained from each patient before inclusion in the study. Archival temporal bones were used in the present study. The temporal bone donors were part of a National Institute of Health (NIH) funded Human Temporal Bone Consortium for Research Resource Enhancement through the National Institute on Deafness and Other Communication Disorders (NIDCD). The medical history for each of the patients who had donated their temporal bones was maintained and preserved in a secured electronic database. Two maculae utricle from 2 individuals with no vestibular or auditory disease were used (62-year-old male, and 82-year-old female). Five maculae utricle from 5 patients with intractable vertigo spells or Tumarkin falls (vestibular induced involuntary drop attacks) in Meniere’s disease were studied (Table 1). References Resident macrophages in the cochlear blood-labyrinth barrier and their renewal via migration of bone-marrow-derived cells. Cell Tissue Res 342, 21–30 (2010). 3. Suzuki, M. & Kaga, K. Development of blood-labyrinth barrier in the semicircular canal ampulla of the rat. Hear Res. 129, 27–34 (1999).hl ( ) 4. Dengerink, H. A., Wright, J. W. Circulation of the inner ear: III. The physiology of cochlear blood flow: implications for treatment In Physiology of the Ear. Jahn AF, and Santos-Sacchi J, editors, Raven Press, New York, pp 327–340 (1988). 15. Fetoni, A. R., Picciotti, P. M., Paludetti, G. & Troiani, D. Pathogenesis of presbycusis in animal models: a review. Exp Gerontol. 46, 413–425 (2011).i 16. Yang, Y. et al. Na+/K+-ATPase a1 identified as an abundant protein in the blood-labyrinth barrier that plays an essential role barrier integrity. PloS One. 6, e16547 (2011). g y ( ) 17. Yamakawa, K. Hearing organ of a patient who showed Meniere’s symptoms. J Otolaryngol Soc Jpn. 44, 2310–2312 (1938). ll k b h h l f ’ d ( f l ) d 18. Hallpike, C. S. & Cairns, H. Observations on the pathology of Meniere’s disease: (section of otology). Proc R Soc Med 31, 131 (1938). 19. Merchant, S. N., Adams, J. C. & Nadol, J. B. Jr. Pathophysiology of Meniere’s syndrome: are symptoms caused by endolymphatic hydrops? Otol Neurotol. 26, 74–81 (2005).h y p 0. Semaan, M. T., Alagramam, K. N. & Megerian, C. A. The basic science of Meniere’s disease and endolymphatic hydrops. Curr Opin Otolaryngol Head and Neck Surgery. 13, 301–7 (2005). y g g y 1. Semaan, M. T. & Megerian, C. A. Contemporary perspectives on the pathophysiology of Meniere’s disease: implications fo treatment. 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All subjects with Meniere’s disease had stage IV definite Meniere’s disease as defined by the American Academy of Otolaryngology- Head and Neck Surgery criteria56 with profound hearing loss and intractable recurrent vertigo spells despite maximum medical treatment. Patients who had previously undergone intratympanic gentamicin or endolymphatic shunt surgery were excluded. 8 Scientific Reports | 7: 253 | DOI:10.1038/s41598-017-00330-5 www.nature.com/scientificreports/ Transmission electron Microscopy. For ultrastructural studies, the specimens are immersed in the fol- lowing solutions: 2% OsO4 and 2% potassium ferricyanide (EMS, Fort Washington, PA), 0.1% thiocarbohy- drazide for 1 hr, 2% OsO4 for 30 minutes, uranyl acetate 1% overnight, and 0.1% lead aspartate for 30 minutes (5 × 3 min washes with double distilled water are made between steps). Tissue was dehydrated in ascending ethyl alcohols and embedded in resin (Epon®, EMS). Half-micrometer-thin serial sections are made with a dia- mond knife (Diatome) on an AO/Reichter Ultracut-E ultramicrotome. When the area of interest was visible, ultrathin 100 nm serial sections were made, and mounted on 200 mesh formvar and carbon coated copper grids (Polysciences). TEM image acquisition. TEM observations and digital image capture were made using a FEI Tecnai trans- mission electron microscope T20 TEM −200 KV (Hillsboro, Oregon USA). Morphological analysis was made in ultrathin sections containing blood vessels through the stroma of the maculae utricle. All sections are systemati- cally analyzed at low (x 3,500-5000) and higher magnification (x 19,000-25,000). All sections were studied for the presence of vesicles and tight junctions in the endothelial cells, pericyte cytoplasmic organization, and perivascu- lar basement membrane alterations (thickening and disruption). References 1. Juhn, S. K., Ryback, L. P. & Prado, S. Nature of blood-labyrinth barrier in experimental conditions. Ann Otol Rhinol Laryngo 135–52 (1981). 2. Juhn, S. K., Rybak, L. P. & Fowlks, W. L. Transport characteristics of blood – perilymph barrier. Am J Otolaryn 3 Shi X Ph i h l f h hl i i l i H i R 282 10 24 (2011) 2. Juhn, S. K., Rybak, L. P. & Fowlks, W. L. Transport characteristics of blood – perilymph barrier. Am J Otolaryngol. 3, 392–339 (1 3. Shi, X. Physiopathology of the cochlear microcirculation. Hearing Res. 282, 10–24 (2011).l 2. Juhn, S. K., Rybak, L. P. & Fowlks, W. L. Transport characteristics of blood perilymph barrier. Am J Otolaryngol. 3, 392 339 (1982). 3. Shi, X. Physiopathology of the cochlear microcirculation. Hearing Res. 282, 10–24 (2011).l 3. Shi, X. Physiopathology of the cochlear microcirculation. Hearing Res. 282, 10–24 (2011).l y p gy g 4. Juhn, S. K., Hunter, B. A. & Odland, R. M. Blood-labyrinth barrier and fluid dynamics of the inner ear. Int Tinnitus Journal. 7, 72–83 (2001).h 5. Salt, A. N., Melichar, I. & Thalmann, R. Mechanisms of endocochlear potential generation by stria vascularis. Laryngoscope. 97, 984–991 (1987). 6. Sakagami, M., Matunaga, T. & Hashimoto, P. H. Fine structure and permeability of capillaries in the stria vascularis and spiral ligament of the inner ear of the guinea pig. Cell Tissue Research. 226, 511–522 (1982). g g p g ( ) 7. Neng, L., Zhang, F., Kachelmeier, A. & Shi, X. Endothelial cell, pericytes, and perivascular macrophage-type melanocyte interactions regulate cochlear instrastrial fluid-blood barrier permeability. JARO. 14, 175–85 (2013). gl y 8. Neng, L. et al. Isolation and culture of endothelial cells, pericytes and perivascular resident macrophage-like melanocytes from the young mouse ear. Nat Protocols. 8, 709–20 (2013). 9. Shi, X. Cochlear pericytes responses to acoustic trauma and the involvement of hypoxia-inducible factor-1alpha and vascular endothelial growth factor. Am J Pathol. 174, 1692–1704 (2009). g 0. Zhang, W. et al. Perivascular-resident macrophage-like melanocytes in the inner ear are essential for the integrity of the instrastria fluid-blood barrier. PNAS USA 109, 10388–93 (2012).l l 11. Zhang, F., Zhang, J., Neng, L. & Shi, X. Characterization and inflammatory response of perivascular resident macrophage-like melanocytes in the vestibular system. JARO. 14, 635–43 (2013). y y 12. Shi, X. Scientific Reports | 7: 253 | DOI:10.1038/s41598-017-00330-5 www.nature.com/scientificreports/ www.nature.com/scientificreports/ 33. Tsukita, S., Furuse, M. & Itoh, M. Molecular dissection of tight junctions. Cell Struct Function 21, 381–5 (1996).h g j 34. Jahnke, K. The blood-perilymph barrier. Arch Otorhinolaryngol. 228, 29–34 (1980). h p y p y g 5. Sepahdari, A. R., Vorasubin, N., Ishiyama, G. & Ishiyama, A. Endolymphatic hydrops reversal following acetazolamide therapy demonstrated with delayed intravenous contrast-enhanced 3D-FLAIR MRI. AJNR Am J Neuroradiol. 37, 151–154 (2016). y 36. Yamazaki, M., Naganawa, M., Tagaya, M., Kawai, H., Ikeda, M., Sone, M., Teranishi, M., Suzuki, H. & Nakashima, T. Comp contrast effect on the cochlear perilymph after intratympanic and intravenous gadolinium injection. AJNR 33, 773–778 (2 y 6. Yamazaki, M., Naganawa, M., Tagaya, M., Kawai, H., Ikeda, M., Sone, M., Teranishi, M., Suzuki, H. & Nakashima, T. Comparison o 36. Yamazaki, M., Naganawa, M., Tagaya, M., Kawai, H., Ikeda, M., Sone, M., Teranishi, M., Suzuki, H. & Nakashima, T. Comparison of contrast effect on the cochlear perilymph after intratympanic and intravenous gadolinium injection. AJNR 33, 773–778 (2012). T k T h d l h h d d bl d l b h b ’ d ft 7. Tagaya, M., Yamazaki, M. & Teranishi, M. Endolymphatic hydrops and blood-labyrinthine barrier in Meniere’s disease. Acta Otolaryngol. 131, 474–479 (2011). y g 38. Barath, K., Schuknecht, B., Naldi, A. M., Schrepter, T., Bockish, C. J. & Hegemann, S. C. Detection and grading of endolymphatic hydrops in Meniere’s disease using MR imaging. AJNR Am J Neuroradiol. 35, 1387–92 (2014). 9. Ishiyama, G., Lopez, I. A., Sepahdari, A. R. & Ishiyama, A. Meniere’s disease: histopathology, cytochemistry, and imaging. Ann NY Acad Sci. 1343, 49–57 (2015). 0. Pakdaman, M. N. et al. Blood-labyrinth barrier permeability in Meniere’s disease and idiopathic sudden sensorineural hearing loss findings on delayed post-contrast 3D-FLAIR MRI. AJNR AM J Neuroradiol. doi:10.3174/ajnr.A4822 (2016).l i g y j 41. LeFloch’h, J. et al. Markers of cochlear inflammation using MRI. J Mag Reson Imaging. 39, 150–161 (2014). l 2. Counter, S. A. et al. Magnetic resonance imaging of membranous labyrinth during in vivo gadolinium uptake in the normal and lesioned cochlea. Neuroreport. 11, 3979–3983 (2000). p 3. Hirose, K., Hartsock, J. J., Johnson, S., Santi, P. & Salt, A. N. Systemic lipopolysaccharide compromises the blood-labyrinth barrier and increases entry of serum fluorescein into the perilymph. JARO. 15, 707–719 (2014).hl l 4. Hirose, K., Li, S. Z., Harsock, J., Salt, A. www.nature.com/scientificreports/ The blood perilymph barrier is vulnerable to systemic inflammation but is minimally affected by cochlear macrophages. 7th International Symposium on Meniere’s disease and Inner Ear Disorders. Abst p13. (2015).f f y p g y p p 45. Nag, S., Manias, J. L., Kapadia, A., Stewart, D. J. Molecular changes associated with the protective effects of angiopoietin-1 du blood-brain barrier breakdown post-injury. Mol Neurobiol., doi:10.1007/s12035-016-9973-4 (2016). j y 46. Calzada, A. P., Lopez, I. A., Beltran Parrazal, L., Ishiyama, A. & Ishiyama, G. Cochlin expression in vestibular endorgans obtained from patients with Meniere’s disease. Cell Tissue Res. 350, 373–84 (2012).h 47. Foster, C. A. & Breeze, R. E. The Meniere’s attack: an ischemia/reperfusion disorder of inner ear sensory tissues. Med Hypotheses. 81 1108–1115 (2013). ( ) 48. Nuttall, A. L. Sound-induced cochlear ischemia/hypoxia as a mechanism of hearing loss. Noise Health. 2, 17–32 (1999). yp g 49. Calabrese, V. et al. Oxidative stress, redox homeostasis and cellular stress response in Meniere’s disease: role of vitagenes. Neurochemical Res. 35, 2208–2217 (2010). 50. Trune, D. R. & Nguyen-Huynh, A. Vascular pathophysiology in hearing disorders. Seminars in Hearing 33, 242–250 (2012). 51. Calzada, A. P., Balaker, A. E., Ishiyama, G., Lopez, I. A. & Ishiyama, A. Temporal bone histopathology and immunoglo 50. Trune, D. R. & Nguyen-Huynh, A. Vascular pathophysiology in hearing disorders. Seminars in Hearing 33, 242–250 (2012). 51. Calzada, A. P., Balaker, A. E., Ishiyama, G., Lopez, I. A. & Ishiyama, A. Temporal bone histopathology and immunoglobulin deposition in Sjogren’s syndrome. Otol Neurotol. 33, 258–266 (2012). p j g y 52. Ruckenstein, M. J., Keithley, E. M., Bennett, T., Powell, H. C., Baird, S. & Harris, J. P. Ultrastructural pathology in the stria vascu of the MRL-Faslpr mouse. Hearing Res. 131, 22–28 (1999). g 3. Lin, D. W. & Trune, D. R. Breakdown of stria vascularis blood – labyrinthine barrier in C3H/lpr autoimmune disease mice Otolaryngol Head Neck Surg. 117, 530–534 (1997). y g g 4. Neng, L., Zhang, L., Yang, F., Lopez, I. A., Dong, M. & Shi, X. Structural changes in the strial blood-labyrinth barrier of aged C57BL/6 mice. Cell Tissue Res. 361, 685–96 (2015). 5. Pakdaman, M. N., Ishiyama, G., Ishiyama, A., Peng, K. A., Kim, H. J., Pope, W. B., Sepahadari, A. R. Blood-Labyrinth barrie permeability in Meniere’s disease and idiopathic sudden sensorineural hearing loss: findings on delayed postcontrast 2DFLAIR MRI., doi:3174/ajnr.A48822.i j 56. Goebel, J. A. Author Contributions G.I., I.A.L., and A.I. conceived the project. G.I., I.A.L., and A.I. wrote the manuscript. I.A.L. and P.I. performed transmission electron microscopy tissue processing and image collection. I.A.L., G.I., H.V.V. performed image interpretation and analysis. www.nature.com/scientificreports/ 2015 Equilibrium committee amendment to the 1995 AAO-HNS guidelines for the definition of Meniere’s dis Otolaryngol H&N Surg. 154, 403–404 (2016). Acknowledgements g Hearing Health Foundation, grant # 20164266 to Gail Ishiyama. g Hearing Health Foundation, grant # 20164266 to Gail Ishiyama. References Tange, R. & Hodde, K. C. The microvasculature of the cochlea and the vestibular system as seen in scanning electron microscopy Clin Otolaryngol. 9, 306 (1984). y g , ( ) 6. McCall, A., Ishiyama, G., Lopez, I. A., Sunita, B. & Ishiyama, A. Histopathological and ultrastructural analysis of vestibula endorgans obtained from patients with Meniere’s disease. BMC Ear Nose Throat Disorders. 9, 4 (2009). h 27. Iurato, S. Blood supply, light microscopic feature. CH III, In Iurato, S. Ed. Submicroscopic structure of the inner ear. Pergamon Press, London, First Ed, pp 18–28 (1976).i pp 8. Wersall, J., Densert, O., Per-Gotthard, L. Studies on fine structure of inner ear vessels. In Darin de Lorenzo, A. J., Editor. University Park Press, Baltimore, London, Tokyo. pp 43–73 (1973). y pp 9. Ishiyama, A., Mowry, S. E., Lopez, I. A. & Ishiyama, G. Immunohistochemical distribution of basement membrane proteins in the human inner ear from older subjects. Hearing Res. 254, 1–14 (2009). j g 30. Suzuki, M., Kitamura, K. & Nomura, Y. Anionic sites of the basement membrane of the labyrinth. Acta Otolaryngol Suppl. 481, 112–5 (1991). 31. Nag, S., Robertson, D. M. & Dinsdale, H. B. Quantitative estimate of pinocytosis in experimental acute hypertension. Acta Neuropathol. 46, 107–16 (1979). p , ( ) 2. Stokum, J. A., Gerzanich, V. & Simard, J. M. Molecular pathophysiology of cerebral edema. J Cerebral Blood Flow & Metabolism. 36 513–538 (2016). 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Validation of the SCID-hu Thy/Liv Mouse Model with Four Classes of Licensed Antiretrovirals
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Validation of the SCID-hu Thy/Liv Mouse Model with Four Classes of Licensed Antiretrovirals doi:10.1371/journal.pone.0000655 itation: Stoddart CA, Bales CA, Bare JC, Chkhenkeli G, Galkina SA, et al (2007) Validation of the SCID-hu Thy/Liv Mouse icensed Antiretrovirals. PLoS ONE 2(8): e655. doi:10.1371/journal.pone.0000655 CA, Bare JC, Chkhenkeli G, Galkina SA, et al (2007) Validation of the SCID-hu Thy/Liv Mouse Model with Four Classes o S ONE 2(8): e655. doi:10.1371/journal.pone.0000655 PLoS ONE | www.plosone.org INTRODUCTION Academic Editor: Linqi Zhang, AIDS Research Center, Chinese Academy of Medical Sciences and Peking Union Medical College, China Academic Editor: Linqi Zhang, AIDS Research Center, Chinese Academy of Medical Sciences and Peking Union Medical College, China Received April 18, 2007; Accepted June 20, 2007; Published August 1, 2007 Copyright:  2007 Stoddart et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This project has been funded in whole or in part with Federal funds from NIAID, NIH, under contract no. N01-AI-05418. Competing Interests: The authors have declared that no competing interests exist. Academic Editor: Linqi Zhang, AIDS Research Center, Chinese Academy of Medical Sciences and Peking Union Medical College, China Validation of the SCID-hu Thy/Liv Mouse Model with Four Classes of Licensed Antiretrovirals Cheryl A. Stoddart1¤*, Cheryl A. Bales1¤, Jennifer C. Bare1, George Chkhenkeli1¤, Sofiya A. Galkina1¤, April N. Kinkade1, Mary E. Moreno1¤, Jose´ M Rivera1¤, Rollie E. Ronquillo1¤, Barbara Sloan1¤, Paul L. Black2 1 Gladstone Institute of Virology and Immunology, University of California at San Francisco, San Francisco, California, United States of America, 2 Targeted Interventions Branch, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America Background. The SCID-hu Thy/Liv mouse model of HIV-1 infection is a useful platform for the preclinical evaluation of antiviral efficacy in vivo. We performed this study to validate the model with representatives of all four classes of licensed antiretrovirals. Methodology/Principal Findings. Endpoint analyses for quantification of Thy/Liv implant viral load included ELISA for cell-associated p24, branched DNA assay for HIV-1 RNA, and detection of infected thymocytes by intracellular staining for Gag-p24. Antiviral protection from HIV-1-mediated thymocyte depletion was assessed by multicolor flow cytometric analysis of thymocyte subpopulations based on surface expression of CD3, CD4, and CD8. These mice can be productively infected with molecular clones of HIV-1 (e.g., the X4 clone NL4-3) as well as with primary R5 and R5X4 isolates. To determine whether results in this model are concordant with those found in humans, we performed direct comparisons of two drugs in the same class, each of which has known potency and dosing levels in humans. Here we show that second-generation antiretrovirals were, as expected, more potent than their first-generation predecessors: emtricitabine was more potent than lamivudine, efavirenz was more potent than nevirapine, and atazanavir was more potent than indinavir. After interspecies pharmacodynamic scaling, the dose ranges found to inhibit viral replication in the SCID-hu Thy/Liv mouse were similar to those used in humans. Moreover, HIV-1 replication in these mice was genetically stable; treatment of the mice with lamivudine did not result in the M184V substitution in reverse transcriptase, and the multidrug-resistant NY index case HIV-1 retained its drug- resistance substitutions. Conclusion. Given the fidelity of such comparisons, we conclude that this highly reproducible mouse model is likely to predict clinical antiviral efficacy in humans. Citation: Stoddart CA, Bales CA, Bare JC, Chkhenkeli G, Galkina SA, et al (2007) Validation of the SCID-hu Thy/Liv Mouse Model with Four Classes of Licensed Antiretrovirals. PLoS ONE 2(8): e655. INTRODUCTION occurs with both molecular clones and clinical isolates of HIV-1 in 3–5 weeks [12–16]. This depletion includes loss of CD4+CD8+ (double-positive, DP) immature cortical thymocytes and a decrease in the CD4/CD8 ratio in the thymic medulla. There is evidence for both indirect, apoptotic destruction of uninfected thymocytes and direct infection and destruction of CD32CD4+CD82 intrathymic T-progenitor cells, which severely disrupts thymocyte maturation [17,18]. Infection of human thymus with HIV-1 induces IFN-a secretion from plasmacytoid dendritic cells, which upregulates MHC class I (MHC-I) expression on DP thymocytes, where expression is normally low [19]. The SCID-hu mouse model, in which human lymphoid organs are implanted into severe-combined immunodeficient (SCID) mice, was designed by McCune et al. [1] to provide a small animal model for the study of human hematopoiesis. It has also facilitated study of the pathogenesis of HIV-1 in human hematolymphoid organs [2–7] and evaluation of anti-HIV-1 compounds in vivo [2,8,9]. In this model, SCID mice are implanted with a variety of human fetal organs, including bone, liver, thymus, lymph node, and spleen. The fetal implants become tolerant of the mouse environment, and re- ciprocally, growth of the human tissue is permitted by the immunocompromised status of the recipient SCID mouse [6]. The administration of nucleoside and nonnucleoside reverse transcriptase (RT) inhibitors to these mice results in dose-dependent The SCID-hu Thy/Liv mouse, first reported by Namikawa et al. in 1990 [10], is generated by coimplanting human fetal thymus and liver beneath the mouse kidney capsule. In a highly reproducible manner, these organs fuse, become vascularized, and grow into a stable organ termed ‘‘Thy/Liv,’’ reaching a total mass of 100–3006106 human cells in 18 weeks. The Thy/Liv implant reproduces the differentiation, proliferation, and function of human hematopoietic progenitor cells derived from the fetal liver within the human thymus. The implants possess histologically normal cortical and medullary compartments that sustain multi- lineage human hematopoiesis for 6–12 months [10,11], generating a continuous source of CD4-expressing thymocytes that can serve as target cells for HIV-1 infection and replication. Importantly for a model of antiviral chemotherapy, 50–60 SCID-hu Thy/Liv mice can be made with tissues from a single fetal donor, and the Thy/ Liv implant is amenable to experimental manipulation and infection with HIV-1. Viruses The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: pNL4-3 [38] from Malcolm Martin and HIV-1 Ba-L [39] from Suzanne Gartner, Mikulas Popovic, and Robert Gallo. A working stock of NL4-3 was prepared in 293T cells by calcium phosphate transfection. A T-20-sensitive NL4-3 (NL4-3 D36G) was altered by site-directed mutagenesis to match the consensus sequence at amino acid position 36 (aspartic acid replaced by glycine) [40,41]. The R5X4 AZT-resistant clinical HIV-1 isolate JD [42] was kindly provided by Mike McCune, and the multidrug-resistant (MDR) R5X4 NY index case isolate [43] by Hiroshi Morhi and Martin Markowitz. The clinical isolates were prepared and titrated by limiting dilution in phytohemagglutin-activated peripheral blood mononuclear cells. The SCID-hu Thy/Liv model has also been employed by us and by others for in vivo studies of HIV-1 pathogenesis. The roles of HIV- 1 nef [24–26], other accessory genes [24], and the Rev–Rev responsive element [27] have been explored as well as the expression of HIV-1 coreceptors (CXCR4 and CCR5) on thymocytes [28,29] and the effects of coreceptor usage on viral tropism and pathogenesis [16,28,30–32]. The CXCR4-utilizing (X4) strain NL4-3 replicates quickly and extensively in thymocytes in the cortex and medulla, causing significant thymocyte depletion [16]. In contrast, the CCR5- utilizing (R5) strain Ba-L initially infects stromal cells, including macrophages, in the thymic medulla without any obvious pathologic consequences for 3–4 weeks, after which the infection slowly spreads through the thymocyte populations and results in slowly progressing thymocyte depletion after 6 weeks. In addition, three R5 viruses isolated from rapid AIDS progressors lacking X4 strains were much less pathogenic than NL4-3 in SCID-hu Thy/Liv mice, suggesting that R5 virus-mediated rapid disease progression is associated with host, not viral, factors [30]. The lab-adapted LAI/IIIB isolate and its associated infectious molecular clones (e.g., HXB2) fail to replicate in Thy/Liv implants, and this impairment has been mapped to a specific amino acid change in the crown region of V3 in Env [33,34]. The model has also been used to study immune reconstitution and recovery of hematopoietic colony-forming activity in HIV-1-infected implants after treatment of mice with potent combination antiretroviral therapy [35–37]. Antiretroviral drugs and mouse dosing The antiretroviral drugs lamivudine (3TC), emtricitabine [(–)- FTC], nevirapine, efavirenz, indinavir, and atazanavir were kindly supplied by Opendra Sharma and the AIDS Reagent Program. Dosing solutions were prepared in sterile water for injection or Dulbecco’s phosphate-buffered saline, and groups of 5–8 mice were treated by oral gavage (200 ml per dose) with an 18-gauge62- inch feeding needle twice daily at 7:00 AM and 6:00 PM for the duration of the infection period (2–6 weeks depending on the HIV-1 isolate). Enfuvirtide (T-20) was purchased from a pharmacy, prepared in sterile water, and administered by twice-daily subcutaneous injection (200 ml per dose). * To whom correspondence should be addressed. E-mail: cheryl.stoddart@ucsf. edu * To whom correspondence should be addressed. E-mail: cheryl.stoddart@ucsf. edu ¤ Current address: Division of Experimental Medicine, Department of Medicine, University of California at San Francisco, San Francisco, California, United States of America The Thy/Liv implants support viral replication after inocula- tion of HIV-1 by direct injection [3], and thymocyte depletion PLoS ONE | www.plosone.org August 2007 | Issue 8 | e655 1 Validation of SCID-hu Model performed in SCID-hu Thy/Liv mouse cohorts each made with tissues from a single donor. We observed that the more recent drugs in each class were significantly more potent than their predecessors. We conclude from such comparisons with licensed antiretrovirals that this well-established model is likely to predict clinical antiviral efficacy in humans for other members of these classes and possibly other drug classes as well. inhibition of HIV-1 replication, prevention of IFN-a-mediated MHC-I upregulation, and protection of CD4+ cells within the implanted human tissue [8,9,20]. The model has been also used to evaluate new classes of HIV-1 inhibitors, such as bicyclam [21] and oligonucleotide [22] inhibitors of HIV-1 entry, the nucleoside analog 29-deoxy-39-oxa-49-thiocytidine (dOTC) [20], and an oxime-piper- idine CCR5 antagonist [23]. This is the first animal model in which the action of candidate anti-HIV-1 compounds can be tested within the setting of an intact HIV-1-infected human target organ. MATERIALS AND METHODS We use a standardized protocol described by Rabin et al. [8] for evaluation of antiviral agents against HIV-1 in the SCID-hu Thy/ Liv mouse model. Drugs can be administered by various routes (oral, subcutaneous, intraperitoneal, or by mini-osmotic pump) before or after virus inoculation and continued for 2–6 weeks. Mice are challenged with titrated inocula of various HIV-1 isolates, and endpoint analyses include quantification of viral protein by p24 ELISA and viral RNA by branched DNA assay, quantification and isolation of replication-competent virus by cocultivation after limiting dilution, and assessment of the effects on human thymocyte subpopulations by flow cytometry [8]. Implantation and inoculation of SCID-hu Thy/Liv mice Human fetal thymus and liver were obtained through services provided by a nonprofit organization (Advanced Bioscience Resources, Alameda, CA) in accordance with federal, state, and local regulations. Coimplantation of thymus and liver fragments under the kidney capsule to create SCID-hu Thy/Liv mice and inoculation of the Thy/Liv implants with HIV-1 was carried out as described [8,10]. Male C.B-17 SCID (model #CB17SC-M, homozygous, C.B-Igh-1b/IcrTac-Prkdcscid) mice were obtained at 6–8 weeks of age from Taconic (Germantown, NY), and cohorts of 50–60 SCID-hu Thy/Liv mice were implanted with tissues from a single donor. Implants were inoculated 18 weeks after implantation with 50 ml of stock virus (500–2,000 TCID50) or RPMI 1640 medium (mock infection) by direct injection after surgical exposure of the implanted kidney of anesthetized mice. Animal protocols were approved by the UCSF Institutional Animal Care and Use Committee. Given the potential correlations between virus-host interactions in the Thy/Liv implant and in humans, we wanted to determine whether the SCID-hu Thy/Liv mouse model predicts antiretro- viral efficacy in humans for representatives of all four classes of currently licensed drugs. Does it, for instance, yield data that reflect the greater potency of more recently developed drugs than their first-generation predecessors in each class? In vivo models are far more stringent than cell culture-based assays for demonstrating drug activity, particularly in the case of orally delivered drugs as they must be absorbed by the gastrointestinal tract without degradation, enter the blood and circulate with sufficient half-lives, and penetrate solid target organs at concentrations high enough to be potently efficacious. The greater potency of the second- generation drugs is likely the result of a combination of greater in vitro potency and superior pharmacokinetics. In vitro virus-cell culture systems would likely predict greater potency, but only in vivo studies can demonstrate sustained plasma half-life or superior oral bioavailability. To determine such relative potency in vivo, head-to-head comparisons of two drugs in the same class were PLoS ONE | www.plosone.org Viral load and prophylactic antiviral efficacy are highly reproducible among mouse cohorts each made with tissues from a single donor g The levels of HIV-1 RNA and p24 in the Thy/Liv implants of untreated- and 3TC-treated SCID-hu Thy/Liv mice were highly reproducible among seven different mouse cohorts infected with the same stock of HIV-1 NL4-3 (Figure 1). In seven consecutive experiments, with mice in a given cohort constructed from human fetal tissue from a single donor, mean cell-associated HIV-1 RNA ranged from 5.2–6.3 log10 copies per 106 cells (Figure 1A), and mean p24 ranged from 640–1,700 pg per 106 cells (Figure 1B). There was also good correspondence between viral RNA and p24 p g p g p p Figure 1. Implant viral loads in untreated- and 3TC-treated SCID-hu Thy/Liv mice are highly reproducible, and 3TC treatment after HIV-1 inoculation is nearly as effective as prophylactic treatment. A, HIV-1 RNA in Thy/Liv implants of untreated mice and mice treated by twice-daily oral gavage with 3TC at 30 mg/kg per day beginning 1 day before inoculation with NL4-3 (means6SEM) Each experiment was performed in a mouse cohort made with tissues from a single donor. B, Implant p24 for the same groups as in panel A. C, Implant HIV-1 RNA in mice treated with 3TC beginning on day 21, day +1, day +3, and day +7 with respect to inoculation with the indicated HIV-1 strains. D, Implant p24 for the same groups as in panel C. Implants were collected 21 days after inoculation for NL4-3, 14 days for JD, and 42 days for Ba-L. *p#0.05, compared with untreated mice for 5–7 mice per group. doi:10.1371/journal.pone.0000655.g001 Figure 1. Implant viral loads in untreated- and 3TC-treated SCID-hu Thy/Liv mice are highly reproducible, and 3TC treatment after HIV-1 inoculation is nearly as effective as prophylactic treatment. A, HIV-1 RNA in Thy/Liv implants of untreated mice and mice treated by twice-daily oral gavage with 3TC at 30 mg/kg per day beginning 1 day before inoculation with NL4-3 (means6SEM) Each experiment was performed in a mouse cohort made with tissues from a single donor. B, Implant p24 for the same groups as in panel A. C, Implant HIV-1 RNA in mice treated with 3TC beginning on day 21, day +1, day +3, and day +7 with respect to inoculation with the indicated HIV-1 strains. D, Implant p24 for the same groups as in panel C. Implants were collected 21 days after inoculation for NL4-3, 14 days for JD, and 42 days for Ba-L. Implant collection and viral load quantification Implant collection and viral load quantification The Thy/Liv implants were collected from euthanized mice, and single-cell suspensions were prepared by dispersing the implant through nylon mesh and processed for p24 ELISA, bDNA assay, and FACS analysis as described [8,20]. Unless specified otherwise, PLoS ONE | www.ploso August 2007 | Issue 8 | e655 PLoS ONE | www.plosone.org 2 Validation of SCID-hu Model implants were collected 21 days after inoculation with NL4-3, 14 days after JD inoculation, and 42 days after Ba-L inoculation. Statistical analysis Results are expressed as the mean6SEM for each mouse group. Nonparametric statistical analyses were performed by use of the Mann-Whitney U test. Data for mice in each group were compared to those for untreated infected mice, and p values #0.05 were considered statistically significant. Flow cytometry Implant cells were stained with phycoerythrin cyanine dye CY7- conjugated anti-CD4 (BD Biosciences, San Jose, CA), phycoerythrin cyanine CY5.5-conjugated anti-CD8 (Caltag Laboratories, Burlin- game, CA), allophycocyanin cyanine CY7-conjugated anti-CD3 (eBiosciences, San Diego, CA), and phycoerythrin-conjugated anti- W6/32 (DakoCytomation, Glostrup, Denmark. Cells were fixed and permeabilized with 1.2% paraformaldehyde and 0.5% Tween 20, stained with fluorescein isothiocyanate-conjugated anti-p24 (Beck- man Coulter, Fullerton, CA), and analyzed on an LSR II (BD Biosciences). After collecting 100,000 total cell events, percentages of marker-positive (CD4+, CD8+, and DP) thymocytes in the implant samples were determined by first gating on a live lymphoid cell population identified by forward- and side-scatter characteristics and then by CD3 expression. W6/32-positive mean fluorescence intensity (MFI) of DP thymocytes was determined, and CD4/CD8 ratios were calculated by dividing the percentage of CD4+ cells by the percentage of CD8+ cells for each individual implant. Head-to-head comparisons of antiretroviral drugs at multiple dosage levels show relative potency p g p y In the course of preclinical antiviral drug development, it is often important to determine whether a newly discovered agent is more potent than an existing, perhaps licensed, drug in the same class. To that end, we produced cohorts of SCID-hu Thy/Liv mice that were large enough for head-to-head comparisons of two drugs at multiple (typically three) dosage levels against one virus isolate. When the nucleoside analogs (–)-FTC and 3TC were compared, (–)-FTC was more potent at the same dosage levels, with greater reductions in viral RNA at 30 mg/kg per day (p= 0.035) (Figure 4A) and greater reductions in p24 at 10 mg/kg per day (p= 0.025) and 30 mg/kg per day (p = 0.009) (Figure 4B). The p24 levels in mice treated with (– )-FTC at 30 mg/kg per day were 1% that of mice treated with 3TC at the same dose (1,300 pg per 106 cells in untreated mice versus 1.2 pg per 106 cells for (–)-FTC and 110 pg per 106 cells for 3TC) (Figure 4B). Good protection from thymocyte depletion was observed at all three dosage levels for both drugs (Figure 4C). Viral load and prophylactic antiviral efficacy are highly reproducible among mouse cohorts each made with tissues from a single donor In the seven experiments, 3TC treatment beginning 1 day before virus inoculation reduced HIV-1 RNA in the implants by 1.2–2.1 log10 (94%–99%) (Figure 1A) and p24 by 77%–92% (Figure 1B) compared to untreated mice in the same experiment. Importantly, HIV-1 NL4-3 replication in these mice was genetically stable over the 21-day infection period; there was no evidence of the RT M184V substitution in viral RNA amplified by RT-PCR and sequenced from 32 of 32 Thy/Liv implants collected from mice treated with 3TC in these seven experiments (data not shown). The lack of resistance development is most likely the result of the limited time before virus-mediated thymocyte depletion becomes severe enough to limit the number of target cells available for infection. We view the genetic stability of the viruses over several weeks as an asset because our results are not confounded by the rapid development of antiviral resistance, particularly against drugs for which only one amino acid substitution is sufficient for high-level resistance. Viral load and prophylactic antiviral efficacy are highly reproducible among mouse cohorts each made with tissues from a single donor *p#0.05, compared with untreated mice for 5–7 mice per group. doi:10.1371/journal.pone.0000655.g001 August 2007 | Issue 8 | e655 PLoS ONE | www.plosone.org 3 Figure 2. 3TC treatment of HIV-1 JD-infected SCID-hu Thy/Liv mice inhibits viral replication and protects implants from virus-mediated thymocyte depletion. Mice were treated by twice-daily oral gavage with 3TC at 300 mg/kg per day beginning on day +1 after inoculation, and Thy/ Liv implants were collected 14, 21, and 28 days after inoculation. Antiviral efficacy was assessed by determining HIV-1 RNA (A), p24 (B), Gag-p24+ thymocytes (C), and MHC-I expression on DP thymocytes (D). Thymocyte protection was assessed by total implant cellularity (E), thymocyte viability Validation of SCID-hu Model Validation of SCID-hu Model Figure 2. 3TC treatment of HIV-1 JD-infected SCID-hu Thy/Liv mice inhibits viral replication and protects implants from virus-mediated thymocyte depletion. Mice were treated by twice-daily oral gavage with 3TC at 300 mg/kg per day beginning on day +1 after inoculation, and Thy/ Liv implants were collected 14, 21, and 28 days after inoculation. Antiviral efficacy was assessed by determining HIV-1 RNA (A), p24 (B), Gag-p24+ thymocytes (C), and MHC-I expression on DP thymocytes (D). Thymocyte protection was assessed by total implant cellularity (E), thymocyte viability (F), percentage of DP thymocytes (G), and CD4/CD8 ratio (H) for 3TC-treated mice versus untreated mice (means6SEM). *p#0.05, compared with untreated mice for 5–7 mice per group. doi:10 1371/journal pone 0000655 g002 August 2007 | Issue 8 | e655 4 Validation of SCID-hu Model thymocytes (Figure 2C), and IFN-a-induced MHC-I expression on DP thymocytes (Figure 2D), compared to untreated mice at all three times. Reductions in viral load by 3TC were accompanied by virtually complete protection of the Thy/Liv implants from thymocyte depletion, in terms of total cellularity (Figure 2E), thymocyte viability (Figure 2F), percentage of DP thymocytes (Figure 2G), and CD4/CD8 ratio (Figure 2H). Flow cytometric analysis of representative implants obtained 28 days after in- oculation with HIV-1 JD showed severe depletion of DP and CD4+ thymocytes in an untreated mouse and nearly complete protection from thymocyte depletion in one mouse treated with 3TC (Figure 3). means of implants in the same groups (i.e., lowest for untreated mice in experiment 6 and highest for those in experiments 3 and 5). Postexposure dosing also reduces viral load and prevents virus-mediated thymocyte depletion Initiation of 3TC treatment 1 or 3 days after NL4-3 inoculation was nearly as effective at reducing implant viral RNA (Figure 1C) and p24 (Figure 1D) as was prophylactic treatment with treatment beginning 1 day before inoculation. This was also true for mice inoculated with the clinical R5X4 isolate JD and treated with 3TC beginning 3 days after inoculation. Treatment of mice inoculated with the R5 isolate Ba-L, which replicates in Thy/Liv implants with slower kinetics than does X4 HIV-1 NL4-3 [16], was highly effective at reducing implant viral RNA and p24 even when initiation was delayed until 7 days after inoculation. In another experiment, the nonnucleoside RT inhibitor efavirenz was directly compared with nevirapine. Efavirenz was approximately 10 times more potent than nevirapine, as demonstrated by similar reductions in viral RNA (Figure 5A) and p24 (Figure 5B) and by thymocyte protection (Figure 5C) in groups treated with 300 mg/kg per day of nevirapine compared to 30 mg/kg per day of efavirenz. To determine whether postexposure dosing also prevents thymocyte depletion, SCID-hu mice were inoculated with HIV- 1 JD and treated twice daily with 3TC at 300 mg/kg per day in a time-course study in which implants were collected at weekly intervals up to 28 days after inoculation. By that point, the implants from untreated mice had become severely depleted of thymocytes. As expected, 3TC-treated mice had significant reductions in viral RNA (Figure 2A), p24 (Figure 2B), Gag-p24+ Finally, the second-generation protease inhibitor atazanavir was 3–10 times more potent than indinavir in NL4-3-infected SCID- Figure 3. 3TC treatment protects HIV-1 JD-infected Thy/Liv implants from thymocyte depletion. Flow cytometric analysis of representative implants stained for CD4 and CD8 obtained 28 days after inoculation with HIV-1 JD shows severe depletion of DP and CD4+ thymocytes in an untreated mouse and nearly complete protection from thymocyte depletion by 3TC treatment (300 mg/kg per day beginning on day +1 after inoculation). doi:10.1371/journal.pone.0000655.g003 Figure 3. 3TC treatment protects HIV-1 JD-infected Thy/Liv implants from thymocyte depletion. Flow cytometric analysis of representative implants stained for CD4 and CD8 obtained 28 days after inoculation with HIV-1 JD shows severe depletion of DP and CD4+ thymocytes in an untreated mouse and nearly complete protection from thymocyte depletion by 3TC treatment (300 mg/kg per day beginning on day +1 after inoculation). Figure 3. 3TC treatment protects HIV-1 JD-infected Thy/Liv implants from thymocyte depletion. Postexposure dosing also reduces viral load and prevents virus-mediated thymocyte depletion Flow cytometric analysis of representative implants stained for CD4 and CD8 obtained 28 days after inoculation with HIV-1 JD shows severe depletion of DP and CD4+ thymocytes in an untreated mouse and nearly complete protection from thymocyte depletion by 3TC treatment (300 mg/kg per day beginning on day +1 after inoculation). doi:10.1371/journal.pone.0000655.g003 doi:10.1371/journal.pone.0000655.g003 PLoS ONE | www.plosone.org August 2007 | Issue 8 | e655 PLoS ONE | www.plosone.org 5 Validation of SCID-hu Model Figure 4. (–)-FTC is more potent than 3TC against HIV-1 NL4-3 in SCID-hu Thy/Liv mice. Mice were treated by twice-daily oral gavage with 3TC or (–)-FTC at 10, 30, and 100 mg/kg per day beginning on day Figure 5. Efavirenz is more potent than nevirapine against HIV-1 NL4-3 in SCID-hu Thy/Liv mice. Mice were treated by twice-daily oral gavage with nevirapine or efavirenz at the indicated dosage levels Validation of SCID hu Model Figure 4. (–)-FTC is more potent than 3TC against HIV-1 NL4-3 in SCID-hu Thy/Liv mice. Mice were treated by twice-daily oral gavage with 3TC or (–)-FTC at 10, 30, and 100 mg/kg per day beginning on day –1. Antiviral efficacy was assessed by determining HIV-1 RNA (A) and p24 (B), and thymocyte protection was assessed by percentage of DP thymocytes (C) (means6SEM). *p#0.05, compared with untreated mice for the number of mice indicated under each bar. doi:10.1371/journal.pone.0000655.g004 Figure 5. Efavirenz is more potent than nevirapine against HIV-1 NL4-3 in SCID-hu Thy/Liv mice. Mice were treated by twice-daily oral gavage with nevirapine or efavirenz at the indicated dosage levels beginning on day 21. Antiviral efficacy was assessed by determining HIV-1 RNA (A) and p24 (B), and thymocyte protection was assessed by percentage of DP thymocytes (C) (means6SEM). *p#0.05, compared with untreated mice for the number of mice indicated under each bar. doi:10.1371/journal.pone.0000655.g005 Figure 4. (–)-FTC is more potent than 3TC against HIV-1 NL4-3 in SCID-hu Thy/Liv mice. Mice were treated by twice-daily oral gavage with 3TC or (–)-FTC at 10, 30, and 100 mg/kg per day beginning on day –1. Antiviral efficacy was assessed by determining HIV-1 RNA (A) and p24 (B), and thymocyte protection was assessed by percentage of DP thymocytes (C) (means6SEM). *p#0.05, compared with untreated mice for the number of mice indicated under each bar. doi:10.1371/journal.pone.0000655.g004 Figure 5. Efavirenz is more potent than nevirapine against HIV-1 NL4-3 in SCID-hu Thy/Liv mice. Postexposure dosing also reduces viral load and prevents virus-mediated thymocyte depletion Inhibitory drug dosage levels are comparable between SCID-hu Thy/Liv mice and humans The SCID-hu Thy/Liv mouse has the advantage of having human, not mouse, cells as the targets of antiretroviral uptake and action, but the pharmacokinetics of antiretrovirals in the mouse have the potential to be significantly different from those in humans. Small mammals usually eliminate drugs faster than large Figure 6. Atazanavir is more potent than indinavir against HIV-1 NL4- 3 in SCID-hu Thy/Liv mice. Mice were treated by twice-daily oral i h i di i i d /k Figure 7. T-20 causes dose-dependent reductions in viral load in HIV- 1 NL4-3 D36G-infected SCID-hu Thy/Liv mice. Mice were treated by twice-daily subcutaneous injection with T-20 at 10, 30, and 100 mg/kg per day beginning on day 21. Antiviral efficacy was assessed by determining cell-associated HIV-1 RNA and p24. Data are expressed as means6SEM; *p#0.05 for treated mice versus untreated mice by the Mann-Whitney U test for the number of mice indicated under each bar. doi:10.1371/journal.pone.0000655.g007 Validation of SCID-hu Model Figure 6. Atazanavir is more potent than indinavir against HIV-1 NL4- 3 in SCID-hu Thy/Liv mice. Mice were treated by twice-daily oral gavage with indinavir or atazanavir at 100, 300, and 1000 mg/kg per day beginning on day 21. Antiviral efficacy was assessed by determining HIV-1 RNA (A) and p24 (B), and thymocyte protection was assessed by percentage of DP thymocytes (C) (means6SEM). *p#0.05, compared with untreated mice for the number of mice indicated under each bar. doi:10.1371/journal.pone.0000655.g006 Figure 7. T-20 causes dose-dependent reductions in viral load in HIV- Figure 7. T-20 causes dose-dependent reductions in viral load in HIV- 1 NL4-3 D36G-infected SCID-hu Thy/Liv mice. Mice were treated by twice-daily subcutaneous injection with T-20 at 10, 30, and 100 mg/kg per day beginning on day 21. Antiviral efficacy was assessed by determining cell-associated HIV-1 RNA and p24. Data are expressed as means6SEM; *p#0.05 for treated mice versus untreated mice by the Mann-Whitney U test for the number of mice indicated under each bar. doi:10.1371/journal.pone.0000655.g007 hu mice, with a 3.1 log10 greater reduction in viral RNA (p = 0.006) with atazanavir than with indinavir at 300 mg/kg per day (5.3 log10 copies per 106 cells in untreated mice versus ,1.5 log10 copies per 106 cells for atazanavir and 4.6 log10 copies per 106 cells for indinavir) (Figure 6A). Postexposure dosing also reduces viral load and prevents virus-mediated thymocyte depletion The p24 levels in mice treated with atazanavir at 100 mg/kg per day were 5% (p = 0.029) that of mice treated with indinavir at the same dose (400 pg p24 per 106 cells in untreated mice versus 19 pg p24 per 106 cells for atazanavir and 380 pg p24 per 106 cells for indinavir) (Figure 6B). There was comparable protection from thymocyte depletion for the drugs at 100 and 300 mg/kg per day (Figure 6C). Figure 6. Atazanavir is more potent than indinavir against HIV-1 NL4- 3 in SCID-hu Thy/Liv mice. Mice were treated by twice-daily oral gavage with indinavir or atazanavir at 100, 300, and 1000 mg/kg per day beginning on day 21. Antiviral efficacy was assessed by determining HIV-1 RNA (A) and p24 (B), and thymocyte protection was assessed by percentage of DP thymocytes (C) (means6SEM). *p#0.05, compared with untreated mice for the number of mice T-20 causes dose-dependent reductions in implant viral load Although we did not perform this experiment as a direct comparison with another HIV-1 fusion inhibitor, we are including these data to complete the interspecies scaling analysis described in the next section with a fourth class of antiretroviral (T-20). Treatment of NL4-3 D36G-infected SCID-hu Thy/Liv mice with T-20 by twice-daily subcutaneous injection resulted in dose- dependent reductions of both HIV-1 RNA and p24, reducing viral RNA by 3.3 log10 copies per 106 cells and p24 to undetectable levels at 100 and 30 mg/kg per day (Figure 7). Postexposure dosing also reduces viral load and prevents virus-mediated thymocyte depletion Mice were treated by twice-daily oral gavage with nevirapine or efavirenz at the indicated dosage levels beginning on day 21. Antiviral efficacy was assessed by determining HIV-1 RNA (A) and p24 (B), and thymocyte protection was assessed by percentage of DP thymocytes (C) (means6SEM). *p#0.05, compared with untreated mice for the number of mice indicated under each bar. doi:10.1371/journal.pone.0000655.g005 August 2007 | Issue 8 | e655 PLoS ONE | www.plosone.org 6 Figure 7. T-20 causes dose-dependent reductions in viral load in HIV- 1 NL4-3 D36G-infected SCID-hu Thy/Liv mice. Mice were treated by twice-daily subcutaneous injection with T-20 at 10, 30, and 100 mg/kg per day beginning on day 21. Antiviral efficacy was assessed by determining cell-associated HIV-1 RNA and p24. Data are expressed as means6SEM; *p#0.05 for treated mice versus untreated mice by the Mann-Whitney U test for the number of mice indicated under each bar. doi:10.1371/journal.pone.0000655.g007 Validation of SCID-hu Model Validation of SCID-hu Model hu mice, with a 3.1 log10 greater reduction in viral RNA (p = 0.006) with atazanavir than with indinavir at 300 mg/kg per day (5.3 log10 copies per 106 cells in untreated mice versus ,1.5 log10 copies per 106 cells for atazanavir and 4.6 log10 copies per 106 cells for indinavir) (Figure 6A). The p24 levels in mice treated with atazanavir at 100 mg/kg per day were 5% (p = 0.029) that of mice treated with indinavir at the same dose (400 pg p24 per 106 cells in untreated mice versus 19 pg p24 per 106 cells for atazanavir and 380 pg p24 per 106 cells for indinavir) (Figure 6B). There was comparable protection from thymocyte depletion for the drugs at 100 and 300 mg/kg per day (Figure 6C). T-20 causes dose-dependent reductions in implant viral load Although we did not perform this experiment as a direct comparison with another HIV-1 fusion inhibitor, we are including these data to complete the interspecies scaling analysis described in the next section with a fourth class of antiretroviral (T-20). Treatment of NL4-3 D36G-infected SCID-hu Thy/Liv mice with T-20 by twice-daily subcutaneous injection resulted in dose- dependent reductions of both HIV-1 RNA and p24, reducing viral RNA by 3.3 log10 copies per 106 cells and p24 to undetectable levels at 100 and 30 mg/kg per day (Figure 7). DISCUSSION The SCID-hu Thy/Liv mouse is an efficient model for the study of HIV-1 infection and for the preclinical evaluation of potential antiretrovirals. Here we show that this model provides a validated platform for reproducible viral infectivity and drug-mediated reductions in viral load in vivo. We also show that the model is Table 1. Antiretroviral drug dosage levels adjusted for difference in surface area-to-body weight ratio between mice and humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Drug Human dosage regimena Human dosageb (mg/kg per day) Mouse equivalent dosagec (mg/kg per day) Calculated reduction in implant HIV-1 RNAd (log10 copies) 3TC 150 mg twice daily 5.0 62 1.9 (–)-FTC 200 mg once daily 3.3 41 2.3 nevirapine 400 mg once daily 6.7 82 0.9 efavirenz 600 mg once daily 10 123 3.7 indinavir 800 mg three times daily 40 490 1.2 atazanavir 400 mg once daily 6.7 82 1.4 T-20 90 mg twice daily 3.0 37 3.2 aFrom ref. [56]. bBased on 60 kg adult human weight and 20 g mouse weight. cHuman dosage612.3 (fold difference in surface area to body weight between mice and humans; from ref. [45,46]. dCalculated by linear regression of the viral RNA data shown in Figures 3–6 at the equivalent mouse dosage level. doi:10.1371/journal.pone.0000655.t001 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Multidrug-resistant NY index case HIV-1 replicates and depletes thymocytes with kinetics comparable to HIV-1 NL4-3 A key test of any new class of antiretroviral drug is a demonstration of potent activity against HIV-1 with known drug-resistance mutations. We reported the activity of the 3TC analog dOTC against 3TC-resistant HIV-1 NL4-3/M184V in the SCID-hu Thy/Liv model [20]. Here we report that the MDR NY index case HIV-1, which was isolated from a patient with highly rapid progression to AIDS [43], replicates with kinetics similar to HIV-1 NL4-3 in SCID-hu Thy/Liv mice (Figure 8A–D) resulting in severe thymocyte depletion by 28 days after inoculation (Figure 8E–H). The lower viral loads for the NY index case isolate compared to NL4-3 at day 28 are most likely the result of somewhat faster thymocyte depletion and thus greater loss of target cells for viral replication. The goal of this study was to validate the SCID-hu Thy/Liv model for use in drug discovery efforts by determining the efficacy of different classes of FDA-approved antiretroviral drugs. Antiviral efficacy in this model is demonstrated by dose-dependent reductions in viral load as well as dose-dependent protection of CD4+ T cells from virus-mediated depletion. Assessment of thymocyte percentage and viability is particularly important for proving that the observed antiviral activity is not merely the result of cytotoxicity to the Thy/Liv implant just as the selectivity index (ratio of cytotoxic to inhibitory concentration) is important for assessing antiviral activity in vitro. Additional evidence that HIV-1 replication is genetically stable within the 28-day infection period was provided by sequence analysis of implant viral RNA showing that the MDR NY index case isolate [43] retained the full complement of drug-resistance amino acid substitutions of the original inoculum (data not shown). These data show that this MDR clinical isolate can readily be used as a challenge virus in the SCID-hu Thy/Liv model for in vivo studies of new classes of antiretroviral drugs with activity against HIV-1 that is resistant to currently licensed antiretrovirals. As new antiretroviral drugs in existing drug classes are developed to improve upon antiviral potency or to limit in vivo toxicity, direct comparisons of the existing drug and the new drug in single SCID-hu mouse cohorts represent a powerful preclinical tool for the prediction of improved efficacy in human patients. To accomplish this, we produced cohorts of SCID-hu Thy/Liv mice that were large enough for comparison of two drugs at three dosage levels against one virus isolate. Inhibitory drug dosage levels are comparable between SCID-hu Thy/Liv mice and humans This factor reflects the 12.3-fold difference in surface area-to-body weight ratio between mice (0.0066 m2/0.02 kg) and humans (1.6 m2/60 kg); consequently, 12.3 times more drug is required in the mouse to be comparable to the dose in humans. When currently approved dosage levels for humans of each antiretroviral drug are adjusted for this difference, the adjusted dosage levels were similar to those that cause 0.9–3.7 log10 reductions in viral RNA in the SCID-hu Thy/Liv mouse (Table 1). Thus, not only is the model predictive of relative potency within these drug classes, it might also be useful in determining approximate dosing ranges for effective use in humans. This factor reflects the 12.3-fold difference in surface area-to-body weight ratio between mice (0.0066 m2/0.02 kg) and humans (1.6 m2/60 kg); consequently, 12.3 times more drug is required in the mouse to be comparable to the dose in humans. When currently approved dosage levels for humans of each antiretroviral drug are adjusted for this difference, the adjusted dosage levels were similar to those that cause 0.9–3.7 log10 reductions in viral RNA in the SCID-hu Thy/Liv mouse (Table 1). Thus, not only is the model predictive of relative potency within these drug classes, it might also be useful in determining approximate dosing ranges for effective use in humans. Multidrug-resistant NY index case HIV-1 replicates and depletes thymocytes with kinetics comparable to HIV-1 NL4-3 For studies of viral pathogenesis and hematopoietic reconstitution, a relatively small number of animals can generate sufficient replicate points for analysis. In contrast, preclinical antiviral evaluations require groups of animals sufficiently large to demonstrate statistical proof of efficacy in vivo. Two-drug, three-dose comparisons are made possible by our unique ability to construct large (50–60-mouse) SCID-hu cohorts with tissues from a single donor, which permits Inhibitory drug dosage levels are comparable between SCID-hu Thy/Liv mice and humans The SCID-hu Thy/Liv mouse has the advantage of having human, not mouse, cells as the targets of antiretroviral uptake and action, but the pharmacokinetics of antiretrovirals in the mouse have the potential to be significantly different from those in humans. Small mammals usually eliminate drugs faster than large mammals [44], and toxic endpoints for therapeutics administered systemically to animals scale well between species when doses are normalized to body surface area [45]. To determine whether the doses found inhibitory in the SCID-hu Thy/Liv mouse bear any relation to those used clinically in humans, an interspecies scaling factor of 12.3 [46] was applied to generate mouse equivalent doses. Figure 6. Atazanavir is more potent than indinavir against HIV-1 NL4- 3 in SCID-hu Thy/Liv mice. Mice were treated by twice-daily oral gavage with indinavir or atazanavir at 100, 300, and 1000 mg/kg per day beginning on day 21. Antiviral efficacy was assessed by determining HIV-1 RNA (A) and p24 (B), and thymocyte protection was assessed by percentage of DP thymocytes (C) (means6SEM). *p#0.05, compared with untreated mice for the number of mice indicated under each bar. doi:10 1371/journal pone 0000655 g006 August 2007 | Issue 8 | e655 7 PLoS ONE | www.plosone.org Validation of SCID-hu Model especially valuable for direct, head-to-head comparisons of the potency and effective dose ranges of existing antiretroviral drugs. In consecutive experiments performed in different SCID-hu mouse cohorts infected with the same stock of HIV-1 NL4-3, we observed a range in mean HIV-1 RNA of only 1.1 log10 copies per 106 cells. Reproducible reductions in viral load were also observed after oral administration of 3TC. Although our standardized protocol calls for prophylactic treatment 1 day before virus inoculation to maximize the potential for discerning antiviral efficacy of new drugs in initial in vivo tests, we show here that postexposure drug initiation (beginning 1, 3, or 7 days after HIV-1 inoculation) also renders potent antiviral efficacy. Importantly, potent therapeutic efficacy, in terms of both inhibition of viral replication and virtually complete protection from virus-mediated cytopathicity and thymocyte depletion, was also demonstrated in 3TC-treated mice infected with the highly cytopathic R5X4 isolate, JD. In addition, we have minimized one potential experimental variable by inoculating the mice at a uniform time point 18 weeks after implantation. DISCUSSION . . dosage levels adjusted for difference in surface area-to-body weight ratio between mice and humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Table 1. Antiretroviral drug dosage levels adjusted for difference in surface area-to-body weight ratio be . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . August 2007 | Issue 8 | e655 PLoS ONE | www.plosone.org 8 Validation of SCID-hu Model Figure 8. Multidrug-resistant (MDR) NY index case HIV-1 replicates and depletes thymocytes with kinetics comparable to HIV-1 NL4-3 in SCID- hu Thy/Liv mice. Viral replication assessed by determining HIV-1 RNA (A), p24 (B), Gag-p24+ thymocytes (C), and MHC-I expression on DP thymocytes (D). Thymocyte protection was assessed by total implant cellularity (E), thymocyte viability (F), percentage of DP thymocytes (G), and CD4/CD8 ratio (H) for NL4-3 versus MDR NY index case HIV-1-infected mice (means6SEM). *p#0.05, compared with untreated mice for 7 mice per group. doi:10.1371/journal.pone.0000655.g008 Validation of SCID-hu Model Figure 8. Multidrug-resistant (MDR) NY index case HIV-1 replicates and depletes thymocytes with kinetics comparable to HIV-1 NL4-3 in SCID- hu Thy/Liv mice. DISCUSSION Viral replication assessed by determining HIV-1 RNA (A), p24 (B), Gag-p24+ thymocytes (C), and MHC-I expression on DP thymocytes (D). Thymocyte protection was assessed by total implant cellularity (E), thymocyte viability (F), percentage of DP thymocytes (G), and CD4/CD8 ratio (H) for NL4-3 versus MDR NY index case HIV-1-infected mice (means6SEM). *p#0.05, compared with untreated mice for 7 mice per group. doi:10.1371/journal.pone.0000655.g008 August 2007 | Issue 8 | e655 PLoS ONE | www.plosone.org 9 Validation of SCID-hu Model six or seven dosing groups of 5–8 mice for optimal statistical power. Given such group sizes and appropriate controls, it is possible to assign effective antiviral dose ranges with statistical precision [8]. Among animal models for HIV-1, this ability is unique: nonhuman primate models (i.e., SIV- or SHIV-infected rhesus macaques) cannot provide dosing groups of such size and cannot achieve the same statistical power and relatively lower cost per animal. There are several recent reports on the infection of humanized Rag22/2cc2/2 (RAG-hu) [47–50] and NOD/SCID BLT mice [51] with HIV-1, but there are no reports yet published on the evaluation of antiretroviral efficacy in these models, and it has not yet been demonstrated that 50–60 mice can be generated with the CD34+ hematopoietic stem cells from a single donor. In a recent review [52], Manz states that one of the challenges these CD34+-reconstituted mouse models face is that usually less than 10 mice can be transplanted from one human graft. once-daily dosing [55]. Taken together, these comparisons of licensed antiretrovirals in each major class suggest that the SCID- hu Thy/Liv model may also predict clinical antiviral efficacy in humans. These results extend an earlier validation of the model with the nucleoside analogs zidovudine and dideoxyinosine [8]. The predictive nature of SCID-hu mouse efficacy is further supported by the dosage levels required to achieve log order reductions in implant viral load when adjusted for the difference in surface area-to-body weight ratio between mice and humans. Upon initial examination, the effective dosage level of 1,000 mg/ kg per day for indinavir in the mice seems overly high until such an adjustment is made and the very high human daily dosage of 2.4 g (800 mg three times a day) is taken into consideration. Predictiveness of the SCID-hu Thy/Liv model for the behavior of HIV-1 in humans can be extended beyond antiretroviral efficacy to simple kinetics of viral replication and virus-mediated cytopathicity. REFERENCES 1. McCune JM, Namikawa R, Kaneshima H, Shultz LD, Lieberman M, et al. (1988) The SCID-hu mouse: murine model for the analysis of human hematolymphoid differentiation and function. Science 241: 1632–1639. 14. Stanley SK, McCune JM, Kaneshima H, Justement JS, Sullivan M, et al. 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Annu Rev Immunol 9: 399–429. 19. Keir ME, Stoddart CA, Linquist-Stepps V, Moreno ME, McCune JM (2002) IFN-alpha secretion by type 2 predendritic cells up-regulates MHC class I in the HIV-1-infected thymus. ACKNOWLEDGMENTS We thank Sandra Bridges for longstanding support of the SCID-hu Thy/ Liv model, Opendra Sharma for supplying multiple-gram quantities of antiretroviral drugs, Tim Schmidt for performing p24 ELISA, and Mike McCune for helpful discussions. Author Contributions Conceived and designed the experiments: CS PB. Performed the experiments: CB JB GC SG AK MM JR RR BS. Analyzed the data: CS MM RR. Wrote the paper: CS. Conceived and designed the experiments: CS PB. Performed the experiments: CB JB GC SG AK MM JR RR BS. Analyzed the data: CS MM RR. Wrote the paper: CS. DISCUSSION AZT-resistant (JD) and MDR NY index case R5X4 clinical isolates replicate and cause thymocyte depletion with kinetics comparable to HIV-1 NL4-3. The data presented here show that drug-resistant clinical isolates can readily be used as challenge viruses in the SCID-hu Thy/Liv model for in vivo studies of new classes of antiretroviral drugs with activity against HIV-1 that is resistant to currently licensed antiretrovirals. In the experiments presented here, the second-generation antiretrovirals in each drug class were more potent than their first-generation predecessors. Thus, (–)-FTC was more potent than 3TC, the nonnucleoside RT inhibitor efavirenz was more potent than nevirapine, and the protease inhibitor atazanavir was more potent than indinavir. The greater potency of the newer drugs in SCID-hu Thy/Liv mice is consistent with their observed greater potency in HIV-1-infected individuals. The nonnucleoside RT inhibitor (–)-FTC has greater potency than 3TC because the (–)- FTC-triphosphate is incorporated 10 times more efficiently than 3TC-triphosphate during HIV-1 RT-catalyzed RNA-dependent DNA synthesis [53]. 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Computational tools for clinical support: a multi-scale compliant model for haemodynamic simulations in an aortic dissection based on multi-modal imaging data
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Keywords: y patient-specific simulation, aortic dissection, computational fluid dynamics, fluid–structure interaction, Windkessel model, moving boundary Computational tools for clinical support: a multi-scale compliant model for haemodynamic simulations in an aortic dissection based on multi-modal imaging data MB, 0000-0001-5548-3298; SB, 0000-0002-6287-1106; JPG, 0000-0002-2861-0914 MB, 0000-0001-5548-3298; SB, 0000-0002-6287-1106; JPG, 0000-0002-2861-0914 Aortic dissection (AD) is a vascular condition with high morbidity and mortality rates. Computational fluid dynamics (CFD) can provide insight into the progression of AD and aid clinical decisions; however, oversimplified modelling assumptions and high computational cost compromise the accuracy of the information and impede clinical translation. To overcome these limit- ations, a patient-specific CFD multi-scale approach coupled to Windkessel boundary conditions and accounting for wall compliance was developed and used to study a patient with AD. A new moving boundary algorithm was implemented to capture wall displacement and a rich in vivo clinical dataset was used to tune model parameters and for validation. Comparisons between in silico and in vivo data showed that this approach successfully captures flow and pressure waves for the patient-specific AD and is able to predict the pressure in the false lumen (FL), a critical variable for the clinical management of the condition. Results showed regions of low and oscillatory wall shear stress which, together with higher diastolic pressures predicted in the FL, may indi- cate risk of expansion. This study, at the interface of engineering and medicine, demonstrates a relatively simple and computationally efficient approach to account for arterial deformation and wave propagation phenom- ena in a three-dimensional model of AD, representing a step forward in the use of CFD as a potential tool for AD management and clinical support. http://dx.doi.org/10.1098/rsif.2017.0632 Received: 30 August 2017 Accepted: 13 October 2017 Received: 30 August 2017 Accepted: 13 October 2017 Subject Category: Life Sciences–Engineering interface Subject Areas: biomedical engineering, biomechanics Keywords: patient-specific simulation, aortic dissection, computational fluid dynamics, fluid–structure interaction, Windkessel model, moving boundary Authors for correspondence: Mirko Bonfanti e-mail: mirko.bonfanti.15@ucl.ac.uk Vanessa Dı´az-Zuccarini e-mail: v.diaz@ucl.ac.uk Electronic supplementary material is available online at https://dx.doi.org/10.6084/m9. figshare.c.3917929. Mirko Bonfanti1, Stavroula Balabani1, John P. Greenwood2,3, Sapna Puppala3, Shervanthi Homer-Vanniasinkam1,3,4 and Vanessa Dı´az-Zuccarini1 Cite this article: Bonfanti M, Balabani S, Greenwood JP, Puppala S, Homer- Vanniasinkam S, Dı´az-Zuccarini V. 2017 Computational tools for clinical support: a multi-scale compliant model for haemodynamic simulations in an aortic dissection based on multi-modal imaging data. J. R. Soc. Interface 14: 20170632. 1Department of Mechanical Engineering, University College London, Torrington Place, London WC1E 7JE, UK 2Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds LS2 9JT, UK 3Leeds Teaching Hospitals NHS Trust, Leeds LS1 3EX, UK g p 4University of Warwick Medical School & University Hospitals Coventry and Warwickshire NHS Trust, Coventry CV2 2DX, UK Authors for correspondence: Mirko Bonfanti e-mail: mirko.bonfanti.15@ucl.ac.uk Vanessa Dı´az-Zuccarini e-mail: v.diaz@ucl.ac.uk Authors for correspondence: Mirko Bonfanti e-mail: mirko.bonfanti.15@ucl.ac.uk Vanessa Dı´az-Zuccarini e-mail: v.diaz@ucl.ac.uk Electronic supplementary material is available online at https://dx.doi.org/10.6084/m9. figshare.c.3917929. & 2017 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited. 1. Introduction Computational fluid dynamics (CFD) has provided significant insight into the haemodynamics of many cardiovascular pathologies, being particularly amen- able to study aortic dissection (AD) due to its complexity and patient-specific nature [1]. AD is characterized by the separation of the layers of the aortic wall. A tear in the intima layer allows blood to flow within the aortic wall inducing the formation of two flow channels, the true (TL) and false lumen (FL), separated by an intimal flap (IF) [2]. Diagnosis, management and treatment of AD are patient specific and difficult; experts claim that ‘difficulty in diagnosis, delayed diagnosis or failure to diagnose are so common as to approach the norm for this disease, even in the best hands. . .’ [3]. Initial management of acute AD focuses on pain control, heart rate and blood pressure management, followed by surgical intervention, typically involving stenting of the entry tear. The supra-aortic branches (left and right subclavian arteries (LSA, RSA), left and right common carotid arteries (LCC, RCC)) and the main visceral branches (coeliac trunk (CT), superior mesenteric artery (SMA), left and right renal arteries (LRA, RRA)) are included. (c) Flow-rate curves at different locations extracted from 2D PC-MRI data: (A) ascending aorta; (B) RCC and LCC; (C) aortic arch, distal to the LSA; (D,E) flow rate in the TL of the dissected aorta at two sections along the descending aorta, above and below the diaphragm, respectively; (F) AbAo, proximal to the iliac bifurcation. Although type B dissections (i.e. AD involving only the des- cending aorta) have lower initial mortality than type A (i.e. AD of the ascending aorta), they carry a poor long-term prognosis, with late-term complications reported in 20–50% of cases within 5 years [4]. the fluid flow by the vessel wall motion and vice versa. In FSI, high uncertainties regarding the wall thickness and mechanical properties of the dissected vessel remain [13], complicating their application to AD studies. Nonetheless, recent FSI work by our group [14] showed that wall motion has an impact on clinically relevant haemodynamic parameters for AD, and thus should be accounted for. Although a reasonable match between computational and experimental data has been found in recent attempts to validate simulation results against in vivo phase-contrast magnetic resonance imaging (PC-MRI) [5,11], discrepancies attributed to a rigid-wall assumption remained. Hence, the need for reliable compliant models of AD is evident. Detailed characterization of complex intra-aortic haemo- dynamic parameters via CFD, which currently cannot be determined in vivo, has the potential to aid clinical decision- making around AD; for instance, by identifying those prone to adverse outcomes and supporting clinicians by simulating different interventional strategies [5–7]. A number of computational studies on AD have been published in the last decade [8]. CFD modelling approaches differ significantly across studies, especially regarding the boundary conditions (BCs) and the treatment of the wall. As the simulation of the whole vascular system is unpractical and patient specific, time-varying pressure and flow wave- forms at all termination branches are usually not available, different strategies have been developed, e.g. via the coupling of zero-dimensional (0D) Windkessel models to the outlets of a three-dimensional (3D) domain, forming together a multi- scale model (referring to the combination of models of different dimensions) representing the vascular system [9]. CT data geometry RCC LCC B LSA RSA C entry tear (18.5 mm2) true lumen true lumen false lumen false lumen intimal flap inlet D B A A C D, E F F E 600 flow (ml s–1) inlet 0 0.2 0.4 0.6 0.8 0 0.2 0.4 0.6 0.8 0 0.2 0.4 0.6 0.8 0 0.2 0.4 0.6 0.8 0 0.2 0.4 t (s) 0.6 0.8 RCC LCC aortic arch plane D-TL plane E-TL AbAo AbAo LRA CT RRA SMA 400 200 0 40 20 0 400 200 –200 0 400 200 200 100 –100 0 –200 0 2D PC-MRI (a) (b) (c) Figure 1. (a) Rendering of the CT data showing the dissected aorta. (b) Three-dimensional model extracted from CT data representing the dissected aorta from the ascending aorta (inlet) to the abdominal aorta (AbAo). The supra-aortic branches (left and right subclavian arteries (LSA, RSA), left and right common carotid arteries (LCC, RCC)) and the main visceral branches (coeliac trunk (CT), superior mesenteric artery (SMA), left and right renal arteries (LRA, RRA)) are included. (c) Flow-rate curves at different locations extracted from 2D PC-MRI data: (A) ascending aorta; (B) RCC and LCC; (C) aortic arch, distal to the LSA; (D,E) flow rate in the TL of the dissected aorta at two sections along the descending aorta, above and below the diaphragm, respectively; (F) AbAo, proximal to the iliac bifurcation. B A C D, E F 600 flow (ml s–1) inlet 0 0.2 0.4 0.6 0.8 0 0.2 0.4 0.6 0.8 0 0.2 0.4 0.6 0.8 0 0.2 0.4 0.6 0.8 0 0.2 0.4 t (s) 0.6 0.8 RCC LCC aortic arch plane D-TL plane E-TL AbAo 400 200 0 40 20 0 400 200 –200 0 400 200 200 100 –100 0 –200 0 2D PC-MRI (c) geometry RCC LCC B LSA RSA C entry tear (18.5 mm2) true lumen false lumen intimal flap inlet D A F E AbAo LRA CT RRA SMA (b) CT data true lumen false lumen (a) (a) A AbAo Figure 1. (a) Rendering of the CT data showing the dissected aorta. (b) Three-dimensional model extracted from CT data representing the dissected aorta from the ascending aorta (inlet) to the abdominal aorta (AbAo). This study aims to tackle some of these challenges through the development of a novel framework for patient- specific simulations, combining multi-scale and compliant AD models as well as dynamic BCs. A flexible and robust method for tuning Windkessel parameters using non-inva- sive patient-specific clinical data is presented. Moreover, the rigid-wall assumption is addressed through the development of a novel and computationally efficient approach to account for the vessel wall motion in CFD simulations. Simulation results are validated against a rich and unique patient- specific clinical dataset from multiple imaging modalities such as two-dimensional (2D) PC-MRI, 2D cine-MRI and computed tomography (CT) scans. However, this coupling is relatively challenging, needing to be handled appropriately and Windkessel parameters must be accurately tuned. With the exception of a few studies [2,5], most often simpler BCs are adopted in AD studies, such as flow-split [10], constant zero-pressure [11] or pressure waveforms taken from the literature [12]. rsif.royalsocietypublishing.org The Navier–Stokes (NS) and continuity equations for 3D time- dependent flows were solved with finite-volume-based CFD solver ANSYS-CFX 17.0 (ANSYS Inc., PA, USA). The Navier–Stokes (NS) and continuity equations for 3D time- dependent flows were solved with finite-volume-based CFD solver ANSYS-CFX 17.0 (ANSYS Inc., PA, USA). Blood was modelled as incompressible with density ¼ 1056 kg m23 and non-Newtonian viscosity described by the Carreau–Yasuda model with parameters taken from Gijsen et al. [16]. Blood flow was considered as laminar, a common assumption in large arteries [2,12]. BCs applied to the fluid boundaries are shown schematically in figure 2a. In particular, uniform velocity pro- files were prescribed at the inlet and LCC and RCC boundaries for which patient-specific flow-rate waveforms were available from PC-MRIdata(i.e.QIN(t),QLCC(t),QRCC(t),figure1c).Thecorrespond- ing mean Reynolds and Womersley numbers, based on the inlet diameter of the reconstructed aorta, were equal to 1408 and 23, respectively. The peak Reynolds number was 5275, which is below the critical Reynolds number for transition to turbulence, based on a dynamic viscosityof 4  1023 Pa s and following Peacocket al. [17]. 2.2.2. MRI data q g p The NS equations were spatiallyand temporally discretized with a high-resolution advection scheme [20] and a second-order implicit backward Euler scheme, respectively, using a uniform time step of 1 ms, good enough for time-step size-independent results. At each anatomical location, a single slice was acquired, for each of the two pulse sequences. The total acquisition time for the whole MRI protocol was approximately 80 min. The lumen cross-sectional area (A) was quantified from 2D cine-MRI sequences using thresholding algorithms available in IMAGEJ (NIH, MD, USA). The normalized cross-sectional area variation (DA*) was calculated as 2.2. Clinical dataset 2.2.1. CT scans org J. R. Soc. Interface 14: 20170632 The entire aorta was imaged with a 16-slice CT scanner (Sensation 16; Siemens AG, Munich, Germany; 120 kV, 380 mA s, rotation time: 0.5 s, field of view (FOV): 284 mm, slice thickness: 1 mm, reconstruction kernel: B20F, contrast agent: Ultravist 300; Bayer AG, Leverkusen, Germany) obtaining 946 slices with in-plane resolution ¼ 0.55 mm and inter-slice distance ¼ 0.7 mm (figure 1a). g The geometry of the dissected aorta was extracted using semi-automated segmentation tools based on thresholding, implemented in ScanIP (Synopsys Inc., CA, USA). Smoothing operations were used on the resulting mask to reduce pixellation artefacts. The IF separating the FL from the TL was identified based on greyscale-value differences in the two lumina; however, because it was difficult to clearly resolve its thickness from CT images, it was approximated as a zero-thickness membrane for modelling purposes. The aortic branches were cropped perpen- dicularly to the vessel’s longitudinal axis to provide a flat surface at the boundaries. The resulting geometry is shown in figure 1b, representing the dissected aorta: from the ascend- ing aorta, just distal to the sinotubular junction of the aortic root, to the abdominal aorta (AbAo) just proximal to the iliac bifurcation [15]. y y g The remaining outlets of the 3D model were coupled to three-element Windkessel models (WK3s), shown as electrical analogues in figure 2a, inset. The flow curve obtained from PC-MRI at the AbAo was not applied as the outflow BC to avoid constraining the wave propagation. The flow (Q) and the mean pressure (P) over these boundaries are related by P ¼ (R1 þ R2)Q  R2C dP dt þ R1R2C dQ dt , ð2:2Þ ð2:2Þ where R1 and R2 represent the proximal and distal resistances, andC is the compliance of the distal vasculature. R1 is used to absorb the incoming waves and reduce artificial wave reflections, as shown by Alastruey et al. [18]. WK3s seem to be the best compromise among other physiologically relevant 0D outflow models to simulate the peripheral vasculature [19], and should be used—instead of purely resistance models—when a significant compliance is located in the modelled distal vasculature [18]. WK3 parameters must be tuned to obtain physiological flow/pressure waveforms. The tuning procedure is described in §2.3.3. 2.2.2. MRI data High spatial and temporal resolution electrocardiogram (ECG)- gated cine-imaging sequences (2D cine-MRI; slice thickness: 10 mm, repetition time: 3.1 ms, echo time: 1.5 ms, FOV: 375 mm, in-plane resolution: 1.1 mm, temporal resolution: 40 phases/cardiac cycle, steady-state free precession) were acquired with an Ingenia 1.5 T MRI scanner (Philips Healthcare, Amsterdam, The Nether- lands) at several sections perpendicular to the aorta (indicated in figure 1b). Additional slices (coronal plane) were acquired to detect IF motion in the distal part of the dissection. Pressure at the model outlet is updated at every solver loop according to equation (2.2), using as input the current flow rate calculated at the outlet of the 3D domain. Derivative terms were discretized with a first-order backward Euler approach using flow and mean pressure values calculated over the boundary at the previous time step. A no-slip condition was applied; the IF was assumed to be rigid. The external aortic wall was allowed to expand and con- tract with pressure fluctuations using a moving boundary (MB) technique accounting for aortic compliance, as described in §2.3.2. A no-slip condition was applied; the IF was assumed to be rigid. The external aortic wall was allowed to expand and con- tract with pressure fluctuations using a moving boundary (MB) technique accounting for aortic compliance, as described in §2.3.2. The NS equations were spatiallyand temporally discretized with a high-resolution advection scheme [20] and a second-order implicit backward Euler scheme, respectively, using a uniform time step of 1 ms, good enough for time-step size-independent results. Additionally, through-plane phase-contrast velocity mapping images (2D PC-MRI) were acquired at the same locations for flow quantification (slice thickness: 6 mm, repetition time: 17 ms, echo time: 1.9 ms, FOV: 400 mm, in-plane resolution: 1.56 mm, temporal resolution: 40 phases/cardiac cycle, velocity encoding: 200 cm s21). At each anatomical location, a single slice was acquired, for each of the two pulse sequences. The total acquisition time for th h l MRI t l i t l 80 i Additionally, through-plane phase-contrast velocity mapping images (2D PC-MRI) were acquired at the same locations for flow quantification (slice thickness: 6 mm, repetition time: 17 ms, echo time: 1.9 ms, FOV: 400 mm, in-plane resolution: 1.56 mm, temporal resolution: 40 phases/cardiac cycle, velocity encoding: 200 cm s21). 2.3.1. Flow model and boundary conditions rsif.royalsocietypublishing.org 2.1. Patient information High complexity and the computational demands of fluid– structure interaction (FSI) simulations means a rigid-wall approximation is widely used, neglecting the effects exerted on Data from a 77-year-old man with a chronic Stanford type B AD were studied. The dissection originated approximately 40 mm 2.3. Computational simulation set-up 3 distal to the left subclavian artery, extended throughout the length of the descending aorta and terminated about 10 mm distal to the coeliac trunk. From CT scans (figure 1a), one entry tear (area approx. 18.5 mm2) was located approxi- mately 10 mm distal to the proximal end of the dissection; no other communication between the TL and FL was evident from CT data, confirmed by a reduced flow in the FL observed in PC-MRI data. 2.3. Computational simulation set-up 2.3.1. Flow model and boundary conditions 2.3.2. Moving boundary method An MB algorithm was employed to account for the compliance of the aorta, capturing wave transmission effects. Details are pre- sented in Bonfanti et al. [21] and summarized here. The motion of the ascending aorta due to the heart motion has been disregarded. DA ¼ A  A0 A0 , ð2:1Þ ð2:1Þ Assuming that the displacement of the aortic wall follows the local surface-normal direction and is linearly related to the fluid pressure (figure 2a, inset), the displacement dn (m) of each mesh node n on the external vessel wall is calculated as where A0 is the minimal cross-sectional area measured during a cardiac cycle. Velocity information was extracted from the PC-MRI sequences using the GTFlow software (GyroTools LCC, Zu¨rich, Switzerland); flow-rate curves measured at different sections are reported in figure 1c. Flow curves A and B are used as BCs, whereas C–F are used for validation. A heart rate of 75 bpm and a stroke volume of 107.6 ml were reported, corresponding to a cardiac output of 8.1 l min21. dn ¼ pn  pext Kn nn, ð2:3Þ dn ¼ pn  pext Kn nn, ð2:3Þ ð2:3Þ where pn (Pa) is the pressure at node n, pext (Pa) is the external pressure set as equal to the mean diastolic pressure over the aortic wall (in this study 76 mmHg), nn is the unit normal vector in the multi-scale model 0D model tuning procedure 0D model: vascular system pressure (x, t) 0D model: rigid aorta + peripheral circulation target: Q– outlet target: Psys, Pdia Csys Ci, R1 i, R2 i " WK3 Ci, R1 i, R2 i " WK3 multi-scale model: 3D rigid aorta + 0D peripheral circulation aortic compliance estimate 2D-MRI data vessel-area variation multi-scale model: 3D compliant aorta + 0D peripheral circulation input: QIN(t) QRCC(t) QLCC(t) 1 2 3 4 WK3 RSA WK3 RRA WK3 CT WK3 CT WK3 SMA WK3 RRA WK3 AbAo WK3 LRA LR8 LR9 LR10 R2 R1 C LR12 LR11 LR L R WK3 WK3-AbAo 0 0.3 0.7 1.0 1.4 CA (mm2 mmHg–1) WK3 LRA WK3 SMA WK3 LSA WK3 LSA LR3 LR4 LR2 LR1 LR7 LR5 LR6 WK3 RSA QRCC QRCC QIN QLCC QLCC QIN A A section A-A dn dn = pn pn – pext Kn pext n (a) (b) (c) nn nn Figure 2. (a) Schematic of the multi-scale model domain and its BCs. 2.3.2. Moving boundary method WK3 indicates the Windkessel models coupled at the outlets (see inset). The colour map shows the area compliance of the aorta (CA ¼ DA/DP), estimated with patient-specific 2D cine-MRI data. The inset shows a cross-section of the ascending aorta and a schematic depicting how the displacement of a node n on the vessel wall is related to the pressure pn calculated in the fluid domain according to the MB method. (b) Schematic of the 0D model (lumped-parameters model) used during the tuning procedure. LR indicates the elementary building block, composed by an inertance (L) and a resistance (R) (see inset), representing a vessel segment: LR1: ascending aorta and arch, LR2: brachiocephalic trunk, LR3: right subclavian artery, LR4: right common carotid, LR5: left common carotid, LR6: left subclavian artery, LR7: descending aorta, LR8: coeliac trunk, LR9: superior mesenteric artery, LR10: right renal artery, LR11: left renal artery, LR12: abdominal aorta. (c) Flowchart of the procedure adopted to tune the parameters of the multi-scale model. Steps 1–4 are described in more detail in the text. tuning procedure 0D model: vascular system pressure (x, t) 0D model: rigid aorta + peripheral circulation target: Q– outlet target: Psys, Pdia Csys Ci, R1 i, R2 i " WK3 Ci, R1 i, R2 i " WK3 multi-scale model: 3D rigid aorta + 0D peripheral circulation aortic compliance estimate 2D-MRI data vessel-area variation multi-scale model: 3D compliant aorta + 0D peripheral circulation input: QIN(t) QRCC(t) QLCC(t) 1 2 3 4 WK3 LRA 11 (c) (c) tuning procedure 0D model multi-scale model (a) 0D model: vascular system .org J. R. Soc. Interface 14: 20170632 2D-MRI data vessel-area variation Figure 2. (a) Schematic of the multi-scale model domain and its BCs. WK3 indicates the Windkessel models coupled at the outlets (see inset). The colour map shows the area compliance of the aorta (CA ¼ DA/DP), estimated with patient-specific 2D cine-MRI data. The inset shows a cross-section of the ascending aorta and a schematic depicting how the displacement of a node n on the vessel wall is related to the pressure pn calculated in the fluid domain according to the MB method. (b) Schematic of the 0D model (lumped-parameters model) used during the tuning procedure. 2.3.2. Moving boundary method The ratio R1-to- Rtot (with Rtot¼ R1 þ R2) was set following Les et al. [23], and Ri tot was calculated as Pi= Qi, where Pi is the mean pressure at WK3 i obtained with the 0D model. Ri tot was adjusted to obtain the target flow distribution. Th 0D d l i l d i h b k d Mesh displacement equations were solved so as to obtain an implicit two-way coupling between mesh motion and fluid dynamics. The compliance Ci was calculated by distributing Csys among each WK3 i proportionally to Qi. The ratio R1-to- Rtot (with Rtot¼ R1 þ R2) was set following Les et al. [23], and Ri tot was calculated as Pi= Qi, where Pi is the mean pressure at WK3 i obtained with the 0D model. Ri tot was adjusted to obtain the target flow distribution. 2.3.2. Moving boundary method LR indicates the elementary building block, composed by an inertance (L) and a resistance (R) (see inset), representing a vessel segment: LR1: ascending aorta and arch, LR2: brachiocephalic trunk, LR3: right subclavian artery, LR4: right common carotid, LR5: left common carotid, LR6: left subclavian artery, LR7: descending aorta, LR8: coeliac trunk, LR9: superior mesenteric artery, LR10: right renal artery, LR11: left renal artery, LR12: abdominal aorta. (c) Flowchart of the procedure adopted to tune the parameters of the multi-scale model. Steps 1–4 are described in more detail in the text. outwarddirectionandKn (N m23) is a measure of thewall stiffness at the location of node n. Under the hypothesis of a circular cross- section, Kn can be related to the vessel areacompliance CA as follows: (1) A WK3 analogue of the entire vascular system was used to determine the total system Csys, following the method used by Les et al. [23]. WK3 parameters Csys, Rsys 1 and Rsys 2 (Rsys 1 =ðRsys 1 þ Rsys 2 Þ ¼ 5:6% [23]) were iteratively varied to obtain the target Psys and Pdia values, using as input QIN(t). The obtained Csys was equal to 0.99 ml mmHg21. outwarddirectionandKn (N m23) is a measure of thewall stiffness at the location of node n. Under the hypothesis of a circular cross- section, Kn can be related to the vessel areacompliance CA as follows: Kn ¼ 2 CA ffiffiffiffiffiffiffiffiffi pA0 p , ð2:4Þ ð2:4Þ where A0 is the vessel cross-sectional area at the location of node n. CA can be estimated using patient-specific 2D cine-MRI sequences, as described in §2.3.3. (2) A 0D model representing the vascular system (figure 2b) was employed to tune R1, R2 and C of each WK3 coupled to the 3D aorta, considered rigid at this stage. The aorta was divided into segments modelled as 0D building blocks made by an inertance and a resistance, as shown in figure 2b. (2) A 0D model representing the vascular system (figure 2b) was employed to tune R1, R2 and C of each WK3 coupled to the 3D aorta, considered rigid at this stage. The aorta was divided into segments modelled as 0D building blocks made by an inertance and a resistance, as shown in figure 2b. The compliance Ci was calculated by distributing Csys among each WK3 i proportionally to Qi. 2.3.5. Mesh Th d The extracted AD geometry (figure 2) was discretized with ICEM- CFD (ANSYS Inc.), adopting a tetrahedral mesh in the core region and seven prism layers at the walls (external aortic wall and IF sides). The resulting mesh consisted of about 506 000 elements, comparable to the grids used in other CFD studies of the aorta [2,14]. In order to select the computational grid, a preliminary mesh independence study was carried out using the rigid model on two additional grids, with 267 000 and 1 045 000 elements. Com- parison between pressure and flow waves obtained at the outlets with the three meshes (coarse, medium and fine) showed a maxi- mum difference of 4.8% and 2.5% for flow and pressure, respectively, between the coarse and medium mesh, and only 1.1% and 0.7% when comparing the medium and fine mesh. Thus, the medium mesh was deemed good enough for simulation purposes. The effect of the mesh on the results was further ana- lysed for the compliant model on the coarse and medium grids. The results showed a maximum difference of 1.9% and 1.3% for the outlets’ flow and pressure waves, respectively. The obtained TAWSS and OSI distributions over the aortic wall compared well qualitatively (electronic supplementary material). The difference between the mean and peak TAWSS and OSI was quantified over selected regions of interest (i.e. areas around the entry tear and FL wall, as shown in the electronic supplementary material), resulting in maximum differences of 7.6% and 5.8% for the peak and mean TAWSS, respectively, and 6.0% and 10% for the peak and mean OSI, respectively. These differences give an upper bound to the errors introduced by using a medium, rather than a fine, mesh in the model. TL and FL cross-sectional areas were considered in the calculation of the distensibility, as in [24]. TL and FL cross-sectional areas were considered in the calculation of the distensibility, as in [24]. y The distensibility of the main aortic branches was calculated from the pressure wave velocity (PWV) as D ¼ r1  PWV2 using the empirical relationship between PWV (m s21) and the vessel diameter (d (mm)) from Reymond et al. [25], PWV ¼ a db , ð2:5Þ ð2:5Þ where a and b are 13.3 and 0.3. Finally, CA (figure 2a) was calculated as CA ¼ D  A0, where A0 is the local vessel cross-sectional area. 2.3.5. Mesh Th d By integrating CA along the vessel length, the compliance associated with the 3D aortic model was obtained (Caorta ¼ 0.41 ml mmHg21). g (4) The parameters of the WK3 models coupled to the compliant aorta needed to be re-tuned to obtain physiological pressure and flow waveforms. Only the compliance attributed to the peripheral circulation (Cper¼ Csys2 Caorta) was distributed among the WK3 models. Although Rtot was kept equal to the one estimated at step 2 for each WK3, R1 was set equal to r  PWV/A0 (i.e. the characteristic impedance of the coupled 3D vessel) in order to reduce the impedance mis- match between the 3D compliant model and the 0D WK3, and thus to minimize the intensity of the non-physiological waves reflected by the Windkessel models [18,25]. WK3 par- ameters used for the multi-scale model (compliant 3D aorta plus 0D WK3 models) are reported in table 2. rsif.royalsocietypublishing.org location mean flow, Q (ml s21) source/expression reference inlet 134.5 2D PC-MRI n.a. RCC 12.7 2D PC-MRI n.a. LCC 8.9 2D PC-MRI n.a. RSA/LSA 9.8 0:5  ðQIN  QRCC  QLCC  QDescÞa n.a. CT 20.7 0:330  ðQDesc  QAbAoÞa [22] SMA 14.0 0:223  ðQDesc  QAbAoÞa [22] RRA/LRA 14.0 0:223  ðQDesc  QAbAoÞa [22] AbAo 30.5 2D PC-MRI n.a. aQDesc ¼ 93:3 ml s1, mean flow at the descending aorta obtained from 2D PC-MRI data (plane E in figure 1). rsif.royalsocietypublishing.org org J. R. Soc. Interface 14: 20170632 CFD-Post (ANSYS Inc.) and Matlab (Mathworks, MA, USA). Time-averaged haemodynamic indices, such as time-averaged wall shear stress (TAWSS) and oscillatory shear index (OSI), were calculated according to Gallo et al. [26]. Table 2. RCR parameters used for the Windkessel models coupled to the 3D compliant model as a result of the tuning procedure. outlet R1 (mmHg s ml21) R2 (mmHg s ml21) C (ml mmHg21) RSA 1.660 9.977 0.051 LSA 1.518 10.110 0.051 CT 1.588 3.858 0.107 SMA 1.488 6.604 0.072 RRA 2.947 5.102 0.072 LRA 2.162 5.919 0.072 AbAo 0.115 3.608 0.157 3.1. Flow rate and pressure waveforms Comparisons between target pressure and mean flow values and simulation results are shown via histograms in figure 3 with good agreement between the two datasets (maximum difference of 1.8%). Flow rates and pressure waveforms obtained with the multi-scale compliant model at the inlet and at selected outlets are shown in figure 3. Pressure waveforms from the aortic root (inlet) to the periphery (AbAo) exhibit typical physiological features, such as peripheral amplification of systolic and pulse pressures, and increase in the wave-foot time delay 2.3.3. Model tuning based on patient-specific data The parameters of the combined 3D aortic model and 0D WK3 models were specifically tuned for the simulated patient. R1, R2 and C were specified for each WK3 to characterize the peripheral circulation, while CA needed to be estimated to mimic the compliance of the aortic model. The 0D model equations were solved with a backward- differentiation scheme using the software 20-sim (Controllab Products B.V., Enschede, The Netherlands). The aim of the tuning procedure was to obtain physiological values for the mean flow rate at the outlets ð QiÞ, and target systolic (Psys) and diastolic (Pdia) blood pressure at the inlet. (3) (3) A CFD simulation (rigid 3D aorta plus 0D WK3 models) was run using the WK3 parameters from step 2. (3) A CFD simulation (rigid 3D aorta plus 0D WK3 models) was run using the WK3 parameters from step 2. The systolic–diastolic pressure variation (DP, obtained from CFD simulations) and DA* (extracted from 2D cine- MRI sequences—equation (2.1)) were used to estimate the distensibility of various aortic segments (aortic arch, ascend- ing, descending and abdominal aorta) as D½Pa1 ¼ DA=DP. In the aortic segments presenting the dissection, both the (3) A CFD simulation (rigid 3D aorta plus 0D WK3 models) was run using the WK3 parameters from step 2. Patient-specific brachial Psys and Pdia (150 and 80 mmHg, respectively) were used as pressure target values. Target values for Qi were set based on available PC-MRI and literature data, as summarized in table 1. The systolic–diastolic pressure variation (DP, obtained from CFD simulations) and DA* (extracted from 2D cine- MRI sequences—equation (2.1)) were used to estimate the distensibility of various aortic segments (aortic arch, ascend- ing, descending and abdominal aorta) as D½Pa1 ¼ DA=DP. In the aortic segments presenting the dissection, both the The workflow illustrated in figure 2c was followed, as described below. Table 1. Mean blood flow at the aortic branches used for tuning the Windkessel parameters. Table 1. Mean blood flow at the aortic branches used for tuning the Windkessel parameters. 5 5 2.3.4. Numerical simulations and post-processing (Online version in colour.) RCC LCC LCC RRA LSA 40 20 0 inlet CT RRA SMA LRA AbAo RSA mean: 115 mmHg 154/76 mmHg mean: 111 mmHg 157/74 mmHg mean: 8.9 ml s–1 peak: 26.6 ml s–1 mean: 13.8 ml s–1 peak: 29.4 ml s–1 Q (ml s–1) Qmean (ml s–1) P (mmHg) 160 120 80 40 20 0 Q (ml s–1) P (mmHg) Pinlet (mmHg) 160 120 80 40 30 20 10 0 0 0.2 0.4 0.6 0.8 0 0.2 0.4% 0% 1.1% 0.2% 1.4% 0.7% –1.8% Sys target values CFD RSA RCC LCC LSA CT SMA RRA LRA AbAo Dia 150 80 0 –0.4% –0.9% –1.6% –1.0% 0.4 0.6 0.8 t (s) 0 0.2 0.4 0.6 0.8 0 0.2 0.4 0.6 0.8 t (s) c) (d) (f) (e) LCC RRA 40 20 0 mean: 115 mmHg 154/76 mmHg mean: 111 mmHg 157/74 mmHg mean: 8.9 ml s–1 peak: 26.6 ml s–1 mean: 13.8 ml s–1 peak: 29.4 ml s–1 Q (ml s–1) P (mmHg) 160 120 80 40 20 0 Q (ml s–1) P (mmHg) 160 120 80 0 0.2 0.4 0.6 0.8 0 0.2 0.4 0.6 0.8 t (s) 0 0.2 0.4 0.6 0.8 0 0.2 0.4 0.6 0.8 t (s) (d) (e) 600 inlet R AbAo mean: 134 ml s–1 mean: 116 mmHg 151/79 mmHg mean: 115 mmHg 168/74 mmHg peak: 510 ml s–1 mean: 30.9 ml s–1 peak: 157 ml s–1 400 200 Q (ml s–1) Q (ml s–1) P (mmHg) P (mmHg) 0 160 120 80 200 100 0 160 120 80 0 0.2 0.4 0.6 0.8 0 0.2 0.4 t (s) 0.6 0.8 0 0.2 0.4 t (s) 0.6 0.8 0 0.2 0.4 0.6 0.8 (a) (c (b) 6 (a) CT RRA SMA LRA AbAo Qmean (ml s–1) Pinlet (mmHg) 40 30 20 10 0 0.4% 0% 1.1% 0.2% 1.4% 0.7% –1.8% Sys target values CFD RSA RCC LCC LSA CT SMA RRA LRA AbAo Dia 150 80 0 –0.4% –0.9% –1.6% –1.0% (f) AbAo t (s) Figure 3. (a,b,d,e) Flow (Q) and pressure (P) waves obtained with CFD simulation at the inlet and selected outlets (c) of the compliant model. P is calculated as the average pressure over the boundary area. (f) Comparison between the flow and pressure target values used for the tuning procedure of the Windkessel parameters and CFD results. 2.3.4. Numerical simulations and post-processing The relative percentage difference between CFD results and target values are reported in the figure, showing a good agreement. (Online version in colour.) pressure velocity 1.00 0.75 0.50 0.25 0 0 1.3 2.5 3.8 >5.0 0 0.1 0.3 0.4 0.5 (m s–1) TAWSS (Pa) OSI 140 130 120 110 100 (mmHg) (a) (b) (c) (d) Figure 4. (a) Velocity magnitude at peak systole (t ¼ 0.12 s). Inset: streamlines passing through the entry tear at peak systole showing the blood flowing into the FL. (b) Colour map of the pressure at peak systole in the ascending aorta and aortic arch. An area of increased pressure can be noted in the proximal FL where the blood flow impinges on the vessel wall. (c) Colour map of the TAWSS. (d) Colour map of the OSI. 0 0.1 0.3 0.4 0.5 OSI (d) (c) (a) Figure 4. (a) Velocity magnitude at peak systole (t ¼ 0.12 s). Inset: streamlines passing through the entry tear at peak systole showing the blood flowing into the FL. (b) Colour map of the pressure at peak systole in the ascending aorta and aortic arch. An area of increased pressure can be noted in the proximal FL where the blood flow impinges on the vessel wall. (c) Colour map of the TAWSS. (d) Colour map of the OSI. 2.3.4. Numerical simulations and post-processing Simulations were run until reaching a periodic steady state. For the multi-scale model, this was achieved within three cardiac cycles after appropriate initialization; the last cycle was used for the analysis of results. Post-processing was done using 600 inlet RCC LCC LCC RRA LSA 40 20 0 inlet CT RRA SMA LRA AbAo RSA AbAo mean: 134 ml s–1 mean: 116 mmHg 151/79 mmHg mean: 115 mmHg 168/74 mmHg mean: 115 mmHg 154/76 mmHg mean: 111 mmHg 157/74 mmHg peak: 510 ml s–1 mean: 8.9 ml s–1 peak: 26.6 ml s–1 mean: 13.8 ml s–1 peak: 29.4 ml s–1 mean: 30.9 ml s–1 peak: 157 ml s–1 400 200 Q (ml s–1) Q (ml s–1) Qmean (ml s–1) Q (ml s–1) P (mmHg) P (mmHg) 0 160 120 80 P (mmHg) 160 120 80 40 20 0 Q (ml s–1) P (mmHg) Pinlet (mmHg) 160 120 80 40 30 20 10 0 200 100 0 160 120 80 0 0.2 0.4 0.6 0.8 0 0.2 0.4 0.6 0.8 0 0.2 0.4% 0% 1.1% 0.2% 1.4% 0.7% –1.8% Sys target values CFD RSA RCC LCC LSA CT SMA RRA LRA AbAo Dia 150 80 0 –0.4% –0.9% –1.6% –1.0% 0.4 0.6 0.8 0 0.2 0.4 t (s) t (s) 0 0.2 0.4 0.6 0.8 0 0.2 0.4 0.6 0.8 t (s) 0.6 0.8 0 0.2 0.4 t (s) 0.6 0.8 0 0.2 0.4 0.6 0.8 (a) (c) (d) (f) (e) (b) Figure 3. (a,b,d,e) Flow (Q) and pressure (P) waves obtained with CFD simulation at the inlet and selected outlets (c) of the compliant model. P is calculated as the average pressure over the boundary area. (f) Comparison between the flow and pressure target values used for the tuning procedure of the Windkessel parameters and CFD results. The relative percentage difference between CFD results and target values are reported in the figure, showing a good agreement. 3.3. Comparison between two-dimensional phase- contrast MRI data and computational fluid d i lt fl t i th di t d in the proximal part of the FL is observed; during systole, a net flow is drawn through the small entry tear into the FL due to the pressure gradient between the TL and the FL (figure 4b) and the dilation of the vessel caused by the increasing pressure. At peak systole, the flow is well organized throughout apart from the proximal FL, where a high-velocity jet-like flow, rolling up to form a vortex, is observed (figure 4a). y Figure 5 compares the blood flow rate measured with PC- MRI and obtained with the CFD model at four different locations: the arch (distally to the LCC), two sections of the TL in the descending aorta, and the abdominal aorta. Peak flow rates are underestimated by the CFD model, with a rela- tive difference between measured and computed flows of 0.1%, 215%, 222% and 29%, respectively. Overall, predicted waveforms agree with those measured in vivo, with a good synchronization between the two waves in the first three locations, even if a delay is present in the AbAo. Blood flowing through the entry tear impinges on the FL wall causing a localized pressure increase (figure 4b), poten- tially leading to further enlargement/rupture of the already structurally compromised FL wall. TAWSS and OSI distributions are shown in figure 4c,d. High TAWSS values (greater than 5 Pa) are observed in the region around the entry tear, which is coherent with the flow description presented previously. Very low TAWSS is seen in the medial/distal part of the FL due to the almost stagnant flow obtained here. 3.2. Aortic dissection haemodynamics (Online version in colour.) (b) (a) mean: 92.3 ml s–1; peak: 400.2 ml s–1 mean: 80.2 ml s–1; peak: 399.9 ml s–1 0.4 0.8 0 0.4 mean: 92.3 ml s–1; peak: 366.9 ml s–1 mean: 87.9 ml s–1; peak: 430.6 ml s–1 0.4 0.8 0 0.4 mean: 92.3 ml s–1; peak: 364.9 ml s–1 mean: 92.9 ml s–1; peak: 467.7 ml s–1 0.4 0.8 0 0.4 mean: 30.2 ml s–1; peak: 151.9 ml s–1 mean: 30.5 ml s–1; peak: 166.7 ml s–1 Figure 5. Comparison between computed and measured flow rates at different locations along the dissected aorta, as indicated in (a). (b) CFD results are presented with the solid lines, while 2D PC-MRI data are shown by the dashed lines. (Online version in colour.) 3.2. Aortic dissection haemodynamics (i.e. point of lowest pressure). The PWV in the aorta was evaluated with the foot-to-foot method (9.21 m s21), in conso- nance with PWV findings reported by Taviani et al. [27] for elderly people (i.e. 60–80 years). Flow characteristics at peak systole are illustrated in figure 4a,b. Figure 4a shows the velocity magnitude in the 3D domain. High blood velocities (greater than 1.0 m s21) can be seen in the TL, where the dissection causes a reduction in cross- sectional area, and in the coeliac trunk and renal arteries, due to low lumen area and high blood flow demand. Velocities in the FL are low in the medial/distal regions (less than 0.05 m s21), due to the absence of secondary communications between the TL and FL. Nonetheless, a region of high velocity In agreement with the literature [23], there is no backflow in the renal arteries, whereas a slight retrograde flow occurs at the abdominal aorta at early diastole. These results suggest that the coupled 3D/0D, compliant and finely tuned model can correctly represent the effects of wave propagation along the vessel. CFD PC-MRI data 400 mean: 92.3 ml s–1; peak: 400.2 ml s–1 mean: 80.2 ml s–1; peak: 399.9 ml s–1 mean: 92.3 ml s–1; peak: 366.9 ml s–1 mean: 87.9 ml s–1; peak: 430.6 ml s–1 mean: 92.3 ml s–1; peak: 364.9 ml s–1 mean: 92.9 ml s–1; peak: 467.7 ml s–1 mean: 30.2 ml s–1; peak: 151.9 ml s–1 mean: 30.5 ml s–1; peak: 166.7 ml s–1 200 0 0 0.4 0.8 0 0.4 0.8 0 0.4 0.8 0 0.4 t (s) 0.8 Q (ml s–1) 400 200 0 Q (ml s–1) 400 200 200 100 –100 0 0 Q (ml s–1) Q (ml s–1) prox. AbAo medial arch (a) (b) Figure 5. Comparison between computed and measured flow rates at different locations along the dissected aorta, as indicated in (a). (b) CFD results are presented with the solid lines, while 2D PC-MRI data are shown by the dashed lines. 3.4. Vessel wall displacement: comparison between computational fluid dynamics results and two-dimensional cine-MRI data Regions with moderate OSI are observed throughout the aorta (figure 4d); although the flow is well organized in systole, it is highly disorganized in diastole. OSI values in the aortic branches are low because the flow is anterograde throughout the cardiac cycle here. Instead, a very high OSI is reported in the distal part of the FL due to the flow wave’s biphasic nature in this region, caused by the alternating expansion and contraction of the FL during systole and diastole (as described in §3.6 for the compliant model). Blood pressure changes during the cardiac cycle drive aortic dilation and contraction. At peak systole, the maximum displacement relative to the undeformed (i.e. diastolic) con- figuration predicted by the simulations is localized in the ascending aorta (approx. 0.74 mm). Subsequently, the pressure peak reaches the AbAo at t ¼ 0.23 s, with a maximum vessel displacement 1.00 20 ascending aorta max DA%: 10.7% max DA%: 11.0% max DA%: 17.2% max DA%: 16.6% 10 DA% 0 0 0.4 abdominal aorta (s) 0.8 0 0.4 CFD 2D cine-MRI t = 0.12 s t = 0.23 s t = 0.78 s (s) 0.8 20 10 DA% 0 0.75 0.50 0.25 0 (mm) (a) (b) Figure 6. (a) Displacement of the vessel wall obtained with the computational model at peak systole (t ¼ 0.12 s), systolic deceleration phase (t ¼ 0.23 s) and end-diastole (t ¼ 0.78 s). (b) Percentage cross-sectional area variation (DA%) during a cardiac cycle at the level of the ascending and abdominal aorta: comparison between CFD results and 2D cine-MRI measurements. DA%¼ (A – A0)/A0, where A is the cross-sectional area of the vessel lumen and A0 is the lowest cross- sectional area of the vessel lumen throughout a cardiac cycle. (b) (a) 20 ascending aorta max DA%: 10.7% max DA%: 11.0% 10 DA% 0 0 0.4 (s) 0.8 max DA%: 17.2% max DA%: 16.6% abdominal aorta (s) 0 0.4 (s) 0.8 20 10 DA% 0 abdominal aorta t = 0.23 s t = 0.12 s t = 0.78 s Figure 6. (a) Displacement of the vessel wall obtained with the computational model at peak systole (t ¼ 0.12 s), systolic deceleration phase (t ¼ 0.23 s) and end-diastole (t ¼ 0.78 s). (b) Percentage cross-sectional area variation (DA%) during a cardiac cycle at the level of the ascending and abdominal aorta: comparison between CFD results and 2D cine-MRI measurements. 3.4. Vessel wall displacement: comparison between computational fluid dynamics results and two-dimensional cine-MRI data DA%¼ (A – A0)/A0, where A is the cross-sectional area of the vessel lumen and A0 is the lowest cross- sectional area of the vessel lumen throughout a cardiac cycle. variation of the transmural pressure across the IF (TMP ¼ PTL 2 PFL, where PTL and PFL represent the pressure in the TL and FL, respectively) at proximal and distal levels is shown in figure 7b. The TMP curves are compared with 2D-MRI sequences at the same locations showing the IF displacement during the cardiac cycle. The TMP is positive (i.e. pressure higher in the TL) during most of systole at both locations, reaching a value of about 30 mmHg, with the distal TMP curve delayed with respect to the proximal one. Instead, a negative TMP (i.e. pressure higher in the FL) is found throughout diastole with a minimum value around 210 mmHg. Comparison of TMP curves with MRI sequences shows that the IF displacement direction agrees well with the computed TMP: a positive TMP corresponds with the motion of the IF through the FL; conversely, a negative TMP relates to the motion of the IF through the TL, indicating reliability of the predicted pressure field while providing further validation of the methodology presented herein. expansion ¼ 1.23 mm (figure 6a). The computed maximum percentage cross-sectional area variation (DA%) is equal to 10.7% and 17.2% at the ascending and abdominal aorta, respectively, and is in agreement with the values extracted from 2D-MRI data (11.0% and 16.6%, respectively; figure 6b). Moreover, the predicted DA% waveforms compare well with the measured ones, suggesting that the MB method can reliably reproduce vessel expansion due to pressure changes during the cardiac cycle. The measured DA% waveform at the ascending aorta presents a peak at early diastole, absent in the computed one, that may be due to the dicrotic wave, which is not completely resolved by the simulation. The dicrotic notch in the pressure wave is caused by the abrupt change of blood flow due to the closure of the aortic valve. However, the flow-rate curve applied at the inlet of the CFD model, which was extracted from PC-MRI data, potentially affected by measurement inaccuracies, lacks such a rapid change at the beginning of the diastole, and thus the production of a dicrotic wave in the simulation may be affected. 3.5. True lumen–false lumen transmural pressure and intimal flap displacement Figure 8 compares the flow rate and pressure waves of a rigid versus a compliant model at a medial section of the dissection. It should be noted that, even if pressure values obtained by both models are of the same order of magnitude, the relative pressure in the two lumina follows an opposite trend: the com- pliant model predicts higher pressures during systole and lower pressures during diastole in the TL than in the FL, while the opposite is found with the rigid model. As previously discussed, the available clinical dataset indicates that the intimal flap displacement Pressure differences between the TL and the FL can provide insight into the actual state and probable progression of the dis- section but cannot usually be assessed in the clinic. For instance, high pressure in the FL can lead to further expansion, or even rupture of the FL, while causing a narrowing of the TL [4]. Simulated pressure distributions at peak systole (figure 7a) show higher pressure in the TL than in the FL. The temporal pressure 30 peak systole initial diastole end-diastole end-diastole TL IF IF TL FL 0.06 s 0.20 s 0.78 s 20 mm FL 0.10 s 0.34 s 0.78 s 10 mm 15 0 –10 30 15 0 –10 0 0.34 systolic acceleration systolic deceleration 0.80 (s) 0 0.34 0.80 (s) systole diastole TMP (mmHg) TMP (mmHg) 142 133 123 113 104 (mmHg) TMP = PTL – PFL * * (a) (b) Figure 7. (a) Pressure colour map at peak systole (t ¼ 0.12 s). (b) Comparison between the TMP across the IF obtained with the CFD compliant model during a cardiac cycle and the displacement of the IF observed from 2D cine-MRI data at different time instances (TMP ¼ PTL 2 PFL, where PTL and PFL are the pressures in the TL and FL averaged over a cross-section—indicated by an asterisk in the figure—located at the same level of the respective 2D cine-MRI slice). It can be noted that the direction of the IF displacement agrees well with the sign of the TMP: a positive TMP corresponds with the motion of the IF through the FL, whereas a negative TMP relates to the motion of the IF through the TL. (a) (b) 30 peak systole initial diastole end-diastole TL IF FL 0.10 s 0.34 s 0.78 s 10 mm 15 0 –10 0 0.34 0.80 ( ) TMP (mmHg) (b) TMP (mmHg) end-diastole IF TL FL 0.06 s 0.20 s 0.78 s 20 mm 30 15 0 –10 systolic acceleration systolic deceleration 0 0.34 0.80 (s) systole diastole TMP (mmHg) diastole Figure 7. (a) Pressure colour map at peak systole (t ¼ 0.12 s). intimal flap displacement (b) Comparison between the TMP across the IF obtained with the CFD compliant model during a cardiac cycle and the displacement of the IF observed from 2D cine-MRI data at different time instances (TMP ¼ PTL 2 PFL, where PTL and PFL are the pressures in the TL and FL averaged over a cross-section—indicated by an asterisk in the figure—located at the same level of the respective 2D cine-MRI slice). It can be noted that the direction of the IF displacement agrees well with the sign of the TMP: a positive TMP corresponds with the motion of the IF through the FL, whereas a negative TMP relates to the motion of the IF through the TL. correct pressure distribution is the one computed by the compliant model, not the rigid one. relative difference with the one computed by the compliant model (TAWSSC). In the proximal part of the FL, TAWSSR is sig- nificantly lower than TAWSSC. The OSI distributions obtained with the two models also differ considerably (figure 9b). Additionally, while the cycle mean flow rate (Q) is approxi- mately the same in both models, its distribution among the cardiac phases is different: the compliant model predicts a lower TL flow during systole and a higher TL flow during diastole than the rigid model. Owing to the aortic compliance, the vessel can store blood during systole and release it during diastole. Following this, 28 ml of blood is accumulated in the aorta during systole due to a pressure increase of 68 mmHg. Consequently, peak flow rates in the compliant model are less pronounced than those obtained with the rigid model. As expected, the flow rate in the FL computed by the rigid model is equal to zero throughout the cardiac cycle. On the other hand, the compliant model predicts a small but non-negligible net flow in the FL resulting from the alternate dilation and contraction of the FL during systole and diastole, respectively. 4. Discussion Although this approach is suit- able for rigid models with a single outlet, it is inadequate for multi-branched or deformable models as flow distribution among the vascular branches strongly depends on the down- stream vasculature. To overcome these limitations, 0D WK3s have been used as BCs in more advanced AD simulations [2,5]. aortic system [12], may be different in pathological cases. More- over, this imposes a constant flow diversion into each branch that can have a major impact on wall shear stress (WSS)- based indices, as shown by Gallo et al. [26]. Additionally, zero-pressure conditions are often used as outflow BCs at the abdominal aortic branches [11]. Although this approach is suit- able for rigid models with a single outlet, it is inadequate for multi-branched or deformable models as flow distribution among the vascular branches strongly depends on the down- stream vasculature. To overcome these limitations, 0D WK3s have been used as BCs in more advanced AD simulations [2,5]. The proposed MB method allowed us to account for aortic wall deformation and compliance in the CFD simulation. A linear, elastic relationship was assumed between wall displa- cement and fluid forces. Although it is well known that the stress–strain relationship of the vessel wall is generally non- linear, experimental evidence suggests that the assumption of a linear constitutive relation for the arterial wall is justified in the range of physiological pressures [28]. It has been shown that the effects of nonlinearity and viscoelasticity on pressure and flow waves are minor in the aorta [25,29]. Also, limited data availability on the viscoelastic properties of the arterial wall in the literature [25] implies that using more advanced con- stitutive relations would introduce additional complexity and parameter uncertainties. Finally, a rigid-wall approximation is commonly adopted. However, as shown in our previous work [14] and also con- firmed here, vessel wall motion has a significant impact on clinically relevant haemodynamic markers for AD. FSI methods, coupling CFD with finite-element modelling of the aortic wall, are subject to uncertainties regarding the mechanical properties ofthetissue, which remain unknown forthedissected aorta and may be patient specific [13]. The high computational cost of FSI simulations can also be prohibitive in the context of CFD models for medical support. Thus, simpler methods to account for wall motion in such applications are necessary. 4. Discussion The present work presents a significant advance of the cur- rent state of the art in CFD simulations of AD for clinical support. The vast majority of computational studies in the lit- erature are limited by simplified assumptions about the treatment of BCs and wall motion. For instance, despite the fact that the cardiac output is patient specific and known to be affected by medical treatment [10], due to the lack of in vivo data, flow waves of healthy sub- jects examined in published studies are commonly employed as inflow BCs [12]. Overly simplified assumptions are often made regarding outflow BCs. A flow split is commonly adopted to prescribe flow to the supra-aortic branches, by diverting 5% of the total inlet flow into each branch during the entire cardiac cycle [12]. This proportion, reported for the healthy human The dissimilar haemodynamics computed by the two models results in different TAWSS distributions; this is particu- larly true in the region around the entry tear, an important indicator of disease progression. Figure 9a shows the TAWSS distribution obtained with the rigid model (TAWSSR) and the 160 TL FL FL TL compliant model rigid model 140 120 P (mmHg) Q (ml s–1) 100 80 600 TL Q: 92.3 ml s–1 Q: 0 ml s–1 Qsys: 182.8 ml s–1 Qsys: 11.8 ml s–1 Qdia: 25.5 ml s–1 Qdia: –8.7 ml s–1 FL TL FL 400 200 0 Q (ml s–1) 600 400 200 0 0 0.34 systole diastole systole diastole (s) 0.80 0 0.34 (s) 0.80 medial plane false lumen false lumen true lumen true lumen 160 140 120 P (mmHg) 100 80 0 0.34 (s) 0.80 0 0.34 (s) 0.80 Q: 93.2 ml s–1 Qsys: 215.4 ml s–1 Qdia: 2.8 ml s–1 Q: 0 ml s–1 Qsys: 0 ml s–1 Qdia: 0 ml s–1 (a) (b) Figure 8. (a,b) Comparison between CFD results obtained with compliant and rigid models. TL results are presented with solid blue lines, whereas FL results are shown by dashed red lines. The pressure results are presented as the averaged values calculated over the cross-sections shown in the schematic in (a), whereas the flow results are calculated as the integral of the velocity over the same sections (a positive flow corresponds to fluid moving towards the abdominal aorta). Q, Qsys and Qdia are the mean flow rates calculated over the entire cardiac cycle, the systole and diastole phase, respectively. 4. Discussion (Online version in colour.) FL TL rigid model 160 140 120 P (mmHg) 100 80 0 0.34 (s) 0.80 160 TL FL compliant model 140 120 P (mmHg) 100 80 0 0.34 (s) 0.80 (b) compliant model 10 (b) (a) medial plane false lumen false lumen true lumen true lumen (a) Q (ml s–1) 600 TL Q: 92.3 ml s–1 Q: 0 ml s–1 Qsys: 182.8 ml s–1 Qsys: 11.8 ml s–1 Qdia: 25.5 ml s–1 Qdia: –8.7 ml s–1 FL 400 200 0 0 0.34 systole diastole (s) 0.80 (s) TL FL Q (ml s–1) 600 400 200 0 systole diastole 0 0.34 (s) 0.80 (s) Q: 93.2 ml s–1 Qsys: 215.4 ml s–1 Qdia: 2.8 ml s–1 Q: 0 ml s–1 Qsys: 0 ml s–1 Qdia: 0 ml s–1 Figure 8. (a,b) Comparison between CFD results obtained with compliant and rigid models. TL results are presented with solid blue lines, whereas FL results are shown by dashed red lines. The pressure results are presented as the averaged values calculated over the cross-sections shown in the schematic in (a), whereas the flow results are calculated as the integral of the velocity over the same sections (a positive flow corresponds to fluid moving towards the abdominal aorta). Q, Qsys and Qdia are the mean flow rates calculated over the entire cardiac cycle, the systole and diastole phase, respectively. (Online version in colour.) MRI. The parameters of the WK3 models coupled to 3D models of the dissected aorta were successfully set, and target values specified on mean flows at model outlets and on blood pressure at the inlet were met. The aortic wall distensibility/ compliance was estimated indirectly from cross-sectional vessel area and pressure changes at different sections along the aorta. At the level of the dissected descending aorta, a dis- tensibility of 0.42  1025 Pa21 was found; this is comparable to values reported by Ganten et al. [24] for a cohort of patients with chronic type B AD. This value is significantly lower than that for healthy patients of the same age [24]. aortic system [12], may be different in pathological cases. More- over, this imposes a constant flow diversion into each branch that can have a major impact on wall shear stress (WSS)- based indices, as shown by Gallo et al. [26]. Additionally, zero-pressure conditions are often used as outflow BCs at the abdominal aortic branches [11]. 4. Discussion other fully rigid AD models, where differences of up to 56% [5] and 28% [11] at peak flow rate are present. simulations. As an example, the computational time reported in our previous work for an FSI simulation of AD was 57 h per cycle [14]. In this context, the MB algorithm is much less compu- tationally expensive and easier to implement, and this is critical for clinical use. Moreover, the model can be easily tuned with patient-specific vessel motion data obtained non-invasively in the clinic (via 2D cine-MRI). This represents an advantage over previous AD studies using traditional FSI methods [14], where uncertainties related to material properties, which are often taken from the literature, can be considerable. The results also show that the use of a rigid-wall approxi- mation requires careful consideration. For instance, the TMP between the two lumina predicted by a rigid model does not agree with the magnetic resonance images. TMP is an important physiological variable that cannot be measured in vivo non-inva- sively and without risk for the patient, but can be predicted instead by CFD simulations; hence, accurate prediction is essential in order to use this parameter in the clinic. The compliant model pre- dicted higher systolic pressure in the TL, and higher diastolic pressure in the FL; these findings compare well with experimen- tal results obtained by Tsai et al. [30] with a model of a chronic typeBADlackingadistalre-entry-tear,similartotheonestudied here. Higher diastolicFL pressure may lead to an increased aortic wall tension and consequent risk of lumen expansion. Comparison between computed and measured blood flows at different sections in the descending aorta showed good agreement; modelling the compliance of the aortic wall allowed amplitude and phase discrepancies between measured and simulated waveforms observed in previous rigid AD studies [5] to be reduced. Therefore, it appears clear that introducing the compliance of the vessel wall in the CFD model allows a better replication of the in vivo fluid dynamics situation, and represents an improvement of this study compared with previous rigid AD models [2,5]. TAWSS is thought to be important for AD initiation, affecting growth and enlargement of the FL [2]. The high TAWSS found by the compliant model in the entry-tear region can be a predictor of further enlargement. 4. Discussion In this study, we developed a patient-specific, compliant CFD model of a chronic type B AD with patient-specific tuned dynamic BCs. A rich in vivo dataset comprising multiple non-invasive imaging modalities was used to inform and vali- date the computational model. A fast procedure for BC tuning and a new, efficient method to model the wall motion have been proposed to tackle some of the aforementioned challenges. The MB method allowed us to capture the essential phy- sics of arterial deformation and wave propagation, as shown in the Results section. The compliant model simulation took 13 h for one cardiac cycle on a desktop computer (Intel Xeon E5, 8 cores, 32 GB RAM) and this is a significant improvement over FSI The model was tuned using brachial blood pressure measurements and flow and displacement data obtained via TAWSS 0 1.3 2.5 3.8 >5.0 <–50 –25 0 25 >50 0 0.1 0.3 0.4 0.5 <–50 –25 0 25 >50 TAWSS: %diff rigid-compliant OSI OSI: %diff rigid-compliant (Pa) (a) (b) Figure 9. Haemodynamic indices in the ascending aorta and aortic arch obtained with the rigid CFD model, and comparison with compliant model results. (a) TAWSS obtained with the rigid model and percentage difference relative to the compliant model results, calculated as: %diff ¼ (TAWSSR 2 TAWSSC)/(5 (Pa)). (b) OSI obtained with the rigid model and percentage difference relative to the compliant model results, calculated as: %diff ¼ (OSIR 2 OSIC)/0.5 (where OSIR and OSIC are the OSI values for rigid and compliant, respectively). The percentage difference figures were normalized by values representing the range of the respective parameter distributions for better visualization. TAWSS 0 1.3 2.5 3.8 >5.0 <–50 –25 0 25 >5 TAWSS: %diff rigid-compliant (Pa) (a) (a) rsif.royalsocietypublishing.org >5.0 <–50 >5.0 <– Figure 9. Haemodynamic indices in the ascending aorta and aortic arch obtained with the rigid CFD model, and comparison with compliant model results. (a) TAWSS obtained with the rigid model and percentage difference relative to the compliant model results, calculated as: %diff ¼ (TAWSSR 2 TAWSSC)/(5 (Pa)). (b) OSI obtained with the rigid model and percentage difference relative to the compliant model results, calculated as: %diff ¼ (OSIR 2 OSIC)/0.5 (where OSIR and OSIC are the OSI values for rigid and compliant, respectively). The percentage difference figures were normalized by values representing the range of the respective parameter distributions for better visualization. 4. Discussion It has been recently reported by Doyle & Norman [4] that areas of low TAWSS may be correlated to regions of rapid local expansion in type B AD. In the present study, the medial and distal parts of the FL exhibit low TAWSS and high OSI, which may indicate elevated risk. It should be noted that there is still a difference between the CFD results and PC-MRI, which may be attributed to factors related to the in vivo data, such as inaccuracies in the segmenta- tion of the lumen contours on magnetic resonance images, noise and the spatial resolution of PC-MRI, and to the model limitations discussed below. The under-estimation of peak flow rates in the TL can affect the derived fluid-dynamic vari- ables, such as TAWSS and OSI, and potentially lead to an under-estimation of the WSS in this region. However, the dis- crepancies reported here are smaller than those obtained in 5. Conclusion This paper presents a novel, patient-specific multi-scale model- ling approach coupled to Windkessel BCs to study AD. The approach accounts for wall compliance in a computationally efficient manner not explored hitherto. Simulation results were compared with patient-specific clinical data as part of a Acknowledgement. The authors thank Prof. D. Rodney Hose for his useful suggestions. J. R. Soc. Interface 14: 20170632 vph-case.eu) and from the Leverhulme Trust through the Senior Research Fellowship ’Exploring the Unknowable Using Simulation: Structural Uncertainty in Multiscale Models’ (fellowship no. RF- 2015-482). Funding. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 642612, VPH-CaSE (www. vph-case.eu) and from the Leverhulme Trust through the Senior Research Fellowship ’Exploring the Unknowable Using Simulation: Structural Uncertainty in Multiscale Models’ (fellowship no. RF- 2015-482). Acknowledgement. The authors thank Prof. D. Rodney Hose for his useful suggestions. References 1. Morris PD et al. 2015 Computational fluid dynamics modelling in cardiovascular medicine. Heart 102, 18–28. (doi:10.1136/heartjnl-2015-308044) 2. Alimohammadi M, Agu O, Balabani S, Dı´az-Zuccarini V. 2014 Development of a patient-specific simulation tool to analyse aortic dissections: assessment of mixed patient-specific flow and pressure boundary conditions. Med. Eng. Phys. 36, 275–284. (doi:10.1016/j.medengphy. 2013.11.003) 3. Elefteriades JA, Barrett PW, Kopf GS. 2007 Litigation in nontraumatic aortic diseases—a tempest in the malpractice maelstrom. Cardiology 109, 263–272. (doi:10.1159/000107790) 4. Doyle BJ, Norman PE. 2016 Computational biomechanics in thoracic aortic dissection: today’s approaches and tomorrow’s opportunities. Ann. Biomed. Eng. 44, 71–83. (doi:10.1007/s10439-015- 1366-8) 5. Dillon-Murphy D, Noorani A, Nordsletten D, Figueroa CA. 2016 Multi-modality image- based computational analysis of haemodynamics in aortic dissection. Biomech. Model. Mechanobiol. 15, 857–876. (doi:10.1007/s10237-015-0729-2) 6. Menichini C, Cheng Z, Gibbs RGJ, Xu XY. 2016 Predicting false lumen thrombosis in patient-specific models of aortic dissection. J. R. Soc. Interface 13, 20160759. (doi:10.1098/rsif.2016.0759) 7. Alimohammadi M, Bhattacharya-Ghosh B, Seshadhri S, Penrose J, Agu O, Balabani S, Dı´az-Zuccarini V. 2014 Evaluation of the hemodynamic effectiveness of aortic dissection treatments via virtual stenting. approaches and tomorrow’s opportunities. Ann. Biomed. Eng. 44, 71–83. (doi:10.1007/s10439-015- 1366-8) 5. Dillon-Murphy D, Noorani A, Nordsletten D, Figueroa CA. 2016 Multi-modality image- based computational analysis of haemodynamics in aortic dissection. Biomech. Model. Mechanobiol. 15, 857–876. (doi:10.1007/s10237-015-0729-2) 6. Menichini C, Cheng Z, Gibbs RGJ, Xu XY. 2016 Predicting false lumen thrombosis in patient-specific models of aortic dissection. J. R. Soc. Interface 13, 20160759. (doi:10.1098/rsif.2016.0759) 7. Alimohammadi M, Bhattacharya-Ghosh B, Seshadhri S, Penrose J, Agu O, Balabani S, Dı´az-Zuccarini V. 2014 Evaluation of the hemodynamic effectiveness of aortic dissection treatments via virtual stenting. 1. Morris PD et al. 2015 Computational fluid dynamics modelling in cardiovascular medicine. Heart 102, 18–28. (doi:10.1136/heartjnl-2015-308044) Int. J. Artif. Organs 37, 753–762. (doi:10.5301/ijao. 5000310) approaches and tomorrow’s opportunities. Ann. Biomed. Eng. 44, 71–83. (doi:10.1007/s10439-015- 1366-8) approaches and tomorrow’s opportunities. Ann. Biomed. Eng. 44, 71–83. (doi:10.1007/s10439-015- 1366-8) Int. J. Artif. Organs 37, 753–762. (doi:10.5301/ijao. 5000310) 8. 8. Sun Z, Chaichana T. 2016 A systematic review of computational fluid dynamics in type B aortic dissection. Int. J. Cardiol. 210, 28–31. (doi:10. 1016/j.ijcard.2016.02.099) 5. 2. Alimohammadi M, Agu O, Balabani S, Dı´az-Zuccarini V. 2014 Development of a patient-specific simulation tool to analyse aortic dissections: assessment of mixed patient-specific flow and pressure boundary conditions. Med. Eng. Phys. 36, 275–284. (doi:10.1016/j.medengphy. 2013.11.003) 2. Alimohammadi M, Agu O, Balabani S, Dı´az-Zuccarini V. J. R. Soc. Interface 14: 20170632 J. R. Soc. Interface 14: 20170632 This study assumes that the IF behaves like a rigid zero- thickness membrane separating the two lumina asthe resolution of CT images did not allow the IF thickness to be estimated with reasonable accuracy. The IF thickness estimated from MRI slices was about 2 mm, which is small compared with the diameter of the aorta; hence, the use of this approximation was deemed appropriate. Moreover, the IF of chronic type B AD is expected to be stiff and not as compliant as in acute settings [30]; this was confirmed by the cine-MRI sequences showing relatively small displacement of the IF (approx. 3 mm maximum displace- ment in the proximal and distal parts, and less than 1 mm in the central segment of the flap). However, in the regions where the IF displacement is at its maximum, the fluid flow can be signifi- cantly affected by the flap motion, and the assumption of a rigid and zero-thickness IF may be the cause of the discrepancy observed between the computed and measured peak flow rates at the locations where the TL and FL coexist. Further work will examine the effect of IF motion on blood flow distribution and pressure in the TL and FL. Ethics. The clinical dataset was acquired as part of an ethically approved protocol (NHS Health Research Authority, ref: 12/YH/ 0551; Leeds Teaching Hospitals NHS Trust, ref: 788/RADRES/16); patient consent was obtained. Data accessibility. The patient-specific geometry of the dissected aorta used in the CFD model is available in .stl format on the Digital Repository Figshare: doi.org/10.6084/m9.figshare.5354359.v1 [15]. Additional supporting data are available as part of the electronic supplementary material. Authors’ contributions. M.B. conceived the study, carried out model devel- opment and numerical simulations, and drafted the manuscript; S.B. provided interpretation of results and helped to draft the manuscript; J.P.G. collected the MRI data; S.P. collected the CT scan data and pro- vided clinical input; S.H.-V. conceived the study and provided clinical input; V.D.-Z. conceived the study, provided resources and coordination, and helped to draft the manuscript. All authors revised the manuscript and gave final approval for publication. Competing interests. We declare we have no competing interests. Funding. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 642612, VPH-CaSE (www. rsif.royalsocietypublishing.org rsif.royalsocietypublishing.org 4.1. Limitations Owing to the difficulties in acquiring in vivo haemodynamic data using non-invasive methods, some model parameters were specified using non-patient-specific information; these include the R1-to-Rtot ratio used to calculate the WK3 parameters coupled to the aortic rigid model, and the popu- lation-based law (equation (2.5)) used to estimate the PWV in the aortic branches. Moreover, the flow distribution among the abdominal branches was specified based on data taken from the literature for healthy patients. Although this assump- tion holds for the subject studied in this paper, in which the visceral branches were not involved in the dissection, it may not be valid in general. For AD cases in which the flow into these arteries is expected to be impaired, it may be useful to acquire additional flow data at the coeliac and infrarenal regions to inform the model. However, for the patient studied, these assumptions allowed us to obtain physiological pressure and flow waves, consistent with the available clinical data. rich dataset from multiple non-invasive imaging modalities, comprising haemodynamics and vessel motion data, and cover- ing the entire extension of the dissection. This work, at the interface of medicine and engineering, was developed in close collaboration with clinicians, who have stressed that the relative good agreement achieved between simulations and the in vivo data demonstrates that the proposed approach can successfully capture the haemodynamics in a chronic type B AD and can potentially lead to a powerful decision-making tool for the clini- cal management of AD. For instance, the model can be used to simulate different interventional strategies (e.g. covering of the entry tear with a stent graft, fenestration of the IF) or medical treatments (e.g. b-blocker therapy) and analyse their effects on the fluid dynamics in the dissection (in particular pressure and blood flow in the FL). Further research from the authors will include the application of the tool on a small cohort of patients. This is work that is well under way with data already collected for a number of patients. 12 References 2014 Development of a patient-specific simulation tool to analyse aortic dissections: assessment of mixed patient-specific flow and pressure boundary conditions. Med. Eng. Phys. 36, 275–284. (doi:10.1016/j.medengphy. 2013.11.003) 5. Dillon-Murphy D, Noorani A, Nordsletten D, Figueroa CA. 2016 Multi-modality image- based computational analysis of haemodynamics in aortic dissection. Biomech. Model. Mechanobiol. 15, 857–876. (doi:10.1007/s10237-015-0729-2) 9. Alastruey J, Xiao N, Fok H, Schaeffter T, Alberto Figueroa C. 2016 On the impact of modelling assumptions in multi-scale, subject- specific models of aortic haemodynamics. J. R. Soc. Interface 13, 20160073. (doi:10.1098/rsif. 2016.0073) 6. Menichini C, Cheng Z, Gibbs RGJ, Xu XY. 2016 Predicting false lumen thrombosis in patient-specific models of aortic dissection. J. R. Soc. Interface 13, 20160759. (doi:10.1098/rsif.2016.0759) 6. Menichini C, Cheng Z, Gibbs RGJ, Xu XY. 2016 Predicting false lumen thrombosis in patient-specific models of aortic dissection. J. R. Soc. Interface 13, 20160759. (doi:10.1098/rsif.2016.0759) 3. Elefteriades JA, Barrett PW, Kopf GS. 2007 Litigation in nontraumatic aortic diseases—a tempest in the malpractice maelstrom. Cardiology 109, 263–272. (doi:10.1159/000107790) 7. Alimohammadi M, Bhattacharya-Ghosh B, Seshadhri S, Penrose J, Agu O, Balabani S, Dı´az-Zuccarini V. 2014 Evaluation of the hemodynamic effectiveness of aortic dissection treatments via virtual stenting. 7. Alimohammadi M, Bhattacharya-Ghosh B, Seshadhri S, Penrose J, Agu O, Balabani S, Dı´az-Zuccarini V. 2014 Evaluation of the hemodynamic effectiveness of aortic dissection treatments via virtual stenting. 10. Chen D, Mu¨ller-Eschner M, Kotelis D, Bo¨ckler D, Ventikos Y, Von Tengg-Kobligk H. 2013 A longitudinal study of type-B aortic dissection and endovascular repair scenarios: computational 10. Chen D, Mu¨ller-Eschner M, Kotelis D, Bo¨ckler D, Ventikos Y, Von Tengg-Kobligk H. 2013 A longitudinal study of type-B aortic dissection and endovascular repair scenarios: computational 4. Doyle BJ, Norman PE. 2016 Computational biomechanics in thoracic aortic dissection: today’s analyses. Med. Eng. Phys. 35, 1321–1330. (doi:10. 1016/j.medengphy.2013.02.006) carotid bifurcation model. J. Biomech. 32, 601–608. (doi:10.1016/S0021-9290(99)00015-9) 24. Ganten MK et al. 2009 Motion characterization of aortic wall and intimal flap by ECG-gated CT in patients with chronic B-dissection. Eur. J. Radiol. 72, 146–153. (doi:10.1016/j.ejrad.2008.06.024) 13 rsif.royalsocietypublishing.org 17. Peacock J, Jones T, Tock C, Lutz R. 1998 The onset of turbulence in physiological pulsatile flow in a straight tube. Exp. Fluids 24, 1–9. (doi:10.1007/ s003480050144) 11. Cheng Z, Juli C, Wood NB, Gibbs RGJ, Xu XY. 2014 Predicting flow in aortic dissection: comparison of computational model with PC-MRI velocity measurements. Med. Eng. Phys. 36, 1176–1184. (doi:10.1016/j.medengphy.2014.07.006) 25. References Reymond P, Merenda F, Perren F, Ru D. 2009 Validation of a one-dimensional model of the systemic arterial tree. Am. J. Physiol. Heart Circ. Physiol. 297, 208–222. (doi:10.1152/ajpheart. 00037.2009.) 18. Alastruey J, Parker KH, Peiro J, Sherwin SJ. 2008 Lumped parameter outflow models for 1-D blood flow simulations: effect on pulse waves and parameter estimation. Commun. Comput. Phys. 4, 317–336. 12. Tse KM, Chiu P, Lee HP, Ho P. 2011 Investigation of hemodynamics in the development of dissecting aneurysm within patient-specific dissecting aneurismal aortas using computational fluid dynamics (CFD) simulations. J. Biomech. 44, 827–836. (doi:10.1016/j.jbiomech.2010.12.014) 26. Gallo D et al. 2012 On the use of in vivo measured flow rates as boundary conditions for image-based hemodynamic models of the human aorta: implications for indicators of abnormal flow. Ann. Biomed. Eng. 40, 729–741. (doi:10.1007/s10439- 011-0431-1) rg J. R. Soc. Interface 14: 20170632 19. Westerhof N, Lankhaar JW, Westerhof BE. 2009 The arterial Windkessel. Med. Biol. Eng. Comput. 47, 131–141. (doi:10.1007/s11517-008-0359-2) 20. ANSYS Inc. 2016 CFX-solver theory guide. Release 17. Canonsburg, PA: ANSYS Inc. 13. Sommer G, Sherifova S, Oberwalder PJ, Dapunt OE, Ursomanno PA, DeAnda A, Griffith BE, Holzapfel GA. 2016 Mechanical strength of aneurysmatic and dissected human thoracic aortas at different shear loading modes. J. Biomech. 49, 2374–2382. (doi:10.1016/j.jbiomech.2016.02.042) 21. BonfantiM, Alimohammadi M, Homer-Vanniasinkam S, Dı´az-Zuccarini V. 2016 Patient-specific simulation of the blood flow in an aortic dissection for clinical support including an efficient method to represent the motion of the intimal flap and vessel wall: a case study. In VPH2016 book of abstracts (ed. AG Hoekstra), pp. 356–359. Amsterdam, The Netherlands: University of Amsterdam. 27. Taviani V, Hickson SS, Hardy CJ, McEniery CM, Patterson AJ, Gillard JH, Wilkinson IB, Graves MJ. 2011 Age-related changes of regional pulse wave velocity in the descending aorta using Fourier velocity encoded M-mode. Magn. Reson. Med. 65, 261–268. (doi:10.1002/mrm.22590) 14. Alimohammadi M, Sherwood JM, Karimpour M, Agu O, Balabani S, Dı´az-Zuccarini V. 2015 Aortic dissection simulation models for clinical support: fluid-structure interaction vs. rigid wall models. Biomed. Eng. Online 14, 34. (doi:10.1186/s12938- 015-0032-6) 28. Zhou J, Fung YC. 1997 The degree of nonlinearity and anisotropy of blood vessel elasticity. Proc. Natl Acad. Sci. USA 94, 14 255–14 260. (doi:10.1073/ pnas.94.26.14255) 22. Moore JE, Ku DN. 1994 Pulsatile velocity measurements in a model of the human abdominal aorta under simulated exercise and postprandial conditions. J. Biomech. Eng. 116, 107. (doi:10.1115/ 1.2895692) 29. References Segers P, Stergiopulos N, Verdonck P, Verhoeven R. 1997 Assessment of distributed arterial network models. Med. Biol. Eng. Comput. 35, 729–736. (doi:10.1007/BF02510985) 15. Bonfanti M, Balabani S, Greenwood JP, Puppala S, Homer-Vanniasinkam S, Dı´az-Zuccarini V. 2017 Data from: computational tools for clinical support: a multi-scale compliant model for haemodynamic simulations in an aortic dissection based on multimodal imaging data. Figshare Digital Repository. (doi:10.6084/m9.figshare.5354359.v1) 23. Les AS, Shadden SC, Figueroa CA, Park JM, Tedesco MM, Herfkens RJ, Dalman RL, Taylor CA. 2010 Quantification of hemodynamics in abdominal aortic aneurysms during rest and exercise using magnetic resonance imaging and computational fluid dynamics. Ann. Biomed. Eng. 38, 1288–1313. (doi:10.1007/s10439-010-9949-x) 30. Tsai TT, Schlicht MS, Khanafer K, Bull JL, Valassis DT, Williams DM, Berguer R, Eagle KA. 2008 Tear size and location impacts false lumen pressure in an ex vivo model of chronic type B aortic dissection. J. Vasc. Surg. 47, 844–851. (doi:10.1016/j.jvs. 2007.11.059) 16. Gijsen FJH, van de Vosse FN, Janssen JD. 1999 The influence of the non-Newtonian properties of blood on the flow in large arteries: steady flow in a
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Biliary dyskinesia. Increasing in Incidence or Better Recognition?
Pediatrics and child health issues
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Biliary dyskinesia. Increasing in Incidence or Better Recognition Kennedy A Sabharwal1*, BA, Michael W Simon2, MD, PhD 1University of Kentucky College of Medicine. 2University of Kentucky Department of Pediatrics, Lexington. Abstract: Gallbladder disease has historically remained uncommon in children. Children with nausea, abdominal pain and vomiting after eating are more likely to be diagnosed with irritable bowel syndrome. When children are evaluated with laboratory studies, ultrasound of gallbladder and HIDA scan, a diagnosis of biliary dyskinesia may be confirmed. Laparoscopic surgery of the gallbladder has been shown to be effective with minimal recovery and complications. Follow-up shows that children unlike adults generally remain symptom-free. *Corresponding author: Kennedy Sabharwal, University of Kentucky College of Medicine 4040 Finn Way Suite 310, Lexington KY 40517. Key Words: biliary dyskinesia; laparoscopic cholecystectomy; gallstones; gallbladder Citation: Kennedy A Sabharwal, Michael W Simon, (2024). “Biliary dyskinesia. Increasing in Incidence or Better Recognition?”. Pediatrics and Child Health Issues, 5(1); DOI: 10.61148/2836-2802/JPCHI/059 Introduction: Copyright: © (2024) Kennedy Sabharwal. 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. Throughout history, gallbladder disease has remained essentially nonexistent in pediatrics other than its association with hemolytic disease especially hereditary spherocytosis. Recently, the incidence of gallstones in the pediatric population has been on the rise, mostly due to the astounding increase in rates of pediatric obesity. (1,2) Other than childhood obesity, gallstones are also common in children who are given total parental nutrition due to short gut syndrome because of the decrease in gastric motility. It is much more common, however, that children with abdominal symptoms of nausea, vomiting and abdominal distress especially after eating have been considered to have other etiologies like irritable bowel syndrome and chronic nonspecific abdominal pain. (2) Gallbladder disease is often overlooked and ignored as a possible etiology, likely because there are no specific diagnostic criteria for children; however, the incidence of gallbladder disease is increasing in children (3). Materials And Methods Enrich your Research Enrich your Research Pediatrics and Child Health Issues Mini Review Pediatrics and Child Health Issues Materials And Methods Children presenting with sharp upper right quadrant abdominal pain, nausea and episodic vomiting after eating were evaluated. Their history was reviewed. If there was clinical suspicion and examination consistent with constipation, they had a flat plate of the abdomen performed. If the abdominal X ray was normal and they had a positive Murphy sign they had collected a comprehensive metabolic panel including gamma glutamyltransferase (GGT), complete blood count (CBC) with a manual differential and received an ultrasound of the abdomen. If the ultrasound showed gallstones, they were referred for surgical evaluation. If the ultrasound and laboratory studies were normal, they received a cholescintigraphy 99mTc hepatobiliary iminodiacetic acid (HIDA) scan with cholecystokinin (CCK) infusion. If the HIDA scan showed an ejection Copy right © Kennedy Sabharwal 1 | P a g e nal discomfo normal they 1 | P a g e nal discomfo normal they 1 inal s no fraction of less than 37% or infusion reproduced abdominal discomfort, they were sent for surgical evaluation. If the HIDA scan was normal they would be referred for gastroenterology evaluation. puberty and the production of estrogens. Estrogens increase the secretion of cholesterol into gallstones, which promotes the formation of gallstones. This could easily lead to increased frequency of cholelithiasis in teenage girls. None of the children within this data were obese. Obesity has been identified as a significant cause of gallbladder disease in children. Results None of the children within this data were obese. Obesity has been identified as a significant cause of gallbladder disease in children. (8) This is likely due to bile saturation with cholesterol seen in obesity, excessive hepatic secretions, and impaired gallbladder motility. (9) Infection with Epstein-Barr virus, streptococci, Hepatitis A, and gram-negative infections may also cause gallbladder disease. (10-12) Since 2015, 33 children, 26 females (12 – 24 years of age, mean 14 years) and 7 males (12 – 20 years of age, mean 16 years), were identified with symptoms consistent with biliary dyskinesia followed by diagnostic confirmation of gallbladder disease. All laboratory studies were normal. One female was taking oral contraceptives. None of these children were obese. One child had stones visualized on ultrasound. None of the children had a thicken gallbladder wall visualized. The HIDA scan with infusion showed a reduced ejection fraction of less than 37% in 24 of the 26 females and all 7 males. The only child who did not have a HIDA scan was the subject with stones identified by ultrasound. For all the children symptoms were reproduced by dye infusion. Indication for surgery may be either symptom-based or chronic cholecystitis. Each child in the researcher’s data had an outpatient laparoscopic cholecystectomy performed. Laparoscopic cholecystectomy has been shown to be a safe surgical approach in children without significant comorbidities. (13) However, the merits and benefits of cholecystectomy as a treatment option for biliary dyskinesia is still debated. Each child recovered uneventfully, and their symptoms abated after the surgery. They were seen at follow-up appointments between 1 month up to 3 years with no complications and no recurrent abdominal symptoms. Discussion The patients reported here had a history of sharp right upper abdominal pain, nausea, and occasional vomiting after eating. For all the children, examination was significant for a positive Murphy sign. These children were otherwise unremarkable for the examination with no fever or other findings. There was no antecedent history of illness. One female was on oral contraceptives. She was not the patient with gallstones. If a provider is suspicious of biliary dyskinesia in a pediatric patient, a positive Murphy's sign with consistent history may be an indication to begin evaluation with screening laboratory studies, ultrasound of the gallbladder and if all normal follow with a HIDA scan with infusion. The author’s observation may reflect an increase in the occurrence of biliary dyskinesia in children or simply a better awareness and recognition of gallbladder disease. Only one child had stones visualized on ultrasound. None of the children had a thicken gallbladder wall visualized. However, HIDA scan with contrast showed a reduced ejection fraction of less than 37% in 24 of the 26 females and all 7 of the males. The only child who did not have a HIDA scan was the subject with stones identified by ultrasound. For all the children symptoms were reproducible by the dye infusion. The HIDA scan is not predictive of underlying gallbladder histopathology, but it may signal dysfunction. A low ejection fraction during the HIDA scan or pain triggered by sincalide biliary infusion does not predict postoperative outcome. (4) Financial disclosures: None References 1. Rothstein, David H., and Carroll M. Harmon. “Gallbladder disease in children.” Seminars in Pediatric Surgery, vol. 25, no. 4, Aug. 2016, pp. 225–231. 2. Pogorelić, Zenon et al. “Gallbladder Disease in Children: A 20-year Single-center Experience.” Indian pediatrics vol. 56,5 (2019): 384-386. Symptoms of gallbladder disease in the pediatric population may present as acute abdominal pain, nausea, vomiting, and/or fatty- food intolerance. (5) The most common of these symptoms is vomiting seen in 60% of children who present with biliary dyskinesia. It is very rare to see a child with biliary dyskinesia presenting with only abdominal pain without vomiting. (6) For these individuals, symptoms had been present for three months up to two years prior to diagnosis. Selected dietary items like pizza and cheese seemed to exacerbate their symptoms, while dietary restrictions eliminating fatty meals improved their symptoms. 3. Goldman, Deborah A. “Gallbladder, Gallstones, and Diseases of the Gallbladder in Children.” Pediatrics in review vol. 41,12 (2020): 623-629. 4. Morris-Stiff, Gareth, et al. “The cholecystokin provocation hida test: Recreation of symptoms is superior to ejection fraction in predicting medium-term outcomes.” Journal of Gastrointestinal Surgery, vol. 15, no. 2, Feb. 2011, pp. 345– 349. Within this data set, there was a gender predisposition for females to have biliary dyskinesia. A previous longitudinal study reported that teenage girls are four times more likely to have gallbladder disease compared to teenage boys. (7) Oral contraceptives may increase the risk of gallstones, but also the female gender as a whole may have an increased susceptibility to gallstones due to 5. Al-Homaidhi, Hossam S et al. “Biliary dyskinesia in children.” Pediatric surgery international vol. 18,5-6 (2002): 357-60. 6. Friesen, C A, and C C Roberts. “Cholelithiasis. Clinical characteristics in children. Case analysis and literature review.” Clinical pediatrics vol. 28,7 (1989): 294-8. Copy right © Kennedy Sabharwal 2 | P a g e 2 | P a g e 2 7. Chilimuri, Sridhar et al. “Symptomatic Gallstones in the Young: Changing Trends of the Gallstone Disease-Related Hospitalization in the State of New York: 1996 - 2010.” Journal of clinical medicine research vol. 9,2 (2017): 117-123. Caused by Streptococcus gallolyticus subspecies pasteurianus: A Case Report.” Microorganisms vol. 10,10 1929. 28 Sep. 2022. Caused by Streptococcus gallolyticus subspecies pasteurianus: A Case Report.” Microorganisms vol. 10,10 1929. 28 Sep. 2022. 12. Ariobimo, Bonfilio Neltio; et al. References Acute “Acalculous Cholecystitis Associated with Hepatitis A Viral Infection.” Biomolecular and Health Science Journal Vol 6,1: 74-77. 8. Zdanowicz, Katarzyna et al. “The Etiology of Cholelithiasis in Children and Adolescents-A Literature Review.” International journal of molecular sciences vol. 23,21 13376. 2 Nov. 2022. 13. Mattson, Anja, et al. “Laparoscopic cholecystectomy in children: A systematic review and meta-analysis.” The Surgeon, vol. 21, no. 3, June 2023. 9. Bonfrate, Leonilde et al. “Obesity and the risk and prognosis of gallstone disease and pancreatitis.” Best practice & research. Clinical gastroenterology vol. 28,4 (2014): 623-35. 14. Santucci, Neha, et al. "Biliary Dyskinesia in Children: A Systematic Review". Journal of Pediatric Gastroenterology and Nutrition, vol. 64, no. 2, February 2017: 186-193. 10. Yi, Dae Yong et al. “Ultrasonographic Gallbladder Abnormality of Primary Epstein-Barr Virus Infection in Children and Its Influence on Clinical Outcome.” Medicine vol. 94,27 (2015): e1120. 15. Ozden, Nuri, and John K DiBaise. “Gallbladder ejection fraction and symptom outcome in patients with acalculous biliary-like pain.” Digestive diseases and sciences vol. 48,5 (2003): 890-7. 11. Shigemori, Tsunehiko et al. “Acute Calculous Cholecystitis Copy right © Kennedy Sabharwal 3 | P a g e 3 | P a g e 3
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https://angeo.copernicus.org/articles/36/641/2018/angeo-36-641-2018.pdf
English
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On application of asymmetric Kan-like exact equilibria to the Earth magnetotail modeling
Annales geophysicae
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Daniil B. Korovinskiy1, Darya I. Kubyshkina1, Vladimir S. Semenov2, Marina V. Kubyshkina2, Nikolai V. Erkaev3,4, and Stefan A Kiehas1 Daniil B. Korovinskiy1, Darya I. Kubyshkina1, Vladimir S. Semenov2, Marina V. Kubyshkin and Stefan A Kiehas1 vinskiy1, Darya I. Kubyshkina1, Vladimir S. Semenov2, Marina V. Kubyshkina2, Nikolai V. E Ki h 1 1Space Research Institute, Austrian Academy of Sciences, Graz, Austria 2Earth’s Physics Department, Saint Petersburg State University, St. Petersburg, Russia 3Institute of Computational Modelling, FRC “Krasnoyarsk Science Center” SBRAS, Krasnoyarsk, Russia 4The Applied Mechanics Department, Siberian Federal University, Krasnoyarsk, Russia 1Space Research Institute, Austrian Academy of Sciences, Graz, Austria 2Earth’s Physics Department, Saint Petersburg State University, St. Petersburg, Russia 3Institute of Computational Modelling, FRC “Krasnoyarsk Science Center” SBRAS, Krasnoyarsk, Russia 4The Applied Mechanics Department, Siberian Federal University, Krasnoyarsk, Russia Correspondence: Daniil B. Korovinskiy (daniil.korovinskiy@oeaw.ac.at) Correspondence: Daniil B. Korovinskiy (daniil.korovinskiy@oeaw.ac.at) Correspondence: Daniil B. Korovinskiy (daniil.korovinskiy@oeaw.ac.at) Received: 2 January 2018 – Discussion started: 17 January 2018 Accepted: 22 March 2018 – Published: 19 April 2018 Received: 2 January 2018 – Discussion started: 17 January 2018 Accepted: 22 March 2018 – Published: 19 April 2018 ground magnetoplasma configuration. In applications to col- lisionless plasma, the background equilibrium is to be de- rived from a solution of the kinetic Vlasov–Maxwell equa- tions. A number of such solutions are derived both numer- ically (e.g., Burkhart et al., 1992; Pritchett and Coroniti, 1992; Cargill et al., 1994, and others) and analytically (e.g., Schindler and Birn, 2002; Yoon and Lui, 2005; Sitnov and Merkin, 2016; Vinogradov et al., 2016). All these solutions describe symmetric planar current sheets; the only approx- imate equilibrium solution for bent CS was introduced in the paper of Panov et al. (2012), where the authors present an analysis of direct THEMIS and GOES observations of plasma sheet evolution near substorm onset. Panov et al. (2012) have found the CS bending to be a source of the tail- ward growing normal magnetic field component Bz (in the present paper we use the reference system with x axis point- ing tailward, y axis pointing dawnward and z axis pointing north). Hence, bending of the current sheet turns out to be an important parameter for the sheet stability, controlled by the sign of the derivative ∂Bz/∂x (e.g., Hau et al., 1989; Erkaev et al., 2007, 2009; Pritchett and Coroniti, 2010) in many in- stances. Abstract. A specific class of solutions of the Vlasov– Maxwell equations, developed by means of generalization of the well-known Harris–Fadeev–Kan–Manankova family of exact two-dimensional equilibria, is studied. Daniil B. Korovinskiy1, Darya I. Kubyshkina1, Vladimir S. Semenov2, Marina V. Kubyshkina2, Nikolai V. Erkaev3,4, and Stefan A Kiehas1 The examined model reproduces the current sheet bending and shifting in the vertical plane, arising from the Earth dipole tilting and the solar wind nonradial propagation. The generalized model al- lows magnetic configurations with equatorial magnetic fields decreasing in a tailward direction as slow as 1/x, contrary to the original Kan model (1/x3); magnetic configurations with a single X point are also available. The analytical solu- tion is compared with the empirical T96 model in terms of the magnetic flux tube volume. It is found that parameters of the analytical model may be adjusted to fit a wide range of averaged magnetotail configurations. The best agreement between analytical and empirical models is obtained for the midtail at distances beyond 10–15 RE at high levels of mag- netospheric activity. The essential model parameters (current sheet scale, current density) are compared to Cluster data of magnetotail crossings. The best match of parameters is found for single-peaked current sheets with medium values of num- ber density, proton temperature and drift velocity. This result is in line with previous findings revealing that the configuration asymmetry can be an important factor of magnetosphere dynamics. Particularly, Kivelson and Hughes (1990) have first suggested that the CS bending may drop down the reconnection onset threshold. This idea was con- firmed later, when Partamies et al. (2009) noticed the sea- sonal variations in the number of substorm events with maxi- 1 Introduction Studies of magnetosphere dynamics, including substorm events, require a relevant current sheet (CS) stability anal- ysis. This in turn requires a proper choice of the back- Ann. Geophys., 36, 641–653, 2018 https://doi.org/10.5194/angeo-36-641-2018 © Author(s) 2018. This work is distributed under the Creative Commons Attribution 4.0 License. Ann. Geophys., 36, 641–653, 2018 https://doi.org/10.5194/angeo-36-641-2018 © Author(s) 2018. This work is distributed under the Creative Commons Attribution 4.0 License. D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets mums in winter and summer periods, when dipole tilt angle is bigger (the known geomagnetic activity maximums, e.g., in the Kp index, are registered contrary around the equinoxes). The paper is organized as follows. In Sect. 2 we describe the analytical solution for bent CS. In Sect. 3 we compare analytical and empirical T96 solutions. In Sect. 4 we present the further generalization of the analytical model, providing more realistic profiles of Bz in the equatorial plane. Then, the model-typical scales are compared with in situ data. Discus- sion and conclusions finalize the paper in Sect. 5. p g y q Later, this effect was investigated in detail in the paper of Kubyshkina et al. (2015), where it was shown that the substorm probability is higher for about 10–25 % during the periods with tilt angle > 15◦, compared to the periods with smaller tilt angles. The direction of the solar wind (SW) flow also affects the substorm probability: it grows for 10–20 % when SW flow direction forces the CS tilt to increase. The statistical analysis has shown that the average substorm in- tensity (defined by AL value during the event) is lower for larger effective tilts (dipole tilt angle plus solar wind flow in- clination). In other words, a large number of weak substorms occur in those time intervals in which effective tilt angles are high, and a smaller number of more intense substorms is observed when tilt angles are small. This also agrees with the results of Nowada et al. (2009), where both AL and AU indices were analyzed for the intervals of negative interplan- etary magnetic field Bz. 2 Analytical solution For two-component (proton + electron) isothermal plasma with Maxwellian distribution functions and constant current velocity the system of Vlasov–Maxwell equations can be re- duced to the 2-D Grad–Shafranov equation (see Schindler, 1972; Yoon and Lui, 2005) for the dimensionless magnetic potential 9 = (0, 9, 0), ∂29 ∂x2 + ∂29 ∂z2 = e−29. (1) (1) The quantity 9 is normalized for (−B0L), where L = 2cTi/(eB0Vi) is the typical scale of CS in the normal di- rection, B0 = √8πn0(Te + Ti) is the lobe magnetic field, n0 = n0e = n0i is the typical number density, Te,i are the electron and ion temperatures, respectively, and Ve,i are the corresponding drift velocities, fulfilling the condition In Kubyshkina et al. (2015), the dependence of magneto- tail lobe magnetic field (as a proxy of the magnetic flux) on the dipole tilt angle was studied by means of empirical mod- eling. The average lobe field was found to be smaller for all radial distances in a case of nonzero tilt angles. The decrease reached 10–20 % for maximum tilt angle. This result is rea- sonable under the assumption that substorm onsets require a lower energy input during the periods of increased dipole tilt. Next, in the paper of Semenov et al. (2015) it was found that there is a clear dependence of the substorm probability on the jumps of the z component of the SW velocity (asym- metric factor), while the jumps of number density or plasma pressure (symmetric factor) turn out to be noneffective. Fi- nally, we should note that the Earth’s dipole tilt angle un- dergoes daily and seasonal variations in the interval of about ±35◦, so that it is equal to zero twice a day within about 4 months a year, and during the other 8 months it is never zero. In addition, the solar wind flow direction varies for about ±6◦. These variations produce CS inclination, bending and shift from the ecliptic plane. Therefore, the simplest so- lar wind–magnetosphere configuration (vertical dipole, pla- nar CS, radial solar wind) adopted by the majority of models, is rather untypical and the development of the relevant bent CS models is in high demand. Vi/Ti + Ve/Te = 0. (2) (2) Vi/Ti + Ve/Te = 0. Equation (2) expresses the condition of the zero electro- static potential. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 642 2 Analytical solution The model of an ion-dominated CS, where |Vi/Ve| > Ti/Te, is considered in the paper of Yoon and Lui (2004). In the case of Maxwellian distribution func- tions condition (2) can be satisfied by means of the proper choice of the reference system, while in the general case of non-Maxwellian distribution functions it cannot be fulfilled (Schindler and Birn, 2002). Equation (2) expresses the condition of the zero electro- static potential. The model of an ion-dominated CS, where |Vi/Ve| > Ti/Te, is considered in the paper of Yoon and Lui (2004). In the case of Maxwellian distribution func- tions condition (2) can be satisfied by means of the proper choice of the reference system, while in the general case of non-Maxwellian distribution functions it cannot be fulfilled (Schindler and Birn, 2002). A series of analytical solutions of Eq. (1) was found by Walker (1915), who showed that the solution may be ex- pressed via an arbitrary generating function g of the complex variable ζ = x + iz, e−29 = 4|g′|2 (1 + |g|2)2 , g′ = dg(ζ) dζ . (3) (3) The first exact solution for two-dimensional (2-D) equi- librium bent CS with nonzero dipole tilt was presented in short notes of Semenov et al. (2015). This solution general- izes the well-known Harris–Fadeev–Kan–Manankova equi- libria family (see Yoon and Lui, 2005). In the present paper we investigate the obtained solution to estimate its relevance for the magnetotail CS modeling and stability analysis. For this end, we compare the analytical solution with the empir- ical Tsyganenko (1995) T96 model and define the analytical model parameters, providing the best agreement. With the solution (3), the equilibrium magnetoplasma con- figuration takes the form 9 = ln 1 + |g|2 2|g′|  , (4) n = exp(−29), p = 0.5exp(−29), (5) (4) (5) where p is the plasma pressure. By definition, the dimen- sionless magnetic field components are Bx = −∂9/∂z and Bz = +∂9/∂x. www.ann-geophys.net/36/641/2018/ Ann. Geophys., 36, 641–653, 2018 D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets Namely, one should set (f = 0, a ̸= 0, b ̸= 0) for the Kan solution; (f ̸= 0, a = b = 0) for the Fadeev solution, and (f = a = b = 0) for the Harris solution. PHI = 30 The solution for a bent CS is developed in the paper of Semenov et al. (2015) by substituting the complex parame- ters a →ia and b →b0eiϕ in Eq. (6). The complex param- eter a controls the shift of the CS in the z direction and ϕ controls the dipole tilt angle. For the case of bent CS with- out plasmoids (Kan-like model, f = 0) the solution (6) takes relatively simple form, 0 5 10 15 20 , (−B0L) −15 −10 −5 0 5 10 15 z, L −10 0 10 20 30 40 50 x, L (c) PHI = 60  (−B L) (d) PHI = 60 9 = ln coshZ∗ √ W  , (7) Z∗= z −b0x sin(ϕ) −b0(z −a)cos(ϕ) R2 , (8) W = 1 + b2 0 + 2b0(x2 −(z −a)2)cos(ϕ) + 4b0x(z −a)sin(ϕ) R4 , (9) (7) (8) 0 5 10 15 20 , (−B0L) −15 −10 −5 0 5 10 15 z, L −10 0 10 20 30 40 50 x, L (d) PHI = 120 1 + b2 0 + 2b0(x2 −(z −a)2)cos(ϕ) + 4b0x(z −a)sin(ϕ) R4 , (9) (9) where R2 = x2 + (z −a)2. Configurations of this type pos- sess a dipole singularity at (x, z) = (0, a) and two additional singularities at (x, z) = (±√b0 sin(ϕ/2), a∓√b0 cos(ϕ/2)), rotating twice as slow as a dipole does. Hence, the effective dipole tilt is equal to ϕ/2. For positive tilt angles the CS is bent and uplifted over the ecliptic plane, and for negative tilts the CS is shifted down. The set of magnetic configurations for dipole tilt angle PHI = {0, 30, 60, 120} degrees clockwise (PHI = −ϕ/2) is shown in Fig. 1. The two first cases (0 and 30◦) can be observed in the Earth’s magnetosphere, and other cases are shown here to illustrate the model behavior. White asterisks in Fig. 1c and d mark the X points (Bx = Bz = 0), being an attribute of the Kan-like solution. D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets 643 , (−B0L) −10 0 10 20 30 40 50 x, L −15 −10 −5 0 5 10 15 z, L −4 −2 0 2 4 6 8 10 12 14 (a) PHI = 0 0 5 10 15 20 , (−B0L) −15 −10 −5 0 5 10 15 z, L −10 0 10 20 30 40 50 x, L (b) PHI = 30  ( ) , (−B0L) −10 0 10 20 30 40 50 x, L −15 −10 −5 0 5 10 15 z, L −4 −2 0 2 4 6 8 10 12 14 (a) PHI = 0 0 5 10 15 20 , (−B0L) −15 −10 −5 0 5 10 15 z, L −10 0 10 20 30 40 50 x, L (b) PHI = 30 0 5 10 15 20 , (−B0L) −15 −10 −5 0 5 10 15 z, L −10 0 10 20 30 40 50 x, L (c) PHI = 60  (−B L) (d) , (−B0L) −10 0 10 20 30 40 50 x, L −15 −10 −5 0 5 10 15 z, L −4 −2 0 2 4 6 8 10 12 14 (a) PHI = 0  The particular choice of the generating function g specifies the particular CS model. In the current paper we consider the family of Harris-like models, including the classical Harris (1962) current sheet, the Fadeev et al. (1965) solution (Har- ris sheet complemented by an infinite chain of magnetic is- lands along the neutral plane), the Kan (1973) solution (Har- ris sheet with quasi-dipole), and the Manankova et al. (2000) solution, representing the combination of all previous mod- els. D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets The last one is specified by the generating function , ( 0 ) −10 0 10 20 30 40 50 x, L −15 −10 −5 0 5 10 15 z, L −4 −2 0 2 4 6 8 10 12 14 (a) PHI = 0 0 5 10 15 20 , (−B0L) −15 −10 −5 0 5 10 15 z, L −10 0 10 20 30 40 50 x, L (b) PHI = 30 0 5 10 15 20 , (−B0L) −15 −10 −5 0 5 10 15 z, L −10 0 10 20 30 40 50 x, L (c) PHI = 60 0 5 10 15 20 , (−B0L) −15 −10 −5 0 5 10 15 z, L −10 0 10 20 30 40 50 x, L (d) PHI = 120 Figure 1. Magnetic potential 9(x,z), calculated from the asymmet- ric Kan model (Eqs. 7–9) with parameters a = 0 and b0 = 8. Solu- tions with dipole tilt angles PHI = {0◦, 30◦, 60◦,120◦} clockwise are plotted on panels (a)–(d), respectively. PHI = −ϕ/2 of the ana- lytical model. Spatial units are normalized for typical CS width L. Magnetic potential is normalized for (−B0L). X points are marked white. PHI = 0 g(ζ) = f + q 1 + f 2 exp  i  ζ − b ζ −a  . (6) (6) Solution (6) contains three real parameters a, b and f , where a specifies the shift along the x axis, b controls the field line elongation, and f defines the current density in the mag- netic islands. Generating functions for other listed models are the special cases of the function (6). Namely, one should set (f = 0, a ̸= 0, b ̸= 0) for the Kan solution; (f ̸= 0, a = b = 0) for the Fadeev solution, and (f = a = b = 0) for the Harris solution. Solution (6) contains three real parameters a, b and f , where a specifies the shift along the x axis, b controls the field line elongation, and f defines the current density in the mag- netic islands. Generating functions for other listed models are the special cases of the function (6). D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets In the symmetric Kan model the X point is located at infinity, but in bent sheets it starts to approach the dipole with increasing tilt angle. This X point is not produced by magnetic reconnection, and it does not break a steady state equilibrium of the CS. However, Figure 1. Magnetic potential 9(x,z), calculated from the asymmet- ric Kan model (Eqs. 7–9) with parameters a = 0 and b0 = 8. Solu- tions with dipole tilt angles PHI = {0◦, 30◦, 60◦,120◦} clockwise are plotted on panels (a)–(d), respectively. PHI = −ϕ/2 of the ana- lytical model. Spatial units are normalized for typical CS width L. Magnetic potential is normalized for (−B0L). X points are marked white. the appearance of the X point can be considered as a mani- festation of potentially unstable configuration. In such a case, the appearance of the X point can be considered as a mani- festation of potentially unstable configuration. In such a case, www.ann-geophys.net/36/641/2018/ 3 Comparison with the T96 model (2009) and veri- fied by in situ data analysis (Sergeev et al., 2014), any bursty bulk flow (BBF), produced by reconnection in the magneto- tail and moving toward the Earth, stops near that particular point where the entropy of the ambient plasma is equal to that inside the BBF. The distribution of entropy along the magnetotail is also an important factor for the stability anal- ysis (Birn et al., 2009) and for the study of wave (oscillation) generation and dissipation (Panov et al., 2016). Figure 3. Flux tube volume R (x,z) (30,−2.4)(dS/B) normalized to the full FTV R (5,1.5) (30,−2.4)(dS/B), in percent, calculated by T96 model (blue) and by the Kan model (red) for the quiet conditions with tilt an- gle PHI = 30◦clockwise (model parameter ϕ = −60). Other model parameters are given in the legend of Fig. 4b. The Earth is on the left. Figure 3. Flux tube volume R (x,z) (30,−2.4)(dS/B) normalized to the full (5 1 5) FTV R (5,1.5) (30,−2.4)(dS/B), in percent, calculated by T96 model (blue) and by the Kan model (red) for the quiet conditions with tilt an- gle PHI = 30◦clockwise (model parameter ϕ = −60). Other model parameters are given in the legend of Fig. 4b. The Earth is on the left. The FTV is determined in the same way for both analyt- ical and empirical models: we integrate dS/B along mag- netic field lines, where dS is the field line length element and B = q B2x + B2z . In the T96 model the location of the flux tube is computed by means of field line tracing; in the an- alytical model this is a curve of constant 9. As one of the first steps, the values of FTV of a single flux tube are com- pared. The model parameters correspond to the quiet magne- tospheric conditions with tilt angle of 30◦clockwise (see the legend of Fig. 4b). FTVs are calculated along the magnetic field line with a node at (x, z) = (30, −2.4). To eliminate singularities, we excluded the near-Earth region x < 5RE, so that the total FTVs are calculated as R (5,1.5) (30,−2.4)dS/B. The re- sults are shown in Fig. 3 by the blue curve for T96 and by the red curve for the Kan model. The values of FTV, normal- ized for total FTV, are plotted as a function of x. 3 Comparison with the T96 model Topologically, magnetic configurations plotted in Fig. 1 are very similar to that of the Earth’s magnetosphere. However, to estimate the relevance of the analytical solution one should compare some important numerical characteristics of the CS model with the corresponding values registered in real ob- servations. This can be done by utilizing empirical magnetic field models, providing realistic averaged magnetospheric configurations at various levels of magnetospheric activity. Of course, we should keep in mind that the real magneto- sphere is an essentially three-dimensional structure. Follow- ing the dipole tilt (and solar wind flow direction) variations, the magnetotail CS bends and shifts from the equatorial plane in the z direction (at most ∼3RE for maximum tilt) and also warps in the y direction. These effects are well pronounced in empirical magnetospheric models, but the 2-D analytical model is evidently unable to reproduce all these complex deformations. Therefore, we restrict our study to the noon– midnight plane y = 0, and the two main effects manifested in that plane: CS bending and shifting in z direction. Figure 2. X-point location vs. the effective tilt angle ϕ/2 in the asymmetric Kan solution (Eqs. 7–9) with a = 0 and parameters b0 = 40 (red), b0 = 15 (blue), and b0 = 9 (green). X, RE 30 25 20 15 10 5 FTV, % 0 20 40 60 80 100 Normalized ∫(dS/B) T96 Kan 90 % 50 % X, RE 30 25 20 15 10 5 FTV, % 0 20 40 60 80 100 Normalized ∫(dS/B) T96 Kan 90 % 50 % Figure 3. Flux tube volume R (x,z) (30,−2.4)(dS/B) normalized to the full FTV R (5,1.5) (30,−2.4)(dS/B), in percent, calculated by T96 model (blue) and by the Kan model (red) for the quiet conditions with tilt an- gle PHI = 30◦clockwise (model parameter ϕ = −60). Other model parameters are given in the legend of Fig. 4b. The Earth is on the left. To explore the appropriateness of the here presented ana- lytical solution for bent CS, we compare the predicted mag- netic flux tube volume (a proxy for the entropy) with that calculated from the empirical model of Tsyganenko (1995) T96. We consider the flux tube volume (FTV) instead of the entropy, since the analytical solution is isothermal. This quantity is chosen due to its importance for the magnetotail dynamics. As was claimed by Birn et al. D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets 644 φ/2, o 45 50 55 60 65 70 75 80 85 90 0 20 40 60 80 100 Distance to the X point versus tilt angle b0 = 40 b0 = 15 b0 = 9 x, L Figure 2. X-point location vs. the effective tilt angle ϕ/2 in the asymmetric Kan solution (Eqs. 7–9) with a = 0 and parameters b0 = 40 (red), b0 = 15 (blue), and b0 = 9 (green). φ/2, o 45 50 55 60 65 70 75 80 85 90 0 20 40 60 80 100 Distance to the X point versus tilt angle b0 = 40 b0 = 15 b0 = 9 x, L www.ann-geophys.net/36/641/2018/ Ann. Geophys., 36, 641–653, 2018 D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets (a) 25 5 PHI=0 0 FTV, R /nT E X, RE 0 30 20 15 10 25 5 6 4 2 pdyn = 2 nPa Dst = -10nT B = 2nT z b = 8.8 a = -0.05 d = 7.4% max d = 5.3% med (b) PHI=30 0 FTV, R /nT E X, RE 0 30 20 15 10 25 5 8 6 4 2 pdyn = 2 nPa Dst = -10nT B = 2nT z b = 8.8 a = -2.35 d = 10.6% max d = 7.7% med (b) (d) 4 3 2 1 PHI=30 0 FTV, R /nT E FTV, R /nT E X, RE X, RE 0 30 20 15 10 25 5 0 30 20 15 10 25 5 8 6 4 2 4 3 2 1 pdyn = 2 nPa pdyn = 3 nPa Dst = -10nT Dst = -50nT B = 2nT z B = -3nT z b = 8.8 b = 14 a = -2.35 a = -2 d = 10.6% max d = 7.2% max d = 7.7% med d = 4.4% med PHI=30 0 (b) (d) (f) 4 3 2 1 PHI 30 FTV, R /nT E FTV, R /nT E FTV, R /nT E X, RE X, RE X, RE X, RE 0 30 20 15 10 25 5 0 30 20 15 10 25 5 0 30 20 15 10 25 5 8 6 4 2 4 3 2 1 1.6 1.2 0.8 0.4 pdyn = 2 nPa pdyn = 3 nPa pdyn = 6 nPa Dst = -10nT Dst = -50nT Dst = -150nT B = 2nT z B = -3nT z B = -7nT z b = 8.8 b = 14 b = 41.5 a = -2.35 a = -2 d = 10.6% max d = 7.2% max d = 3.7% max d = 7.7% med d = 4.4% med d = 1.9% med a = -1.84 (b) (d) (f) 4 3 2 1 FTV, R /nT E FTV, R /nT E FTV, R /nT E X, RE X, RE X, RE X, RE 0 30 20 15 10 25 5 0 30 20 15 10 25 5 0 30 20 15 10 25 5 8 6 4 2 4 3 2 1 1.6 1.2 0.8 0.4 pdyn = 2 nPa pdyn = 3 nPa pdyn = 6 nPa Dst = -10nT Dst = -50nT Dst = -150nT B = 2nT z B = -3nT z B = -7nT z b = 8.8 b = 14 b = 41.5 a = -2.35 a = -2 d = 10.6% max d = 7.2% max d = 3.7% max d = 7.7% med d = 4.4% med d = 1.9% med a = -1.84 (b) (a) (d) (f) 4 3 2 1 FTV, R /nT E FTV, R /nT E X, RE X, RE X, RE X, RE 0 30 20 15 10 25 5 0 30 20 15 10 25 5 4 3 2 1 1.6 1.2 0.8 0.4 pdyn = 3 nPa pdyn = 6 nPa Dst = -50nT Dst = -150nT B = -3nT z B = -7nT z b = 14 b = 41.5 a = -2 d = 7.2% max d = 3.7% max d = 4.4% med d = 1.9% med a = -1.84 (c) (d) (e) Figure 4. D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets Flux tube volumes: analytical solution (red curves) and T96 (black curves) for quiet (a, b), substorm (c, d) and storm (e, f) conditions are plotted for tilt angles 0◦(a, c, e) and PHI = 30◦clockwise (b, d, f). Input parameters for the T96 model (black text), for the Kan-like model (red text), and SD normalized for average FTV (blue text) are given in legends. The Earth is on the left. 90% of FTV are provided by the farther half of the tube, x ∈[15, 30]RE, and 50% of FTV are concentrated in the most distant interval within 3–4 RE in the x direction from the tube node. sets of parameters are taken to specify the quiet magneto- tail {Dst = −10, pdyn = 2 nPa, Bsw z = 2 nT}, substorm con- ditions {Dst = −50, pdyn = 3 nPa, Bsw z = −3 nT} and storm {Dst = −150, pdyn = 6 nPa, Bsw z = −7 nT}. Magnetic field component Bsw y was set equal to zero. Parameters a and b0 of the analytical solution (7–9) are found numerically to min- imize the SD between two models. The results (FTV vs. x co- ordinate of the flux tube node) are presented in Fig. 4, where red lines plot analytical solutions and black ones plot the T96 Then, FTVs, calculated by means of analytical and em- pirical models, are compared at different levels of magne- tospheric activity, characterized by input parameters of the T96 model (Dst index, the SW dynamical pressure, pdyn, and SW magnetic field components Bsw y and Bsw z ). Three D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets 645 y q y (a) (b) (c) (d) (e) (f) 25 5 4 3 2 1 PHI=0 0 PHI=30 0 FTV, R /nT E FTV, R /nT E FTV, R /nT E FTV, R /nT E FTV, R /nT E FTV, R /nT E X, RE X, RE X, RE X, RE X, RE X, RE X, RE 0 30 20 15 10 25 5 0 30 20 15 10 25 5 0 30 20 15 10 25 5 0 30 20 15 10 25 5 0 30 20 15 10 25 5 0 30 20 15 10 25 5 6 4 2 8 6 4 2 3 2 1 4 3 2 1 1.6 1.2 0.8 0.4 1.6 1.2 0.8 0.4 pdyn = 2 nPa pdyn = 2 nPa pdyn = 3 nPa pdyn = 3 nPa pdyn = 6 nPa pdyn = 6 nPa Dst = -10nT Dst = -10nT Dst = -50nT Dst = -50nT Dst = -150nT Dst = -150nT B = 2nT z B = 2nT z B = -3nT z B = -3nT z B = -7nT z B = -7nT z b = 8.8 b = 8.8 b = 22.13 b = 14 b = 51 b = 41.5 a = -2.35 a = -0.05 a = -2 a = -0.03 a = -0.02 d = 7.4% max d = 10.6% max d = 7.2% max d = 4.2% max d = 3.6% max d = 3.7% max d = 5.3% med d = 7.7% med d = 2.9% med d = 4.4% med d = 1.9% med d = 1.9% med a = -1.84 Figure 4. Flux tube volumes: analytical solution (red curves) and T96 (black curves) for quiet (a, b), substorm (c, d) and storm (e, f) conditions are plotted for tilt angles 0◦(a, c, e) and PHI = 30◦clockwise (b, d, f). Input parameters for the T96 model (black text), for the Kan-like model (red text), and SD normalized for average FTV (blue text) are given in legends. The Earth is on the left. 3 Comparison with the T96 model It is seen that two models demonstrate rather close results. A total of the X-point motion towards the dipole with increasing tilt an- gle could mean that CS evolves toward an unstable state. Ac- cording to the solution (Eqs. 7–9), the X-point location also depends on the CS width L and model parameter b0. The X- point position as a function of ϕ is plotted in Fig. 2 for three values of b0, corresponding to three different levels of geo- magnetic activity (see Fig. 4, right column). It is seen that for tilt angles |ϕ/2| < 45◦the X point stays very far beyond 60RE for any realistic value of L and b0, e.g., for b0 = 8 (the value, corresponding to quiet magnetotail) and |ϕ/2| = 45◦ the X point stays as far as ≈340 L. For |ϕ/2| = 60◦(almost 2 times more than the Earth maximal dipole tilt) an approach to 8.5 L is achieved. www.ann-geophys.net/36/641/2018/ Ann. Geophys., 36, 641–653, 2018 D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets 645 rovinskiy et al.: Kan-like equilibria for asymmetric current sheets D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets 646 σ / <FTV> (a) σ / <FTV> σ / <FTV> (a) (b) Figure 5. Comparison of analytical and empirical models for symmetric (dipole tilt 0◦, a) and bent (30◦clockwise, b) current sheets. Standard deviations, σ, normalized for average FTV, are shown as functions of x0, where x0 is the center of the region under consideration [x0 −2.5, x0 + 2.5], for quiet (black curves), substorm (blue curves) and storm (red curves) conditions. The Earth is on the left. σ / <FTV> (b) Figure 5. Comparison of analytical and empirical models for symmetric (dipole tilt 0◦, a) and bent (30◦clockwise, b) current sheets. Standard deviations, σ, normalized for average FTV, are shown as functions of x0, where x0 is the center of the region under consideration [x0 −2.5, x0 + 2.5], for quiet (black curves), substorm (blue curves) and storm (red curves) conditions. The Earth is on the left. results. The left column shows the symmetrical case (zero dipole tilt), and the right column corresponds to the dipole tilt angle of 30◦clockwise. ations are bigger for the more quiet magnetosphere environ- ment, and (3) deviations are smaller for a tilt angle of 30◦. Compared to results of the large interval analysis (Fig. 4), dependence on the activity level is the same, and dependence on the tilt angle demonstrates opposite behavior. Overall, an- alytical and empirical models show good agreement beyond 15RE, improving with growing activity. One can see that the agreement between two models is quite good, with the maximal SDs varying within 2–11 %. The values of dmax = σmax/⟨FTV⟩are given in legends of Fig. 4, where σ is the SD and ⟨FTV⟩is the average FTV. The better agreement is achieved for disturbed magnetospheric conditions, i.e., the analytical model describes the stretched CSs even better than the thicker ones. It is found that minimal difference between two models is obtained when parameter a is very close to the medium neutral sheet position determined from the empirical model. The best-fit value of the parameter b0, controlling the field lines stretching and the CS thinning, depends on the level of activity and the dipole tilt angle. It grows from 8.8 for the quiet magnetosphere to 51 for storm conditions. At any fixed distance, the stretching of field lines makes the FTV decrease with growing magnetospheric activ- ity. D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets For example, at the distance of x = 30RE it changes from ≈5RE/nT for “quiet” conditions to ≈3RE/nT for “sub- storm” conditions and to the ≈1.6RE/nT for “storm-time” conditions. On the contrary, the asymmetric deformation of CS (dipole tilt angle) forces the FTV to increase. www.ann-geophys.net/36/641/2018/ www.ann-geophys.net/36/641/2018/ Ann. Geophys., 36, 641–653, 2018 D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets 647 2 4 6 8 10 12 14 −500 0 500 1000 1500 x, L Beq −1 1/Bz, B0 −1 b=1.0, k=1.0, n=1.00 b=1.0, k=2.0, n=0.99 b=1.0, k=0.1, n=0.99 b=1.0, k=1.0, n=0.99 b=0.5, k=1.0, n=0.98 b=1.0, k=1.0, n=0.98 b=2.0, k=1.0, n=0.98 b=1.0, k=1.0, n=1.10 Figure 7. Profiles 1/Bz(x,0) for symmetric Kan-like CS, calcu- lated from an analytical model (Eqs. 11–16). Parameters {a1 = 0, a2 = 0, ϕ = 0, f = 0} are the same. Other parameters are {b0 = 1, k = 1, n = 1} (red), {b0 = 1, k = 2, n = 0.99} (dark-green dash- dotted), {b0 = 1, k = 0.1, n = 0.99} (dark-green dashed), {b0 = 1, k = 1, n = 0.99} (dark-green solid), {b0 = 0.5, k = 1, n = 0.98} (violet dash-dotted), {b0 = 1, k = 1, n = 0.98} (violet solid), {b0 = 2, k = 1, n = 0.98} (violet dashed), and {b0 = 1, k = 1, n = 1.1} (cyan). Red curve shows the original Kan solution. Units are nor- malized for CS typical width L and for B−1 0 . 2 4 6 8 10 12 14 −500 0 500 1000 1500 x, L Beq −1 1/Bz, B0 −1 b=1.0, k=1.0, n=1.00 b=1.0, k=2.0, n=0.99 b=1.0, k=0.1, n=0.99 b=1.0, k=1.0, n=0.99 b=0.5, k=1.0, n=0.98 b=1.0, k=1.0, n=0.98 b=2.0, k=1.0, n=0.98 b=1.0, k=1.0, n=1.10 z, L −2 −1.5 −1 −0.5 0 0.5 1 1.5 2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 (a)  0L) (-B x, L 2 3 4 5 6 7 8 9 10 n = 0.95 −0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 (b) z, L −2 −1.5 −1 −0.5 0 0.5 1 1.5 2 x, L 2 3 4 5 6 7 8 9 10  0L) (-B n = 1.00 −0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 (c) z, L −2 −1.5 −1 −0.5 0 0.5 1 1.5 2 x, L 2 3 4 5 6 7 8 9 10  0L) (-B n = 1.05 x, L 2 3 4 5 6 7 8 9 10 −0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 (d) z, L −2 −1.5 −1 −0.5 0 0.5 1 1.5 2  0L) (-B n = 1.10 Figure 6. D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets It is seen that the Kan solution (n = 1) is the only degenerated case when the first term of W turns to 1 and its derivative to zero; hence, in the distant tail Bz ∼(1/x2+k) due to the rightmost term of expression (14). For any n ̸= 1 we have (∂W/∂x)/W →O(1/x). For symmetric Kan-like CS without plasmoids (a = 0, f = 0, ϕ = 0), the quantity Bz at the x axis takes the simple form Bz(x,0) = −(∂W/∂x)/(2W). It is seen that the Kan solution (n = 1) is the only degenerated case when the first term of W turns to 1 and its derivative to zero; hence, in the distant tail Bz ∼(1/x2+k) due to the rightmost term of expression (14). For any n ̸= 1 we have (∂W/∂x)/W →O(1/x). Figure 6. The values of magnetic potential 9(x,z), calculated from analytical model (Eqs. 11–16), are shown by color for model parameter n = {0.95, 1, 1.05, 1.1} on panels (a)–(d), respec- tively. Other parameters {a = 0, b0 = 1, ϕ = 0, f = 0, k = 1} are the same. Magnetic field lines are plotted by white curves. Panel (b) shows the original Kan solution. Parameter n controls flaring of magnetic field lines; values of n > 1 force strong convergence of the CS field lines to- ward the x axis, and hence the location of the X-line is dras- tically dependent on n. This feature is illustrated in Fig. 6, where four symmetric magnetic configurations with (a = 0, ϕ = 0, f = 0, b0 = 1, k = 1) and n = {0.95, 1, 1.05, 1.1} are plotted. In Fig. 7 reverse values of the equatorial magnetic field, B−1 z (x,0), are plotted for several sets of the model pa- rameters. The set of green curves illustrates contribution of the parameter k. The set of violet curves shows the effect of the parameter b0 variation. The set of solid curves demon- strates the parameters n impact. It is seen that (a) all curves except the red one (original Kan solution, n = 1) tend to Assuming {f, n, k} to be real values, a = a1 + ia2, and b = b0 exp(iϕ), we derive 9 = ln f cosX∗+ p 1 + f 2 coshZ∗ √ W ! D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets The values of magnetic potential 9(x,z), calculat from analytical model (Eqs. 11–16), are shown by color f model parameter n = {0.95, 1, 1.05, 1.1} on panels (a)–(d), respe tively. Other parameters {a = 0, b0 = 1, ϕ = 0, f = 0, k = 1} a the same. Magnetic field lines are plotted by white curves. Panel ( shows the original Kan solution. z, L −2 −1.5 −1 −0.5 0 0.5 1 1.5 2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 (a)  0L) (-B x, L 2 3 4 5 6 7 8 9 10 n = 0.95  L) ( B x, L −0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 (b) z, L −2 −1.5 −1 −0.5 0 0.5 1 1.5 2 x, L 2 3 4 5 6 7 8 9 10  0L) (-B n = 1.00 (c)  0L) (-B Figure 7. Profiles 1/Bz(x,0) for symmetric Kan-like CS, calcu- Figure 7. Profiles 1/Bz(x,0) for symmetric Kan-like CS, calcu- lated from an analytical model (Eqs. 11–16). Parameters {a1 = 0, a2 = 0, ϕ = 0, f = 0} are the same. Other parameters are {b0 = 1, k = 1, n = 1} (red), {b0 = 1, k = 2, n = 0.99} (dark-green dash- dotted), {b0 = 1, k = 0.1, n = 0.99} (dark-green dashed), {b0 = 1, k = 1, n = 0.99} (dark-green solid), {b0 = 0.5, k = 1, n = 0.98} (violet dash-dotted), {b0 = 1, k = 1, n = 0.98} (violet solid), {b0 = 2, k = 1, n = 0.98} (violet dashed), and {b0 = 1, k = 1, n = 1.1} (cyan). Red curve shows the original Kan solution. Units are nor- malized for CS typical width L and for B−1 0 . n = 1.05 Z∗= rn sin(nϑ) + b0 Rk sin(k2 −ϕ), (13) W = n2r2(n−1) + b2 0k2 R2(k+1) + 2nkb0 rn−1 Rk+1 cos[(n −1)ϑ + (k + 1)2 −ϕ], (14) r = p x2 + z2, ϑ = arctan  z x  , (15) R = p (x −a1)2 + (z −a2)2, θ = arctan  z −a2 x −a1  . (16) (13) For symmetric Kan-like CS without plasmoids (a = 0, f = 0, ϕ = 0), the quantity Bz at the x axis takes the simple form Bz(x,0) = −(∂W/∂x)/(2W). 4 Normal magnetic component and current density The results of the previous section show that parameters of the asymmetric Kan-like model may be adapted to provide rather good agreement with the magnetotail CS, especially in a distant tail beyond 15–20 RE, and especially for bent cur- rent sheets. However, until now the practical usage of this model encountered a substantial obstacle, related to the be- havior of the normal magnetic field component. It can be easily checked that in the distant tail the Kan model yields Bz ∼1/x3, while in reality Bz decreases as 1/x or even slower (e.g., Behannon and Ness, 1966; Mihalov et al., 1968; Behannon, 1970; Wang and Lyons, 2004; Yue et al., 2013). For plane and axially symmetric current sheets the solution with Bz ∼1/xα with arbitrary α is found in Vasko et al. (2013). For Kan-like models considered in the current paper the Bz problem may be solved by introducing one more pa- rameter in the generating function g(ζ). With the additional parameter n, general asymmetric model takes the form (com- pare to Eq. 16 of Yoon and Lui, 2005) Figure 4 shows a comparison of two models within the large interval x ∈[5, 30]. To detect the best-matching region we performed the same analysis for eight short overlapping intervals x ∈[7.5, 12.5] + 2.5n, where n = 0, 1,...,8. The normalized SD as a function of x0n, where x0n is the cen- ter of corresponding interval, is shown in Fig. 5, where three features are observed: (1) SD grows toward the Earth and exceeds 10% for x < 15RE for all activity levels, (2) devi- g(ζ) = f + q 1 + f 2 exp  i  ζ n − b (ζ −a)k  . (10) (10) www.ann-geophys.net/36/641/2018/ Ann. Geophys., 36, 641–653, 2018 D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets 647 D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets −1.2 −1 −0.8 −0.6 −0.4 −0.2 20 30 40 50 60 70 80 x, L 10 b) −2 −1.5 −1 −0.5 0 0.5 1 1.5 2 z, L Jy, J0 Jy J0 ) 20 30 40 50 60 70 80 x, L 10 −2 −1.5 −1 −0.5 0 0.5 1 1.5 2 z, L a) −1.2 −1 −0.8 −0.6 −0.4 −0.2 −1.4 Jy, J0 Jy J ) 20 30 40 50 60 70 80 x, L 10 c) −2 −1.5 −1 −0.5 0 0.5 1 1.5 2 z, L −1.2 −1 −0.8 −0.6 −0.4 −0.2 −1.4 Jy, J0 ) 20 30 40 50 60 70 80 x, L 10 d) −2 −1.5 −1 −0.5 0 0.5 1 1.5 2 z, L −1.2 −1 −0.8 −0.6 −0.4 −0.2 Jy, J0 ) , 20 30 40 50 60 70 80 x, L 10 f) −2 −1.5 −1 −0.5 0 0.5 1 1.5 2 z, L −1.2 −1 −0.8 −0.6 −0.4 −0.2 −1.4 Jy, J0 ) Figure 8. Current density Jy(x,z) by an analytical model (Eqs. 11–16) for {a1 = 0, a2 = −0.03, b = 22.13, f = 0, k = 1} is shown for three values of the parameter n for plane sheets (a, c, e, ϕ = 0) and curved sheets (b, d, f, ϕ = 60). In panels (a, b), n = 0.995. In (c, d) n = 1. In (e, f), n = 1.005. Panel (c) corresponds to the plane substorm sheet (see Fig. 4c). Units are normalized for CS typical width L and J0 = cB0/(4πL). O(x), and (b) numerical values of Bz are highly variable de- pending on different combinations of parameters {b0, k, n}. tion of the CS width and 20% growth of the peaking current density. In two dimensions, contributions of parameters n and ϕ are shown in the next two plots. Figures 8 and 9 present Jy(x,z) and Bz(x,z), respectively, for six sets of the model param- eters, where parameters a1 = 0, a2 = −0.03, f = 0, b0 = 22.13, k = 1 are the same. Panels (a) show the solutions for (n = 0.995, ϕ = 0). Panels (b) show the solutions for the bent sheet (n = 0.995, ϕ/2 = 30). On panels (c) solutions for a plane substorm CS model (see Fig. D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets , (11) X∗= rn cos(nϑ) −b0 Rk cos(k2 −ϕ), (12) (11) (12) www.ann-geophys.net/36/641/2018/ www.ann-geophys.net/36/641/2018/ Ann. Geophys., 36, 641–653, 2018 www.ann-geophys.net/36/641/2018/ Ann. Geophys., 36, 641–653, 2018 www.ann-geophys.net/36/641/2018/ D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets 648 20 30 40 50 60 70 80 x, L 10 −2 −1.5 −1 −0.5 0 0.5 1 1.5 2 z, L a) −1.2 −1 −0.8 −0.6 −0.4 −0.2 −1.4 Jy, J0 −1.2 −1 −0.8 −0.6 −0.4 −0.2 20 30 40 50 60 70 80 x, L 10 b) −2 −1.5 −1 −0.5 0 0.5 1 1.5 2 z, L Jy, J0 20 30 40 50 60 70 80 x, L 10 c) −2 −1.5 −1 −0.5 0 0.5 1 1.5 2 z, L −1.2 −1 −0.8 −0.6 −0.4 −0.2 −1.4 Jy, J0 20 30 40 50 60 70 80 x, L 10 d) −2 −1.5 −1 −0.5 0 0.5 1 1.5 2 z, L −1.2 −1 −0.8 −0.6 −0.4 −0.2 Jy, J0 20 30 40 50 60 70 80 x, L 10 e)e) −2 −1.5 −1 −0.5 0 0.5 1 1.5 2 z, L −1.2 −1 −0.8 −0.6 −0.4 −0.2 −1.4 Jy, J0 20 30 40 50 60 70 80 x, L 10 f) −2 −1.5 −1 −0.5 0 0.5 1 1.5 2 z, L −1.2 −1 −0.8 −0.6 −0.4 −0.2 −1.4 Jy, J0 ) ) ) ) ) ) Figure 8. Current density Jy(x,z) by an analytical model (Eqs. 11–16) for {a1 = 0, a2 = −0.03, b = 22.13, f = 0, k = 1} is shown for three values of the parameter n for plane sheets (a, c, e, ϕ = 0) and curved sheets (b, d, f, ϕ = 60). In panels (a, b), n = 0.995. In (c, d) n = 1. In (e, f), n = 1.005. Panel (c) corresponds to the plane substorm sheet (see Fig. 4c). Units are normalized for CS typical width L and J0 = cB0/(4πL). D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets −2 −1.5 −1 −0.5 0 0.5 1 1.5 2 z, L −0.05 −0.04 −0.03 −0.02 −0.01 0 0.01 0.02 0.03 20 30 40 50 60 70 80 x, L b) b) Bz, B0 ) 20 30 40 50 60 70 80 x, L −2 −1.5 −1 −0.5 0 0.5 1 1.5 2 z, L −5 −4 −3 −2 −1 0 1 2 3 4 5 x 10−3 a)a) Bz, B0 x, L −2 −1.5 −1 −0.5 0 0.5 1 1.5 2 z, L 20 30 40 50 60 70 80 x, L −5 −4 −3 −2 −1 0 1 2 3 4 x 10−3 c)c) Bz, B0 L ) , −3 −2 −1.5 −1 −0.5 0 0.5 1 1.5 2 z, L 20 30 40 50 60 70 80 x, L −0.05 −0.04 −0.03 −0.02 −0.01 0 0.01 0.02 0.03 d) d) Bz, B0 ) x, L −3 −2 −1.5 −1 −0.5 0 0.5 1 1.5 2 z, L 20 30 40 50 60 70 80 x, L −0.04 −0.03 −0.02 −0.01 0 0.01 0.02 0.03 f)f) Bz, B0 ) Figure 9. Magnetic field component Bz(x,z) by an analytical model (Eqs. 11–16) for {a1 = 0, a2 = −0.03, b = 22.13, f = 0, k = 1} is shown for three values of the parameter n for plane sheets (a, c, e, ϕ = 0) and curved sheets (b, d, f, ϕ = 60). In panels (a, b), n = 0.995. In (c, d), n = 1. In (e, f) n = 1.005. Panel (c) corresponds to the plane substorm sheet (see Fig. 4c). Units are normalized for CS typical width L and B0. sented in Table 1 of Runov et al. (2006). Assuming Vi = q V 2x + V 2y + V 2z and B0 = BL, the quantities L and J0 are calculated. The plot of J0(L) is shown in Fig. 10. Most of the points, which we call “regular”, lie within the interval of L ∈[2, 8]103 km and J0 ∈[3, 15]nAm−2 (red asterisks). Other points represent extremely small values of CS param- eters, such as very low ion temperature (Ti < 2keV, blue crosses), drift velocity (Vi < 35kms−1, blue diamonds) and number density (ni < 0.2cm−3, blue asterisks). A single case an of extremely high value of Vi = 659kms−1 is marked by a magenta circle. which are mostly single-peaked current sheets. D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets 4c) with n = 1 and ϕ = 0 are shown; the bent sheet (n = 1, ϕ/2 = 30) quantities are plotted on panels (d). On panels (e) parameter n = 1.005 and ϕ = 0, and on panels (f) n = 1.005 and ϕ/2 = 30. Figure 9 shows that even so weak a variation of parame- ter n affects the distribution of Bz, mostly near to the sheet center. The range of appropriate values of n is restricted from above by the solution geometry (X-point location). Say, for current model parameters and with n = 1.01, the X point is located at x ≈65 in a plane sheet, and it approaches x ≈50 for ϕ/2 = 30. As expected, the increase of tilt angle ϕ en- hances the value of Bz, so that for ϕ/2 = 30◦Bz is growing 10 times. The solution (Eqs. 11–16) is written in normalized units, where the magnetic field is normalized for the lobe value B0, and normalization constants for the length scale and current density are Figure 8 demonstrates that the CS width is almost uniform on x and is not affected by tilt angle, controlling only the sheet location (vertical shift may be recouped by the proper choice of parameter a2). With increasing parameter n, the sheet is thinning and, correspondingly, the peaking current density is growing. The same effect is produced by enhanced geomagnetic activity. Comparison of current densities for quiet and storm conditions (not shown) reveal 20% reduc- L = 2cTi eB0Vi = 2 · 103 · Ti [keV] B0 [nT]Vi [kms−1], 103 km, (17) J0 = cB0 4πL = 0.8 · B0 [nT] L[103 km] , nAm−2. (18) (17) (18) (18) To estimate the relevance of this scaling, we make use of Cluster data of magnetotail CS crossings, pre- Ann. Geophys., 36, 641–653, 2018 www.ann-geophys.net/36/641/2018/ rovinskiy et al.: Kan-like equilibria for asymmetric current sheets D. B. D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets The analyti- cal model (Eqs. 11–16) preserves basic features of the initial Harris solution, and hence it is unable to resolve the com- plex CS structure, such as bifurcated or embedded current sheets (see, e.g., Hoshino et al., 1996; Nakamura et al., 2006; Runov et al., 2006; Artemyev et al., 2009; Petrukovich et al., 2015). It means that the cross-sheet profiles of current den- sity in our model (not shown) resemble the Harris profiles, shown in Figs. 2 and 3 of Runov et al. (2006). Hence, analyt- ical estimates of the CS width usually exceed the real values. However, in some cases (e.g., cases 3, 27 and 28) the Harris profiles may be more or less relevant to real current sheets. Figure 11 shows the model normalization constant J0 vs. peaking observed perpendicular current density (blue curve in Fig. 2 of Runov et al., 2006). It is seen that analytical es- timates and measured values of J0 mismatch in all extreme cases of Fig. 10. In other cases (“regular” points, red aster- isks) the model estimate agrees with observed values with an accuracy up to a coefficient k ∈[0.5, 2] (except for the cases 4 and 23 of Runov et al., 2006, when the discrep- ancy increases by 2.5 times). Thus, the best match of current densities is found for cases {1–3, 5–7, 11, 15–18, 22, 25–28}, D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets 649 20 30 40 50 60 70 80 x, L −2 −1.5 −1 −0.5 0 0.5 1 1.5 2 z, L −5 −4 −3 −2 −1 0 1 2 3 4 5 x 10−3 a)a) Bz, B0 −2 −1.5 −1 −0.5 0 0.5 1 1.5 2 z, L −0.05 −0.04 −0.03 −0.02 −0.01 0 0.01 0.02 0.03 20 30 40 50 60 70 80 x, L b) b) Bz, B0 −2 −1.5 −1 −0.5 0 0.5 1 1.5 2 z, L 20 30 40 50 60 70 80 x, L −5 −4 −3 −2 −1 0 1 2 3 4 x 10−3 c)c) Bz, B0 −2 −1.5 −1 −0.5 0 0.5 1 1.5 2 z, L 20 30 40 50 60 70 80 x, L −0.05 −0.04 −0.03 −0.02 −0.01 0 0.01 0.02 0.03 d) d) Bz, B0 −2 −1.5 −1 −0.5 0 0.5 1 1.5 2 z, L 20 30 40 50 60 70 80 x, L −5 −4 −3 −2 −1 0 1 2 3 4 x 10−3 e)e) Bz, B0 −2 −1.5 −1 −0.5 0 0.5 1 1.5 2 z, L 20 30 40 50 60 70 80 x, L −0.04 −0.03 −0.02 −0.01 0 0.01 0.02 0.03 f)f) Bz, B0 ) ) ) ) ) Figure 9. Magnetic field component Bz(x,z) by an analytical model (Eqs. 11–16) for {a1 = 0, a2 = −0.03, b = 22.13, f = 0, k = 1} is shown for three values of the parameter n for plane sheets (a, c, e, ϕ = 0) and curved sheets (b, d, f, ϕ = 60). In panels (a, b), n = 0.995. In (c, d), n = 1. In (e, f) n = 1.005. Panel (c) corresponds to the plane substorm sheet (see Fig. 4c). Units are normalized for CS typical width L and B0. D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets If two plasma components give zero contribution in the current velocity, Vi2 −Ve2 = 0, Eq. (1) stays valid for nonuniform plasma tempera- ture (Voronina and Kan, 1993). The four-component-plasma model could be probably appropriate for magnetotail stud- ies at high levels of geomagnetic activity. Indeed, in the quiet magnetotail the population of ions {O+, O++, He++}, penetrating from the ionosphere, is less than 1% (Lennarts- son et al., 1986), and hence the approximation of “pro- ton + electron” plasma is relevant. With the growth of ge- omagnetic activity, the O+ contribution becomes essential during the main and recovery phases of intensive storm events. However, practical application of the nonisothermal model requires thorough studies, going beyond the scope of the present paper. Figure 10. Analytical model units: current density J0 vs. spa- tial scale L from Eqs. (17) and (18) for Cluster data of cur- rent sheet crossings, presented in Table 1 of Runov et al. (2006). Blue crosses show cases of the lowest ion temperature, Ti < 2keV (8, 9, 12, 13, 14 in Table 1 of Runov et al., 2006); blue dia- monds show cases of the lowest ion drift velocity, Vi < 35kms−1 (8, 9, 10, 19, 21, 29, 30); and blue asterisks show cases (20, 24) of the lowest ion number density, ni < 0.2cm−3. Magenta circle shows the case 20 of extremely high velocity, Vi = 659kms−1. All other “regular” cases are shown by red asterisks. The red line plots the fitting curve y = 1/(0.045x −0.01). plasma, CS stability analysis and numerical simulations of magnetotail dynamics. So far these studies were restricted by purely symmetric background equilibria. In this paper we present the extension of the well-known family of exact ki- netic Harris–Fadeev–Kan–Manankova solutions to the 2-D bent CS. This extension is really important, since the Earth dipole is tilted most of the time. The constancy of the proton temperature is not reflected in observations (e.g., Kissinger et al., 2012; Wang et al., 2012), and hence the isothermal model may be considered as a first approximation only, although for some local anal- ysis it seems to be rather suitable due to the small (∼10– 20 %) cross-cut variations of proton temperature, detected in observations of central-peaked current sheets (see Fig. 5 in Runov et al., 2006). D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets 650 0 5 10 15 20 0 5 10 15 20 25 30 35 40 L, 1000 km J , nA m 0 -2 Regular Ti < 2 keV Vi < 35 km/s V = 659 km s i ni < 0.2 cm−3 The curve y = 1 / (0.045x − 0.01) −1 Figure 10. Analytical model units: current density J0 vs. spa- tial scale L from Eqs. (17) and (18) for Cluster data of cur- rent sheet crossings, presented in Table 1 of Runov et al. (2006). Blue crosses show cases of the lowest ion temperature, Ti < 2keV (8, 9, 12, 13, 14 in Table 1 of Runov et al., 2006); blue dia- monds show cases of the lowest ion drift velocity, Vi < 35kms−1 (8, 9, 10, 19, 21, 29, 30); and blue asterisks show cases (20, 24) of the lowest ion number density, ni < 0.2cm−3. Magenta circle shows the case 20 of extremely high velocity, Vi = 659kms−1. All other “regular” cases are shown by red asterisks. The red line plots the fitting curve y = 1/(0.045x −0.01). and parameter ϕ specifies the CS bending. For further stud- ies the more general model (Eqs. 11–16) can be considered, where additional parameters n and k provide the more accu- rate adjustment of the magnetoplasma quantities. Moreover, for sub-Alfvénic plasma, i.e., for the low-activity periods, all model parameters may be treated as time-dependent quanti- ties (Wolf, 1983; Semenov et al., 2015). The time-dependent approach in such a modeling approach is not appropriate for the periods of explosive activity, such as storms and sub- storms, when BBFs with Alfvénic speed are produced. 0 5 10 15 20 0 5 10 15 20 25 30 35 40 L, 1000 km J , nA m 0 -2 Regular Ti < 2 keV Vi < 35 km/s V = 659 km s i ni < 0.2 cm−3 The curve y = 1 / (0.045x − 0.01) −1 Of course, the suggested analytical model is still far from universality. One significant limitation of this model is re- lated to the isothermal constraint. This constraint may be released for four-component (two positive + two negative) plasma with bi-Maxwellian distribution functions for each particle species (Kan, 1973; Voronina and Kan, 1993). In such a case the condition (2) takes the form Vik/Tik + Vek/Tek = 0, where k = {1, 2}. 5 Discussion and conclusions In empirical models (T89, T96, T01, TS05, etc.) magnetic field configurations with any plasma populations are not force-balanced since ∇× [j × B] ̸= 0, or there is no ∇P to balance Ampere’s force (Zaharia et al., 2003). That is why we crucially need kinetic force-balanced CS models for many magnetospheric studies, such as wave generation in www.ann-geophys.net/36/641/2018/ Ann. Geophys., 36, 641–653, 2018 D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets In such sheets, inaccuracy of the constant-temperature estimate does not exceed the model in- accuracy in current density or CS width. To validate the obtained analytic solution for bent CS we performed a comparison with the T96 model, used as a proxy of realistic averaged magnetospheric configuration. It is shown that the proposed model provides a reasonable ap- proximation for the magnetotail CS in a wide range of dipole tilt angles and geomagnetic activity levels. Particularly, the parameters of the analytical model can always be adjusted to fit the behavior of the magnetic FTV with an accuracy of about 10% for all distances from 5 to 30 RE tailward. For short segments (5RE) of the CS, located beyond 15RE, the agreement may be improved up to 5% (except the case of the bent CS at quiet magnetospheric conditions). The agree- ment between analytical and empirical models is found to be better for the stretched magnetic configuration, i.e., for the pre-substorm conditions. Other model limitations are the two-dimensionality and isotropy of the plasma pressure. Even with these limitations, the model stays appropriate for a wide class of problems, mentioned in the beginning of the current section. Particu- larly, we lay hopes that application of the presented model can stimulate investigations on the magnetotail CS stability to resolve the questions suggested by Kivelson and Hughes (1990): why can symmetric CS accumulate magnetic flux en- ergy more effectively, and does the threshold of substorm- initiating instability depend on degree of the CS bending? Notably, such a good agreement is obtained for the sim- plest three-parametric Kan-like model (Eqs. 7–9), where pa- rameter a controls the CS displacement from the equatorial plane, parameter b0 controls magnetic field lines stretching, Our findings are summarized as follows: www.ann-geophys.net/36/641/2018/ Ann. Geophys., 36, 641–653, 2018 rovinskiy et al.: Kan-like equilibria for asymmetric current sheets D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets 651 0 5 10 15 20 25 0 5 10 15 20 25 30 35 40 J , nA m , Runov et al. (2006) 0 -2 J , nA m , model 0 -2 Regular Ti < 2 keV V < 35 km s i V = 659 km s i ni < 0.2 cm−3 The curve y = x 4 23 −1 Figure 11. Typical current density J0 of the analytical model vs. peaking perpendicular current density from Fig. Competing interests. The authors declare that they have no conflict of interest. Competing interests. The authors declare that they have no conflict of interest. – Parameters of the asymmetric model may be adjusted to reproduce the realistic distribution of the magnetic flux tube volume at any level of geomagnetic activity; with enhancing activity the model relevance improves. The model-typical scales for CS width and current density match the corresponding parameters of the in situ regis- tered single-peaked current sheets with medium values of number density, proton temperature and drift veloc- ity; disagreement does not exceed a factor of 2. – Parameters of the asymmetric model may be adjusted to reproduce the realistic distribution of the magnetic flux tube volume at any level of geomagnetic activity; with enhancing activity the model relevance improves. The model-typical scales for CS width and current density match the corresponding parameters of the in situ regis- tered single-peaked current sheets with medium values of number density, proton temperature and drift veloc- ity; disagreement does not exceed a factor of 2. Acknowledgements. This study has been supported by the Austrian Science Fund (FWF), P 27012-N27 and I 3506-N27, and by Rus- sian Science Foundation (RSF) grant no. 18-47-05001. The authors thank Anna V. Egorova for her help with preparation of the images, and reviewers for their help in improving the paper. The topical editor, Elias Roussos, thanks two anonymous refer- ees for help in evaluating this paper. – The asymmetric solution does not contain any limitation for the tilt angle values, and hence the model is appro- priate for any Earth-like magnetosphere with arbitrary dipole inclination. D. B. Korovinskiy et al.: Kan-like equilibria for asymmetric current sheets 2 of Runov et al. (2006). Blue crosses show cases of the lowest ion temperature, Ti < 2keV (8, 9, 12, 13, 14 in Table 1 of Runov et al. (2006)); blue diamonds show cases of the lowest ion drift velocity, Vi < 35kms−1 (8, 9, 10, 19, 21, 29, 30); and blue asterisks show cases (20, 24) of the lowest ion number density, ni < 0.2cm−3. Magenta circle shows the case 20 of extremely high velocity, Vi = 659kms−1. All other “regular” cases are shown by red asterisks. The red line plots y = x. Black arrows mark cases 4 and 23, demonstrating the largest (amongst red points) discrepancy of the observed and model values. 0 5 10 15 20 25 0 5 10 15 20 25 30 35 40 J , nA m , Runov et al. (2006) 0 -2 J , nA m , model 0 -2 Regular Ti < 2 keV V < 35 km s i V = 659 km s i ni < 0.2 cm−3 The curve y = x 4 23 −1 Figure 11. Typical current density J0 of the analytical model vs. peaking perpendicular current density from Fig. 2 of Runov et al. (2006). Blue crosses show cases of the lowest ion temperature, Ti < 2keV (8, 9, 12, 13, 14 in Table 1 of Runov et al. (2006)); blue diamonds show cases of the lowest ion drift velocity, Vi < 35kms−1 (8, 9, 10, 19, 21, 29, 30); and blue asterisks show cases (20, 24) of the lowest ion number density, ni < 0.2cm−3. Magenta circle shows the case 20 of extremely high velocity, Vi = 659kms−1. All other “regular” cases are shown by red asterisks. The red line plots y = x. 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Breeding methods Dead seedlings in Malaysian ricefield, 3 wk after transplanting. fertile and sterile. The same pheno- menon was observed in the H93-106 family line. Thirteen out of 18 family lines showed the same phenomenon. All five lines of the H92-150 family line showed more seedling deaths than the lines previously mentioned. TG-43 and TG-44 had a few sterile plants. All plants were fertile in the next generation of TG-43 and TG-44. Two out of 18 family lines showed the same phenomenon. Death of thermosensitive genic male sterile seedlings in Malaysian ricefields H. Kato and M. Sobri, Japan International Research Center for Agricultural Sciences, Ohwashi 1-2, Tsukuba, Ibaraki 305, Japan; and Guok Hup Ping, Malaysian Agricultural Research and Development Institute (MARDI), 13200 Kepala Batas, Seberang Perai, Penang, Malaysia One hundred and fifty-four thermo- sensitive genic male sterile (TGMS) lines were introduced in Malaysia from the National Agricultural Research Center in Japan. The lines were bred from indica and japonica crosses and comprised 31 family lines, ranging from B 1 F 9 to F 4 . These TGMS lines were transplanted in ricefields at MARDI during the 1994-95 dry season (Nov- Feb) and the 1995 wet season (Apr- Aug). Some of the plants in 18 family lines died during the dry season (see figure). This type of seedling death rate has not been observed in nonTGMS lines introduced from Japan, and the phe- nomenon has never occurred in Japan. The 30-yr average temperature during the seedling growth month for Tokyo is 18.4 °C and that for Kuala Lumpur is 25.9 °C. Dead seedlings in Malaysian ricefield, 3 wk after transplanting. the next generation of TG-3, which originated from the fertile plants of the TG-3 line, plants were segregated into fertile and sterile. This trait came from PL12, which contains a single and recessive TGMS gene. The segregation ratio of the TGMS plants, however, was less than the expected ratio (1/4). Three out of five lines of the H92-62 family line also had seedling deaths. TG-22, TG-23, TG-24, and TG-25 had some sterile plants. In the next generation of TG-23, plants were segregated into Seedling death was observed only during high temperatures. Germina- tion of TGMS seeds was also poor. We suspect that the high temperature caused the TGMS seedlings to die. can be explained as follows: all the TGMS seedlings died from high tem- perature, so no TGMS plants lived to flower in the hot dry season. But the TGMS gene in heterozygous plants was expressed and both fertile and sterile plants were produced in the next gener- ation. The phenomenon of H92-62 was different from that of H91-4. Perhaps only a few of the TGMS seedlings died from high temperature. For H92-150, we believe most of the TGMS seedlings and the fertile plants, including the TGMS heterozygous plants, died from the high temperature. Death of thermosensitive genic male sterile seedlings in Malaysian ricefields The phenomenon observed in H91-4 Seedlings died in four of the five lines of the H91-4 family line, and no sterile plants were found (see table). In Dead seedlings in four TGMS family lines. MARDI, Penang, Malaysia. 1994-95. Line Family line Combination Generation Dead/total Sterile plants Next generation seedlings (no.) TG-1 H91-4 H89-1//H89-1/ B 1 F 9 1/20 0 TG-2 Mangetsumochi 0/20 0 TG-3 3/20 0 Fertlle; sterile TG-4 1/20 0 segregation TG-5 2/20 0 TG-21 H92-62 H89-1/X88 F 10 0/20 0 TG-22 6/20 2/14 TG-23 3/10 2/7 Fertile; sterile TG-24 12/20 2/8 segregation TG-25 4/20 6/16 TG-41 H92-150 H89-1/Mansoek//H87-35 F 9 4/20 0 TG-42 8/20 0 TG-43 11/20 1/9 All fertile plants TG-44 10/20 1/10 All fertile plants TG-45 4/20 0 TG-56 H93-106 PL12/90SL495 F 7 7/20 3/13 Fertile, sterile TG-57 5/20 3/15 segregation TG-58 2/20 0 TG-59 5/20 0 TG-60 2/20 1/18 Dead seedlings in four TGMS family lines. MARDI, Penang, Malaysia. 1994-95. The conclusions drawn from this study are speculative and need further confirmation through studies con- ducted with controlled temperature. Family lines with dead seedlings, how- ever, cannot be used to produce hybrid seed. Further selection has to be done to eliminate this trait. Thirteen out of 31 family lines did not show this pheno- menon and can therefore be used as parents for hybrids.
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Exploring interdependencies, vulnerabilities, gaps and bridges in care transitions of patients with complex care needs using the Functional Resonance Analysis Method
BMC health services research
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© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Exploring interdependencies, vulnerabilities, gaps and bridges in care transitions of patients with complex care needs using the Functional Resonance Analysis Method Ann‑Therese Hedqvist1,2*   , Gesa Praetorius3,4    and Mirjam Ekstedt1,5 Hedqvist et al. BMC Health Services Research (2023) 23:851 https://doi.org/10.1186/s12913-023-09832-7 Hedqvist et al. BMC Health Services Research (2023) 23:851 https://doi.org/10.1186/s12913-023-09832-7 BMC Health Services Research Open Access Abstract Background  Hospital discharge is a complex process encompassing multiple interactions and requiring coordi‑ nation. To identify potential improvement measures in care transitions for people with complex care needs, intra- and inter-organisational everyday work needs to be properly understood, including its interdependencies, vulnerabili‑ ties and gaps. The aims of this study were to 1) map coordination and team collaboration across healthcare and social care organisations, 2) describe interdependencies and system variability in the discharge process for older people with complex care needs, and 3) evaluate the alignment between discharge planning and the needs in the home. Methods  Data were collected through participant observations, interviews, and document review in a region of southern Sweden. The Functional Resonance Analysis Method (FRAM) was used to model the discharge process and visualise and analyse coordination of care across healthcare and social care organisations. Results  Hospital discharge is a time-sensitive process with numerous couplings and interdependencies where healthcare professionals’ performance is constrained by system design and organisational boundaries. The greatest vulnerability can be found when the patient arrives at home, as maladaptation earlier in the care chain can lead to an accumulation of issues for the municipal personnel in health and social care working closest to the patient. The possibilities for the personnel to adapt are limited, especially at certain times of day, pushing them to make trade-offs to ensure patient safety. Flexibility and appropriate resources enable for handling variability and responding to uncertainties in care after discharge. Conclusions  Mapping hospital discharge using the FRAM reveals couplings and interdependencies between vari‑ ous individuals, teams, and organisations and the most vulnerable point, when the patient arrives at home. Resilient performance in responding to unexpected events and variations during the first days after the return home requires a system allowing flexibility and facilitating successful adaptation of discharge planning. Keywords  Complexity, Care transitions, Hospital discharge, Patient safety, Resilience, Functional Resonance Analysis Method (FRAM) *Correspondence: Ann‑Therese Hedqvist anntherese.hedqvist@lnu.se Full list of author information is available at the end of the article *Correspondence: Ann‑Therese Hedqvist anntherese.hedqvist@lnu.se Full list of author information is available at the end of the article Background Healthcare can be classified as a complex adaptive system (CAS), as its components have multiple interde- pendencies and work in nonlinear ways [8, 9]. Complex systems have fuzzy boundaries and co-evolve with other systems [10]. A CAS has intrinsic laws or principles, such as self-organisation, emergent behaviour and the capac- ity to learn, making it difficult to predict how its compo- nents will interact temporally in a given situation [11]. This implies that the view of the horizon for each person in the system is quite limited – and the further away it is, the more unpredictable the consequences of any actions taken become. Safety problems are thus not necessarily a direct result of lacking knowledge or limited effort on the part of the healthcare professionals – rather, they are usually a result of the complexity of the system. f Poor healthcare quality is often related to the inher- ent complexity of the healthcare system, characterised by silos, multiple stakeholder interactions and a signifi- cant degree of performance variability within and across system levels [19–21]. Care transitions, especially hos- pital discharges, are complex [15, 22] and require exten- sive collaboration and information exchange. Previous research indicates the hazards associated with hospital discharge and the need of interventions to improve care transitions for people with complex care needs [15, 23– 25]. The vulnerable exchange points in care transitions may increase both the need of healthcare services and healthcare costs [26]. Previous studies on care transitions highlight the importance of applying a systems thinking perspective instead of linear thinking to investigate hos- pital discharge as a process [27–30]. Understanding and improving care transitions is considered to be of major importance [31] and is an international healthcare prior- ity [26, 32], as these are error-prone situations [33]. For- mer studies describing the discharge planning process, using the FRAM, have mapped the variability and WAD up to the discharge [27–30]. One common failure identi- fied [30] was a lack of teamwork collaboration across hos- pital and community care, something the current study has focused on. One recent study [28] evaluated both pre-discharge processes, follow-up, and readmission. An illustrative example of complexity in healthcare is the multidisciplinary teamwork in coordinated discharge planning [12] and the coordination of care for older peo- ple with complex care needs. Background and favour teamwork over professional and organisa- tional boundaries. Advancements in modern medicine and technology have led to new medical interventions and promote high- quality care, creating possibilities to effectively treat acute diseases and leading to a decline in mortality and increased life expectancy [1]. The advancements also lead to increased specialisation and differentiation which may create gaps in communication and knowledge transfer between different healthcare professions and care pro- viders. Patients with chronic illnesses and complex care needs, requiring care from both healthcare and social care providers [2, 3], are especially vulnerable to frag- mentation of care as they will meet different profession- als working for several care providers, whether seeking help for chronic or acute conditions [4, 5]. Coordinating care between different care providers is recognised as dif- ficult [6] and is complicated by the diversity of actors and organisations involved. Due to the burden of increased chronic illness in the aging population, healthcare sys- tems worldwide experience challenges in providing safe and efficient care to older people with complex care needs [3, 7]. As outlined above, care transitions can be under- stood as processes in a CAS. To reflect this, care transi- tions can be analysed using systemic safety models that promote a focus not only on the individuals, but on the interactions between the people, the technology and the organisations involved. Systemic models aid the identifi- cation and understanding of potential vulnerabilities at the system level instead of focusing on individuals as the root causes of any failure (or ‘scapegoats’) [16]. However, there is often a mismatch between how work is presumed to be performed and how it is actually performed. This mismatch is also referred to as the discrepancy between work-as-imagined (WAI) and work-as-done (WAD) (16, 17). When the concepts of WAI and WAD are related to the framework of WAx [17], work may also be rep- resented by the perspectives of work-as-prescribed (in procedures and guidelines), work-as-normative (in rules or laws), work-as-observed (by the analyst), and work- as-disclosed (by the sharp-end or blunt-end operator). Depending on the dimension being investigated, as well as the perspective used for the analysis, work may thus be represented differently [18]. Regardless of this, when performance of tasks is adapted to changing operational conditions across organisations, such as constraints in time or resources, variability will occur due to trade-offs. © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Hedqvist et al. BMC Health Services Research (2023) 23:851 Page 2 of 28 The Functional Resonance Analysis Method (FRAM)hf The Functional Resonance Analysis Method (FRAM) The FRAM [36, 39] offers an approach to map and visual- ise variability and interdependencies in complex systems and examine relationships between individual processes and elements. The FRAM has been used in numerous studies within healthcare [40], providing deeper under- standing on how different components interact and drive non-linear series of actions in complex healthcare processes such as transitional care [15, 28] and hospital discharge [27, 29]. In the FRAM, the discharge process can be modelled as consisting of activities that occur daily within and between organisations. The purpose of a FRAM analysis is to describe how a system should work to achieve the intended goals and to understand how potential variability can prevent this from happening or enhance functionality [36]. It is first necessary to map and construct a model of the system (as in the current study) and then instantiate the model [41] by analysing patient scenarios from WAD (to be presented in a sepa- rate study). The aims were to 1) map coordination and team collab- oration across healthcare and social care organisations, 2) describe interdependencies and system variability in the discharge process for older people with complex care needs, and 3) evaluate the alignment between discharge planning and the needs at home. Background Achieving safe care transi- tions from hospital to home for patients with complex care needs is a complicated process requiring success- ful completion of a number of tasks, e.g., coordinating care with healthcare personnel outside hospital [13]. The structural gaps between services and settings involved in these care transitions create safety gaps [14] and a health- care system design with highly specialised care units may raise walls that obscure a patient’s care pathway and hide safety risks [15]. In a CAS, the known and the unpredict- able exist side by side. During discharge planning, this creates a need for adaptability and flexibility, where each system unit may need to relinquish some of its autonomy Hedqvist et al. BMC Health Services Research (2023) 23:851 Hedqvist et al. BMC Health Services Research (2023) 23:851 Page 3 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 Fig. 1  Study design with multiple convergent data collection methods However, that study focused on the frail older patients’ health status between admission and potential readmis- sion to identify challenges they faced during the tran- sition process. The current study differs from former studies in that it incorporates multi-professional and cross-organisational team collaboration and coordination processes from all care providers involved (hospital, pri- mary care, municipality), related to both pre- and post- discharge processes, to evaluate how discharge planning matches the needs arising when the patient returns home. This study sought to provide a detailed understanding of how coordination of care at hospital discharge is organ- ised, including inherent interdependencies and potential system variability where avoidable adverse events may occur. Furthermore, we hoped to find how discharge planning in cross-organisational collaboration aligns with the needs that arise when the patient is enrolled in or returned to home healthcare and social care. Therefore, it was considered to be valuable to map the critical situa- tions where alignments between demands and resources occur and where there were misalignments between how was described in procedures and intended to be done, and how it was done in practice.h Fig. 1  Study design with multiple convergent data collection methods Design and study setting We conducted a qualitative study using multiple con- vergent data collection methods [34, 35] to gather data on care transitions and then analysed the data with the Functional Resonance Analysis Method (FRAM) [36] (Fig. 1). Qualitative methods comprising document review, observations and interviews with healthcare and social care professionals (HSCPs) were used for data triangulation [34]. This study focused on couplings and interdependencies in care transitions, delimited to hospi- tal admission, in-patient care, hospital discharge and the first 72 h at home. In the FRAM, a function refers to the means, acts or activities that are necessary to achieve a goal or pro- duce a certain result [36, 42]. However, a function can also refer to something the organisation does, such as ‘treat patients’, or something that a technical system does automatically or after manual input. Each function is described by six aspects – Input, Output, Precondition, Resource, Control and Time [43] – as illustrated in Fig. 2. An example of how this can be realised is presented in Table 1. i The study is part of a research project performed in col- laboration with a region and municipalities in southern Sweden. The region encompasses three small to medium- sized hospitals, 40 primary care centres and twelve municipalities. Sweden has a decentralised healthcare system managed and run by regions and municipalities. The regions are responsible for primary, secondary and specialist care, while the municipalities provide health- care and social care in nursing homes and home care ser- vices [37, 38]. The output of one function interacts or ‘couples’ with other functions. In a visual representation of a FRAM model, this is illustrated by lines or ‘couplings’ connect- ing the functions. Functional upstream–downstream couplings create the basis for functional resonance [41], as they mean that the variability of a function may Page 4 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 Fig. 2  The six aspects of a function in the FRAM model [41] information. They in turn informed their personnel. All participants were informed prior to observations and interviews and gave informed consent to participate in the study. The sample consisted of 60 HSCPs from in-hospital care, ambulance care, primary care and municipal care. The participants had a range of professions and roles: registered nurses, physicians, care coordinators, occu- pational therapists, physiotherapists, social service officers, assistant nurses, ambulance nurses, as well as first-line managers. Data collection and procedureh The data collection was performed from June 2020 to October 2021, as an iterative and reflexive process, con- verging data from observations, interviews, and docu- ment review. Our use of multiple data sources allowed for convergence, to achieve comprehensive data and to expand and enrich the understanding of the complex care transition processes [34]. The data collection encom- passed a total of 45 interviews, eleven participant obser- vations and five meeting observations (Table 2). be amplified or dampened by other system functions. Internal variability relates to the nature of a function, for example bias in decision-making or assessment or the quality and effectiveness of communication in an organisation. External variability emerges from the vari- ability of the conditions or work environment in which the function is performed, affected by unspoken norms and expectations or by organisational factors such as regulations and legal constraints. By creating a visual of the intra- and inter-organisational complexity and vari- ability of the care transition process, the FRAM may aid discovery of potential interdependencies, vulnerabilities and gaps where discontinuities occur, thereby potentially contributing to improved patient safety. Design and study setting This provided a multidisciplinary and cross-stakeholder perspective on collaboration in care transitions. Fig. 2  The six aspects of a function in the FRAM model [41 Document review First, to develop observation protocols and questions for interviews, written materials were collected by search- ing and accessing available documents on government websites as well as digital and printed versions used in everyday work at the units being observed. Docu- ments included in the review were national laws [45–48], regional guidelines (n = 3) and local routines from in- hospital care (n = 2) and municipal care (n = 2) describ- ing the discharge process. The documents were analysed using document review, mapping the discharge process according to regulations and routines (i.e., WAI [41]). Sampling and participants A purposive sampling strategy was adopted [44] to recruit participants from various professions and care providers involved in care transitions from hospital to home, to maximise data variation [44]. The extensive sampling enabled capture of the intra- and inter-organ- isational perspectives of stakeholders involved in the delivery of healthcare and social care in such transitions. The operation managers from each organisation received an introduction to the project through written and verbal Participants a total of 86  h, with personnel shadowed in their daily work. Various forms of meetings were observed, includ- ing coordination meetings, care planning meetings and discharge conversations between patients and physicians. Fieldnotes taken in association with the observations were rewritten later the same day. background) was present and recognisable to the par- ticipants, but not an active participant with a role in the social context, enabling observation and occasional inter- actions leading to a high level of involvement while main- taining detachment [35]. Before meeting observations, all participants were informed and gave consent. To protect the privacy of all parties, as well as to avoid gathering sen- sitive information like patient data, none of the meetings were recorded. Instead, the observer wrote fieldnotes, in line with participant observation methods [35]. Observations To gain an understanding of how the everyday work was carried out [36], participant observations were per- formed [35] in multiple contexts. The researcher (nurse Table 1  Example of aspects of a function (‘Initiate planning of coordinated care’) in the FRAM model Aspect Description Example Input That which starts the function. Link to previous/upstream functions What a function uses to produce the output Need of coordinated care Outputs The result of a function. Constitutes links to subsequent/downstream functions Initiated coordination Precondition Conditions that should be satisfied for a function to be executed Consent from patient Resource What is needed or consumed to execute a function Care contact Control What regulates or monitors a function Policies for coordinated care Time Temporal constraints and requirements affecting the function Within 24 h Hedqvist et al. BMC Health Services Research (2023) 23:851 Page 5 of 28 Table 2  Data collection and participants 2a) Data collection n Interviews 45 Participant observations 11 Meeting observations: 5   Coordination meeting 2   Care planning meeting 2   Discharge conversation 1 2b) Occurrences in Participants n (60) Interviews (45) Participant observations (11) Meeting observations (24) Medical and geriatric hospital wards (12) (7) (6) (12)   Registered nurse 5 2 4 3   Care coordinator 4 4 2 4   Physician 3 2 1 Ambulance care (5) (5)   Ambulance nurse 5 5 Primary care (8) (3) (2) (2)   Care coordinator 6 3 2 4   Physician 2 2 Municipal care (35) (27) (3) (14)   Occupational therapist 5 4 2   Physiotherapist 6 4 3   Registered nurse 8 7 2   Social service officer 5 2 4   Care coordinator 5 4 3 3   Assistant nurse 4 4   First-line manager 2 2 Table 2  Data collection and participants 2b) Occurrences in Participants n (60) Interviews (45) Participant observations (11) Meeting observations (24) Medical and geriatric hospital wards (12) (7) (6) (12)   Registered nurse 5 2 4 3   Care coordinator 4 4 2 4   Physician 3 2 1 Ambulance care (5) (5)   Ambulance nurse 5 5 Primary care (8) (3) (2) (2)   Care coordinator 6 3 2 4   Physician 2 2 Municipal care (35) (27) (3) (14)   Occupational therapist 5 4 2   Physiotherapist 6 4 3   Registered nurse 8 7 2   Social service officer 5 2 4   Care coordinator 5 4 3 3   Assistant nurse 4 4   First-line manager 2 2 Interviewsh The knowledge gleaned from the document review was expanded through interviews with first-line managers. These interviews related to the work performed at the management level and what tasks were supposed to be performed in the discharge process, contributing to the understanding of WAI. The interviews were recorded and transcribed verbatim. A semi-structured observation guide was used, build- ing on the understanding of WAI gained from the docu- ment review. It encompassed themes relevant to care transitions and hospital discharge, such as coordination, communication, and information exchange. Observa- tions were complemented with contextual inquiries [35] to understand what was happening (for example ‘Could you tell me about what you are doing there?’). These observations were performed in hospital wards (n = 6), primary care (n = 2) and community settings (n = 3) for To create a more in-depth view of the research prob- lem, and to study different ways of understanding the work processes in care transitions, the observations were complemented with semi-structured interviews with personnel from throughout the care trajectory. Page 6 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 Hedqvist et al. BMC Health Services Research (2023) 23:851 The interviews were conducted on site, via telephone or via Skype/Zoom and lasted 32–75  min (mean 57.8, SD 21.2). An interview guide was developed and revised during the process, reflecting the analyti- cal process and addressing the gaps in understanding remaining after the observations. The interviews thus offered a deeper, clarifying perspective on actions already observed. The interviews were recorded and then transcribed verbatim. how the process could be performed. Then, a preliminary chronology was drafted to position the functions in order based on their internal couplings. The functions were revised until final consensus was reached. i Next, functions that – at an abstract level – made up the discharge process were identified. These functions were stepwise extended into a comprehensive model of everyday work in care transitions from hospital to home, visualised using the ‘FRAM Model Visualizer’ [42]. The model was validated through expert audits by managers and HSCPs involved in discharge process [15] with recognition and affirmation of the process, confirming the relevance of the results and the man- agement of contradictory views. Hence, no revision of the model was needed. Interviewsh Then, the potential variability of function output was assessed [41] based on what can reasonably be expected to happen in the clinical setting, based on observational and interview data. The variability determined the quality of the output, which in turn influenced aspects of other functions in the system. The FRAM model was thus used to under- stand how variability and subsequent adjustments can affect other functions and thus the discharge process as a whole. Based on different phenotypes such as timing, precision, and duration, previously suggested as vari- ability manifestations [41], two analytical clusters (tim- ing and precision) emerged in this process. The clusters characterised how the interdependence of functions could potentially lead to unexpected results, depending on how the outputs of each function could vary in tim- ing (too early, on time, too late, not at all) and precision (precise, acceptable, imprecise), from the perspective of the needs of downstream functions. This step was fol- lowed by identification of functional dependencies and potential for functional resonance that could affect the system’s ability to perform the discharge process suc- cessfully. The couplings made it possible to analyse and describe how variability of the output from one func- tion could affect other functions, without any claims of a cause-effect perspective. Lastly, the couplings and dependencies between functions informed propos- als on ways to manage possible occurrences of uncon- trolled performance emerging from implementation of changes that either dampened negative effects and absorbed variability or reinforced positive effects and amplified variability [51]. Data analysis First, materials from the document review and inter- views with managers were analysed using manifest con- tent analysis [49], mapping WAI. The materials were coded and then combined by comparing and contrast- ing them, in order to further abstract the codes into cat- egories describing the different steps in the discharge process [50].h The four-step approach described by Hollnagel [41] was applied for the analysis of WAD, preceded by a prep- aration step (Step 0), as follows: Step 0: Define the purpose of the FRAM analysis. i Step 1: Identify vital functions that are required for everyday work to succeed. Step 2: Determine and describe the system’s potential for variability. Step 3: Identify functional dependencies and poten- tial for functional resonance that may affect the sys- tem. Step 4: Propose ways to manage possible occurrences of uncontrolled performance variability. Data analysis and creation of the FRAM model of WAD were performed through an iterative and reflexive process of identifying patterns in the transcribed data and fieldnotes from observations, to find a foundation in which existing work and communication processes could be recognised as functions. The datasets were combined by comparing and contrasting the data, with contextual factors investigated and taken into considera- tion. Differences and similarities in the process descrip- tion were identified and discussed by the researchers to allow emerging contradictions to promote a deeper understanding and breadth of perspectives from the par- ticipants, which further illuminated the complexity of the phenomenon.hi Results Th The functions identified were then listed in a Microsoft Excel spreadsheet. For each function, a detailed descrip- tion including as many aspects (time, control, input, output, resources, and preconditions) as possible was identified and described, to create an understanding of The results of this study are presented in five subsec- tions. The first subsection relates to the discharge pro- cess as described in regulations and policy documents (WAI), while the three subsequent subsections present Page 7 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 in-hospital stay subsequently leading to a hospital dis- charge. During this initial phase, the patient is treated at the hospital ward and the potential need of coordi- nated care after discharge is assessed and identified, constituting the function ‘Identify the need of coordi- nated care’. If appropriate and the patient consents, the care coordination process is initiated by the function ‘Initiate planning of coordinated care’. During the ‘Plan and Agree’ phase, the future needs of home care, social support, and care aids are planned for and agreed upon, leading to the function ‘Co-create plan of coordinated care’. A coordinated individual planning meeting is per- formed (‘Execute discharge planning’) or scheduled to be held at the patient’s home after discharge. Next, the care transition is facilitated by transferring informa- tion between stakeholders (‘Transfer information from in-hospital care’) and the list of medicines is updated. To facilitate for home care personnel to administer new or changed medicines to the patient in the first days after their return home, the medicines in question are sent along with the patient by the hospital nurse. The next phase is ‘Transfer’, where the patient is dis- charged and returns home. Here, the function ‘Execute care transition’ marks the point in the process where a transition in both the physical location and caregiver responsibility is performed. There is thus a transfer of responsibility where primary care (re-)assumes medi- cal responsibility for the patient, guided by any refer- rals from the treating physician at hospital. During the ‘Implementation’ phase, the patient is received at home, initiating ‘Execute coordinated care’ where care aids are installed and care needs are handled with avail- able resources. After the return home, during the ‘Re- evaluate’ phase, coordinated care is re-evaluated to consider new or altered care needs or exacerbations in pre-existing illnesses. Results Th If possible, actions may be taken to respond to the altered needs with available resources through the functions ‘Adapt home care’, ‘Adapt social care’, or ‘Adapt care aids’. If this adaptation is insufficient WAD, using the FRAM steps to outline potential gaps within the care transition process. Based on the results of the analysis (steps 1–3), measures to manage variability, risks, and gaps are suggested in the final step. The discharge process as described in regulations and policy documentsh The review of laws, regulations, and policy documents showed that the discharge process was intended to be achieved through eight overarching steps involving mul- tiple stakeholders from several professions, as well as the patient and family. The steps, listed in Table 3, represent WAI and serve to achieve the goal of safe and secure dis- charge from in-patient care.hii The final step of re-evaluating also includes identifi- cation of increased or altered needs for coordination, which may lead to the beginning of a new cycle of care transition. Vital functions required for everyday work to succeed The next step is then to discharge the patient so they can go home. If there is a need for coordinated care and the patient needs new or altered care efforts at home, the physician may delay discharge until preparations for the efforts are completed Function (i.e., event) Description d A patient is assessed as needing inpatient care. If there are hospital beds available, the patient is admitted to the ward and treated and cared for by healthcare professionals Perceived need of prehospital assessment and care Need of inpatient care Emerging needs Inadequate possibility of care in home Adequate care efforts in home Individual plan of care Medical plan at exacerbation Medical assessment or dialogue Vital functions required for everyday work to succeed Vital functions required for everyday work to succeed Hospital discharge as performed in practice was mod- elled as a complex multi-stakeholder process (Table  4), describing relevant coordination and activities that occurred daily within and between different parts of the organisations, constituting WAD. The comprehensive model encompassed a sizeable number of functions (n = 52) required for the discharge process to succeed. In the visual presentation (Fig. 3), the functions have been clustered to follow the patient and their location through the different phases that constitute the discharge process (below the FRAM model). Addi- tionally, WAD is illustrated in relation to the phases of the transition process in WAI (above the FRAM model), which is described by eight steps (Table 3). Furthermore, different colours show the range of actors involved in each function. The model begins with the function ‘Admit the patient to the ward’ located in the ‘Identify and Initi- ate’ phase, as this function indicates the start of an Table 3  The eight steps describing the hospital discharge process as work-as-imagined Step in process Description of action Identify Identify patients in need of coordinated care Initiate Initiate coordination process Plan Plan and prepare for discharge Agree Jointly determine and agree that the patient is fit for discharge Facilitate Facilitate the care transition from hospital by supplying medicines, lists and care aids Transfer Transfer information and care responsibility from one care provider to another Implement Implement and perform the coordinated efforts in the home Re-evaluate Re-evaluate and reconsider the coordinated efforts in light of perceived needs after initiating the planned efforts Table 3  The eight steps describing the hospital discharge process as work-as-imagined Step in process Description of action Identify Identify patients in need of coordinated care Initiate Initiate coordination process Plan Plan and prepare for discharge Agree Jointly determine and agree that the patient is fit for discharge Facilitate Facilitate the care transition from hospital by supplying medicines, lists and care aids Transfer Transfer information and care responsibility from one care provider to another Implement Implement and perform the coordinated efforts in the home Re-evaluate Re-evaluate and reconsider the coordinated efforts in light of perceived needs after initiating the planned efforts Page 8 of 28 Hedqvist et al. Vital functions required for everyday work to succeed BMC Health Services Research (2023) 23:851 Table 4  Work-as-done in the hospital discharge process as modelled by the FRAM Function (i.e., event) Description Admit patient to ward A patient is assessed as needing inpatient care. If there are hospital beds available, the patient is admitted to the ward and treated and cared for by healthcare professionals Identified aspects: Input Patient to hospital Output Patient to inpatient care During inpatient care Precondition Need of inpatient care Resource Availability to beds on ward Control Decision on admission Decision to re-admit patient Adapt aids at home When there is an altered need of aids, the aids supplied may be altered, depending on availability Identified aspects: Input Altered need of aids Output Adequate aids in home Adapt home care When medical or care needs are altered, the home care efforts may be adapted to better suit the patient’s needs Identified aspects: Input Altered need of home care Output Adequate home care efforts Adapt social care at home When there is an altered need of social care, the social care efforts may be altered to better suit the patient’s needs Identified aspects: Input Altered need of social care Output Adequate social care efforts in home Assess ADL and mobility If the patient needs coordinated care after discharge, an assessment of ADL and mobility is conducted to get an under‑ standing of what support and aids the patient may need after discharge. This is done by rehab personnel such as physical therapists or occupational therapists at hospital, depending on availability. This assessment is otherwise performed by nurses on the ward Identified aspects: Input Initiated care coordination process Output Rehab assessment Resource Availability to rehab personnel Assess patient in pre‑ hospital setting If there is a perceived need of prehospital assessments and care, the municipal staff, family or patient may call emergency services. The ambulance personnel then conduct an assessment in the prehospital setting Identified aspects: Input Perceived need of prehospital assessment and care Output Need of inpatient care Emerging needs Inadequate possibility of care in home Resource Adequate care efforts in home Control Individual plan of care Medical plan at exacerbation Medical assessment or dialogue Assess patient ready for discharge When the medical treatment is completed, the patient is assessed as medically fit for discharge and the physician can assess whether the patient is ready for discharge. Assess patient ready for discharge Table 4  (continued) Function (i.e., event) Description Primary care designates a person as a care contact for the patient. This person can call for a CIP (Coordinated Individual Plan‑ ning) Care contact Care contact e The permanent care contact in primary care calls for a CIP at home no later than three days after the discharge notice Identified aspects: Need for CIP in home Call for CIP Patient ready for discharge Care contact Plan for evaluation Within 3 days The physician checks if there are beds available on the ward in question. If the patient has a great need of inpatient care and there are no beds available, they might be placed on a different ward Resource Control Assess patient ready for discharge When the medical treatment is completed, the patient is assessed as medically fit for discharge and the physician can assess whether the patient is ready for discharge. The next step is then to discharge the patient so they can go home. If there is a need for coordinated care and the patient needs new or altered care efforts at home, the physician may delay discharge until preparations for the efforts are completed Identified aspects: Input Output Identified aspects: Input Output Before discharge Page 9 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 Table 4  (continued) Function (i.e., event) Description Assign care contact Primary care designates a person as a care contact for the patient. This person can call for a CIP (Coordinated Individual Plan‑ ning) Identified aspects: Output Care contact Call for a CIP in home The permanent care contact in primary care calls for a CIP at home no later than three days after the discharge notice Identified aspects: Input Need for CIP in home Output Call for CIP Precondition Patient ready for discharge Resource Care contact Control Plan for evaluation Time Within 3 days Check bed availability The physician checks if there are beds available on the ward in question. If the patient has a great need of inpatient care and there are no beds available, they might be placed on a different ward Identified aspects: Output Availability to beds on ward Co-create plan of coordinated care There is an ongoing dialogue between the various units involved in the patient’s case. Regular status updates are com‑ municated from healthcare professionals in inpatient care. Questions are asked between the units to plan and coordinate efforts and aids before the patient returns home. Time Collaborate within and between organizations A lot of planning and dialogue takes place within and between the various healthcare organizations, though the patient and family do not appear to be represented Resource Control A preliminary plan for the care efforts is written after dialogue between all the parties involved Identified aspects: Input Initiated care coordination process Output Prepared plan for care in home Plan for evaluation Plan for aid tools in home Possibility of dialogue Precondition Consent from patient Rehab assessment Need of coordinated care Resource Knowledge of patient Knowledge of family Interprofessional collaboration Digital communication for collaboration Control Needs of patient Needs of family Time During inpatient care Collaborate within and between organizations A lot of planning and dialogue takes place within and between the various healthcare organizations, though the patient and family do not appear to be represented Identified aspects: Output Interprofessional collaboration Conduct admis‑ sion conversation with patient When admitting a patient, the physician and the registered nurse on the ward conduct an admission conversation with the patient to acquire knowledge of their situation, symptoms and needs. At this juncture, family can also be involved or contacted. The patient’s requests for care are listened to. Patients and family are offered the chance to share their views on assumed needs when returning home and whether there is a need for coordination of care Identified aspects: Input Patient in inpatient care Output Requests from patient or family Needs of patient Collaborate within and between organizations Co-create plan of coordinated care Output Co-create plan of coordinated care There is an ongoing dialogue between the various units involved in the patient’s case. Regular status updates are com‑ municated from healthcare professionals in inpatient care. Questions are asked between the units to plan and coordinate efforts and aids before the patient returns home. A preliminary plan for the care efforts is written after dialogue between all the parties involved There is an ongoing dialogue between the various units involved in the patient’s case. Regular status updates are com‑ municated from healthcare professionals in inpatient care. Questions are asked between the units to plan and coordinate efforts and aids before the patient returns home. A preliminary plan for the care efforts is written after dialogue between all the parties involved Identified aspects: Output When admitting a patient, the physician and the registered nurse on the ward conduct an admission conversation with the patient to acquire knowledge of their situation, symptoms and needs. At this juncture, family can also be involved or contacted. The patient’s requests for care are listened to. Patients and family are offered the chance to share their views on assumed needs when returning home and whether there is a need for coordination of care Identified aspects: Input Output Patient in inpatient care Requests from patient or family Needs of patient Patient in inpatient care Requests from patient or family Needs of patient Needs of patient Page 10 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 Table 4  (continued) Function (i.e., event) Description Consult physician in primary care When there is a need for medical assessment of the patient, for example in case of exacerbation of a chronic illness, a dia‑ logue with the primary care physician responsible for patient care is requested Identified aspects: Input Need of medical assessment or dialogue Output Decision to re-admit patient Medical assessment or dialogue Resource Individual plan of care Decide on admission The physician decides whether to admit the patient to the ward Identified aspects: Output Decision on admission Precondition Need of inpatient care Resource Availability to beds on ward Decide upon level of care An increased need of medical care leads to a decision about what may be the most appropriate level of care. To inform this decision, the medical plan and agreed plan for care at home are used. Resource Control Updated list of medicines Resource Patient ready to go home Control Agreed upon time for homecoming Enclose medicines at discharge New medicines that have been added to the medication list are enclosed for the first few days at home. This is to ensure that the patient gets the medicines even if the pharmacy is closed, for example during discharge on the weekend Identified aspects: Input Patient goes home Output Updated list of medicines Escalate care If the situation at home is untenable despite care efforts or when the patient needs inpatient care, the patient is readmitted to hospital Identified aspects: Input Inadequate possibility of care in home Output Decision to re-admit patient Patient to hospital Get consent to share information Healthcare professionals contact the patient (or family if the patient does not have the ability to consent) to get consent to share information across caregiver boundaries. Without such consent, the coordination process cannot proceed Identified aspects: Output Consent from patient Output The patient should preferably receive adapted care efforts at home, but if this is not possible, an escalation of care may be needed, where the patient is admitted to hospital Identified aspects: Input Increased need of medical care Output Inadequate possibility of care in home Need of medical assessment or dialogue Perceived need of prehospital assessment and care Resource Adequate care efforts in home Control Medical plan at exacerbation Decision on medical care in home Agreed upon plan for care in home Individual plan of care Discharge patient The attending physician discharges the patient from inpatient care, thus terminating their inpatient trajectory in this case Identified aspects: Input Patient ready for discharge Output Patient discharged Continuous medical plan in primary care Discharge letter to patient Updated list of medicines Resource Patient ready to go home Control Agreed upon time for homecoming Enclose medicines at discharge New medicines that have been added to the medication list are enclosed for the first few days at home. This is to ensure that the patient gets the medicines even if the pharmacy is closed, for example during discharge on the weekend Identified aspects: Input Patient goes home Output Updated list of medicines Escalate care If the situation at home is untenable despite care efforts or when the patient needs inpatient care, the patient is readmitted to hospital When there is a need for medical assessment of the patient, for example in case of exacerbation of a chronic illness, a dia‑ logue with the primary care physician responsible for patient care is requested Identified aspects: Need of medical assessment or dialogue Decision to re-admit patient Medical assessment or dialogue Individual plan of care Decision on admission Need of inpatient care Availability to beds on ward An increased need of medical care leads to a decision about what may be the most appropriate level of care. To inform this decision, the medical plan and agreed plan for care at home are used. The patient should preferably receive adapted care efforts at home, but if this is not possible, an escalation of care may be needed, where the patient is admitted to hospital Identify need of coor‑ dinated care Resource f the situation at home is untenable despite care efforts or when the patient needs inpatient care, the patient is readmitted to hospital Consent from patient Page 11 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 Table 4  (continued) Function (i.e., event) Description Handle needs with available resources When new or altered needs emerge, they may be handled by family or through self-care, if their abilities are sufficient to achieve adequate care Identified aspects: Input Emerging needs Output Adequate care efforts in home Resource Support from family Self-care Have capacity for care and social support The capacity and ability of the family to provide or aid in care and social support of the patient Identified aspects: Output Ability to provide care and social support Have capacity for self- care The patient’s knowledge, experience and ability for self-care Identified aspects: Output Ability in self-care Identify altered care needs During the continued care at home, the patient, family and healthcare professionals pay attention to any changes that may indicate altered care needs and act accordingly Identified aspects: Input Monitoring of care efforts Attention to patient health status Assessment of efforts in home according to plan Output Altered need of social care Al d d f h Table 4  (continued) Function (i.e., event) Description When new or altered needs emerge, they may be handled by family or through self-care, if their abilities are sufficient to achieve adequate care Emerging needs Adequate care efforts in home Support from family Self-care Ability to provide care and social support During the continued care at home, the patient, family and healthcare professionals pay attention to any changes that m indicate altered care needs and act accordinglyi Monitoring of care efforts Attention to patient health status Assessment of efforts in home according to plan Altered need of social care Altered need of home care Altered need of aid tools Emerging needs Increased need of medical care Output Emerging needs Increased need of medical care An admission conversation is conducted with the patient where the physician or other healthcare professional determine the need for coordinated care after returning home. Admission to hospital may have been preceded by a referral from pri‑ mary care which may clearly state that there is a need for coordinated care. Alternatively, the patient may have been assessed by a registered nurse in home care before hospitalization, who has made clear that there is a need for coordinated care. The patient or family can also communicate their wishes or needs for coordination of care to the healthcare professionals. A prerequisite to considering coordination of care is that the patient consents to information sharing between healthcare providers Identified aspects: Input Output Precondition Resource Identified aspects: Input Patient in inpatient care Output Need of coordinated care Within 24 h Precondition Consent from patient Resource Requests from patient or family Referral from municipal or primary care Knowledge of patient Knowledge of family Control Needs of patient Needs of family Inform family of dis‑ charge No later than the day the patient is returning home, the family of the patient is contacted by the ward and informed of this Identified aspects: Input Patient ready to go home Output Family ready to receive patient at home Agreed upon time for homecoming Time Before discharge Table 4  (continued) Table 4  (continued) Monitor and evaluate care efforts at home Function (i.e., event) Description Inpatient healthcare professionals make an admission announcement in the digital system Cosmic Link, where the estimated date of discharge is presented. Further, healthcare professionals assess and announce the need for efforts and describe the status of the patient. A coordination case is created and units with ongoing efforts for the patient are linked to the case Initiate planning of coordinated care of coordinated care date of discharge is presented. Further, healthcare professionals assess and announce the need for efforts and describe the status of the patient. A coordination case is created and units with ongoing efforts for the patient are linked to the case Identified aspects: Input Need of coordinated care Output Initiated care coordination process Precondition Consent from patient Resource Care contact Control Policies for coordinated care Time Within 24 h Install aids at home Rehab personnel meet with the patient upon their return home. The staff test and install the necessary aids. Some aids and adaptations may take longer than others to implement and therefore the patient may be at home for several days before they are used Identified aspects: Input Patient goes home Output Aid tools in home Precondition Rehab assessment Aid tools available Control Plan for aid tools in home Time Patient goes home Invite patient and family in dialogue When care efforts for the return home are planned, patients and family are invited to participate in this dialogue, for instance in discharge planning meetings Identified aspects: Output Needs of family Needs of patient Knowledge of patient Invite patient and family in dialogue Time Within 24 h Install aids at home me Rehab personnel meet with the patient upon their return home. The staff test and install the necessary aids. Some aids and adaptations may take longer than others to implement and therefore the patient may be at home for several days before they are used Identified aspects: Input Output Precondition Patient goes home Aid tools in home Rehab assessment Aid tools available Install aids at home Notify receiving units of pending discharge Control Plan for aid tools in home Patient goes home Invite patient and family in dialogue When care efforts for the return home are planned, patients and family are invited to participate in this dialogue, for instance in discharge planning meetings Needs of family Needs of patient Knowledge of patient Knowledge of family Knowledge of family Monitor and evaluate care efforts at home An evaluation of the planned and performed care efforts is made in the patient’s home within a couple of days, together with the patient, family and relevant healthcare professionals. Based on what has worked well and less well, adjustments can be made Identified aspects: Input Patient goes home Output Monitoring of care efforts Assessment of efforts in home according to plan Precondition Execution of efforts in home according to plan Resources Call for CIP Control Individual plan of care Plan for evaluation Time Days after homecoming Notify receiving units of pending discharge As soon as possible after the decision has been made that the patient is ready for discharge, the receiving units are notified of this, so they can start preparing to receive the patient Identified aspects: Input Patient ready for discharge Output Units informed of discharge Within 3 days Need for CIP in home Time As soon as possible An evaluation of the planned and performed care efforts is made in the patient’s home within a couple of days, together with the patient, family and relevant healthcare professionals. Based on what has worked well and less well, adjustments can be made Identified aspects: Input Output Input Control Policies for coordinated care Control Before discharge Page 12 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 Identified aspects: Consent from patient Care contact Time As soon as possible after the decision has been made that the patient is ready for discharge, the receiving units are notified of this, so they can start preparing to receive the patient Identified aspects: Input Output Input Output Within 3 days Need for CIP in home As soon as possible Page 13 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 Table 4  (continued) Function (i.e., event) Description Resource Perform CIP Perform CIP Self-care and care efforts are carried out at home by patients, family and social care along with registered nurses from the municipality. The intended care is based on the medical plan from primary care and the agreed plan devised dur‑ ing the care planning before discharge. When returning home from hospital, the patient receives information both verbally and in writing from the hospital regarding their care and treatment, such as medicine changes Function (i.e., event) Description The care transition from hospital to home takes place in connection with the patient being discharged by the attending physician. Healthcare professionals on the ward ensure that the patient comes home to their accommodations and any transport needed is ordered Perform care transi‑ tion Precondition Prepared municipal home care efforts Resource Information from inpatient care Discharge letter to patient Perform CIP A coordinated individual plan is made, usually within the first few days of homecoming. Several professionals collaborate in this meeting together with patient and family, to make sure the care efforts and aids are appropriate for the current needs Identified aspects: Input Call for CIP Output Individual plan of care Time Days after homecoming Perform coordinated care at home Self-care and care efforts are carried out at home by patients, family and social care along with registered nurses from the municipality. The intended care is based on the medical plan from primary care and the agreed plan devised dur‑ ing the care planning before discharge. When returning home from hospital, the patient receives information both verbally and in writing from the hospital regarding their care and treatment, such as medicine changes Identified aspects: Input Patient goes home Output Execution of efforts in home according to plan Attention to patient health status Precondition Medical plan and treatment in primary care Adequate aid tools in home Adequate social care efforts in home Adequate home care efforts Consent from patient Updated list of medicines Medicines for homecoming Adequate care efforts in home Patient met in home Resource Information from inpatient care Booked social care personnel Aid tools in home Summary of medical care Care contact Self-care Support from family Control Agreed upon plan for care in home Decision on medical care in home Identified aspects: Input Patient discharged Output Patient goes home Days after homecoming Precondition Patient ready to go home Municipal efforts ready Family ready to receive patient at home Updated list of medicines Medicines for homecoming Prepared municipal home care efforts Resource Information from inpatient care Discharge letter to patient A coordinated individual plan is made, usually within the first few days of homecoming. Several professionals collaborate in this meeting together with patient and family, to make sure the care efforts and aids are appropriate for the current needs Identified aspects: Resource Control Page 14 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 Table 4  (continued) Identified aspects: Output Resource Self-care Ability in self-care Information from inpatient care Rehab personnel plan what aids may be needed at home after discharge. This depends on the assessment of the patient’s ADL and mobility. The rehab personnel order the aids needed and make sure they are available when the patient returns home Identified aspects: Input Output Precondition Aid tools available Rehab assessment Agreed upon plan for care in home When a decision is made regarding when it is believed the patient will be discharged and go home, the care coordinator may plan for home visits and schedule this for the social care personnel. The social care personnel are then also informed that the patient is on their way home and what interventions will be needed. The earlier this is done, the greater the chances of bringing in enough staff to perform all these tasks while still having time for other assignments already planned Function (i.e., event) Description Before the patient is discharged, a discharge planning is performed. This is often performed as a hybrid meeting where the patient (and if possible, a member of the family) and the nurse from the ward or a designated care coordinator attend physically at the hospital, while professionals from primary care and municipal care attend via video. At this meeting, the patient is informed of the prepared plan for care in home and this is agreed upon in dialogue with the family Perform discharge planning Control Policies for coordinated care Time Before discharge Perform self-care The patient performs self-care at home depending on their own ability and physical condition. The self-care is guided by the information from inpatient care Identified aspects: Output Self-care Resource Ability in self-care Control Information from inpatient care Plan and order aids Rehab personnel plan what aids may be needed at home after discharge. This depends on the assessment of the patient’s ADL and mobility. The rehab personnel order the aids needed and make sure they are available when the patient returns home Identified aspects: Input Patient ready for discharge Output Aid tools available Precondition Rehab assessment Agreed upon plan for care in home Plan and schedule social care personnel When a decision is made regarding when it is believed the patient will be discharged and go home, the care coordinator may plan for home visits and schedule this for the social care personnel. The social care personnel are then also informed that the patient is on their way home and what interventions will be needed. The earlier this is done, the greater the chances of bringing in enough staff to perform all these tasks while still having time for other assignments already planned Identified aspects: Input Units informed of discharge Output Booked social care personnel Informed social care personnel Precondition Agreed upon plan for care in home Plan municipal home care The municipal home care nurses plan for what will be needed when the patient returns home. Examples could include wound dressing or distribution and administration of medicines Identified aspects: Input Patient ready for discharge Output Prepared municipal home care efforts Precondition Agreed upon plan for care in home Time Before discharge The patient performs self-care at home depending on their own ability and physical condition. The self-care is guided by the information from inpatient carei Plan and schedule social care personnel Table 4  (continued) Table 4  (continued) Table 4  (continued) Function (i.e., event) Description Care units, patients and families are prepared for the discharge and the patient to return home. A discharge planning meet‑ ing is held where the patient and family talk to healthcare professionals in the care units that are discharging and receiving the patient at home. Aids are ordered and planned to be installed and provided in the patient’s home upon their return home. Social care personnel are notified that the patient is coming home, what time they should meet them and what meas‑ ures are to be taken. Registered nurses in home care set up care plans and medical records for the patient and plan visits, medicine administration and home care efforts for when the patient comes home Prepare care transition to home Identified aspects: Identified aspects: Input i Input Patient ready for discharge Units informed of discharge Output Patient ready to go home Latest the same day as discharge Municipal efforts ready Before discharge Medicines for homecoming Precondition Consent from patient Informed patient and family Resource Plan for aid tools in home Informed social care personnel Control Agreed upon plan for care in home Home care personnel or rehab personnel meet the patient upon their arrival at home after discharge. This requires that the municipal efforts are ready and that social care personnel are both scheduled and informed about the patient’s status and needsi Resource Control Agreed upon plan for care in home Home care personnel or rehab personnel meet the patient upon their arrival at home after discharge. This requires that the municipal efforts are ready and that social care personnel are both scheduled and informed about the patient’s status and needs Receive patient in home Control Identified aspects: Input Output Precondition Prepare care transition to home Precondition The municipal home care nurses plan for what will be needed when the patient returns home. Examples could include wound dressing or distribution and administration of medicines Patient ready for discharge Prepared municipal home care efforts Agreed upon plan for care in home Input Output Precondition Page 15 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 Policies for coordinated care When the medical care is concluded in inpatient care, the physician writes a medical summary that is made available digi‑ tally, but is also printed and handed to the patient to take home Patient discharged Summary of medical care Medical plan at exacerbation Summary of medical care Take part of infor‑ mation from other caregivers Resource Physicians at the hospital can contact primary care or read medical records to get information, see plans, etc. about the patient’s long-term care. Admission to a hospital can also be initiated after assessment and a referral by a regis‑ tered nurse in home care or a physician in primary care Function (i.e., event) Description Function (i.e., event) Description Support care at home In addition to the self-care performed by the patient, the family may also support care at home, depending on their abilities. This support can be used as a dampening factor if care is not functioning or coordinated, with family stepping in instead Identified aspects: Output Support from family Precondition Information from inpatient care Resource Ability to provide care and social support Take part of infor‑ mation from other caregivers Physicians at the hospital can contact primary care or read medical records to get information, see plans, etc. about the patient’s long-term care. Admission to a hospital can also be initiated after assessment and a referral by a regis‑ tered nurse in home care or a physician in primary care Identified aspects: Output Referral from municipal or primary care Transfer information from inpatient care Information is transferred between inpatient and primary care, municipal health and social care, and to patients and family. The information is provided both verbally and in writing Identified aspects: Input Patient ready to go home Output Information from inpatient care Time Latest the same day as discharge Treat patient in hos‑ pital The medically responsible physician on the ward treats the patient. It is possible to review documentation and medical records from the patient’s previous and ongoing care and treatment in primary care. The physician at the hospital can also contact the physician responsible for the patient in primary care, to discuss the focus of care, treatment and plans Identified aspects: Output Medically treated patient Precondition Patient in inpatient care Resource Documentation from primary care Update list of medi‑ cines At discharge, the current list of medicines needs to be updated by the attending physician. This list is then printed and handed to the patient to take home. Time Digital systems are used for healthcare and social care professionals to communicate in writing and via video meetings to update each other on the status of the patient and the presumed needs at home There are explicit guidelines and laws regarding discharge from inpatient care and what steps should be performed and by whom. There are also criteria regarding when, where and how coordination should take place Output Information from inpatient care Function (i.e., event) Description The list is also made available digitally, to be accessed by primary care and munici‑ pal home care nurses Identified aspects: Input Patient ready for discharge Output Updated list of medicines Use digital systems for collaboration Digital systems are used for healthcare and social care professionals to communicate in writing and via video meetings to update each other on the status of the patient and the presumed needs at home Identified aspects: Output Digital communication for collaboration Use policies on coor‑ dination of care There are explicit guidelines and laws regarding discharge from inpatient care and what steps should be performed and by whom. There are also criteria regarding when, where and how coordination should take place Identified aspects: Output Policies for coordinated care Write medical sum‑ mary When the medical care is concluded in inpatient care, the physician writes a medical summary that is made available digi‑ tally, but is also printed and handed to the patient to take home Identified aspects: Input Patient discharged Output Summary of medical care Medical plan at exacerbation Support care at home In addition to the self-care performed by the patient, the family may also support care at home, depending on their abilities. This support can be used as a dampening factor if care is not functioning or coordinated, with family stepping in instead Identified aspects: Treat patient in hos‑ pital Identified aspects: Support from family Output Information is transferred between inpatient and primary care, municipal health and social care, and to patients and fam The information is provided both verbally and in writing Update list of medi‑ cines Function (i.e., event) Description Function (i.e., event) Description Availability to rehab personnel The physician in primary care receives a referral from the treating physician in inpatient care and thereby gets a medical assignment for follow-up or further medical measures to be performed after inpatient care. This can also determine parts of the care performed at home Referral to primary care Page 16 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 Table 4  (continued) Function (i.e., event) Description Potential variability in the system in relation to the patient’s needs, the physician in pri- mary care may be consulted or an ambulance called to provide prehospital assessment, care, and transport back to hospital if required. The model of the discharge process reveals normal eve- ryday changeability in the functional output, reflect- ing potential variability. Variability with the potential to cause impacts on other parts of the process is most Page 17 of 28 Page 17 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 s-done in relation to the discharge process and the status of the patient Fig. 3  Work-as-done in relation to the discharge process and the status of the patient Page 18 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 Hedqvist et al. BMC Health Services Research (2023) 23:851 Table 5  Examples of potential for variability in the hospital discharge process Table 5  Examples of potential for variability in the hospital discharge process Function Output Potential for variability Expressions of variability Timing Precision Initiate planning of coordinated care Initiated care coordination process Too early Input possibly missed, risk of lacking information for correct decision On time No effect or possible damping of fur‑ ther variability Too late / Omission Delayed or non-initiation of coordi‑ nation may mean delaying the case or other healthcare providers being given a very short time to communi‑ cate and plan Precise Possible damping of further variability Acceptable No effect Imprecise Misjudging the need for coordi‑ nated care (the patient is excluded from the subsequent process and dis‑ charged without intervention) Co-create plan of coordinated care Agreed upon plan for care at home Too early A care plan created too early in the patient’s hospital care risk becoming imprecise due to the dynamic conditions of recov‑ ery. Prepare care transition to home Monitor and evaluate care efforts at home Assessment of efforts in home according to plan Potential variability in the system However, when a patient is discharged prema‑ turely, the patient may not actually be ready for discharge increasing the workload for the home care per‑ sonnel with the risk of readmissions if the situation becomes untenable in the home On time Possible damping of variability Too late A discharge late in the afternoon presses the home care personnel to receive the patient with less person‑ nel on duty and limited availability to contact managers in need of added resources Omission May contribute to resource issues in the hospital ward if the patient could have been discharged success‑ fully but is now occupying a hospital bed Precise Possible damping of variability Acceptable No effect Imprecise Increased variability in downstream functions to compensate for missed aspects Execute coordinated care at home Execution of efforts in home accord‑ ing to plan Too early - On time Dampening of variability Too late Delayed activity affecting the care of the patient, ad hoc solutions Omission Ad hoc solutions or potential omitted care of the patient Precise Possible dampening of variability Acceptable No effect Imprecise If the preparation of care efforts does not correspond to the actual needs, it may mean a lack of care and social support for the patient and "firefight‑ ing" by the various actors to solve this Monitor and evaluate care efforts at home Assessment of efforts in home according to plan Too early If the follow-up occurs too early, the patient has not had time to set‑ tle at home or to know what works and what does not On time Dampening of variability, promot‑ ing a safe and secure care adapted to the patient’s care needs Too late / Omission If it takes too long or the evalua‑ tion is omitted, there may emerge urgent needs or problems due to the care measures not being adapted to the current care needs and resources The discharge of a patient frees up a hospital bed, making it avail‑ able to a new patient. Potential variability in the system Depending on the status of the patient, the care plan may need to be re-created On time Possible damping of further variability Too late / omission Delayed or non-initiation of coordi‑ nated care plan may mean delaying the case or other healthcare providers being given a very short time to com‑ municate and plan Precise Possible damping of variability as a precise care plan will aid in situa‑ tions that may arise downstream Acceptable No effect or increased variability due to ambiguity Imprecise Incorrect depiction of current care efforts and aid tools at home may misrepresent the support available to the patient when discharged Prepare care transition to home Patient ready to go home Too early Increased variability as the patient may not be ready for discharge or the prep‑ arations made will not be relevant, the function may need to be repeated On time Possible damping of further variability Too late / omission Ad hoc solutions are used to prepare the patient with limited time Precise Possible damping of variability Acceptable Increases variability downstream to compensate for flaws in preparation Imprecise In lack of accurate preparation, the correct medications, adapted aid tools or care efforts will not be avail‑ able when the patient comes home. The municipal staff will then need to devote considerable time to try to solve this, taking time from other patients and assignments Co-create plan of coordinated care Agreed upon plan for care at home Too early Co-create plan of coordinated care Agreed upon plan for care at home Too early On time Too late / omission Page 19 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 Hedqvist et al. BMC Health Services Research (2023) 23:851 Table 5  (continued) Function Output Potential for variability Expressions of variability Timing Precision Execute care transition Patient goes home Too early The discharge of a patient frees up a hospital bed, making it avail‑ able to a new patient. Execution of efforts in home accord‑ ing to plan Potential variability in the system apparent in functions related to the planning and prepa- ration for the care transition (during the ‘Plan and Agree’ and ‘Facilitate’ phases) and the evaluation at home (the ‘Re-evaluate’ phase) based on timing and precision of performance. The potential for internal variability relates to bias in assessments of what care aids a patient may need on arrival at home (originating from the func- tions ‘Assess ADL and mobility’ and ‘Plan and order care aids’), or whether a patient is truly ready for dis- charge (‘Assess patient ready for discharge’). The quality of communication via digital systems can also be a fac- tor complicating the assessment of care needs. External variability is exemplified by the legal constraints limiting the transfer and sharing of patient information between the stakeholders involved in a care transition or by the limited personnel resources available when a patient is discharged late in the afternoon. Examples of potential variability related to timing and precision in a selection of functions are presented in Table 5. One of the functions with the most couplings upstream and downstream is ‘Execute coordinated care at home’. The quality of the output is affected by numer- ous upstream functions that will amplify or dampen the variability, depending on the timing and precision of their performance. Consequently, when healthcare per- sonnel perform coordinated care in a patient’s home, the quality of the outcome depends on the previous performance of functions in the planning and prepara- tion for hospital discharge and care transition. When necessary, adjustments are made by municipal person- nel to get the work done. If the care needs of a patient are more extensive than the assessment and planning had prepared for, the personnel will be unable to fol- low protocol. Instead, they must resort to using ad hoc solutions or workarounds to get the work done, which may trigger functional resonance, as the variability in output of the functions creates a suboptimal basis for downstream functions. The functional dependencies and potential for func- tional resonance are here clustered into the perspec- tives of timing and precision. Functional dependencies and potential for functional resonanceh The complexity of the system becomes apparent as functional interdependencies that embody the multidis- ciplinary teamwork in care transitions. For example, the variability in the function ‘Execute care transition’ as performed by hospital personnel can affect the circum- stances for municipal personnel to execute downstream functions, creating the basis for functional resonance. Potential variability in the system However, when a patient is discharged prema‑ turely, the patient may not actually be ready for discharge increasing the workload for the home care per‑ sonnel with the risk of readmissions if the situation becomes untenable in the home Execution of efforts in home accord‑ ing to plan Too early On time Too late Omission Execute coordinated care at home Monitor and evaluate care efforts at home Monitor and evaluate care efforts at home Page 20 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 Function Output Potential for variability Expressions of variability Timing Precision Handle needs with available resources Adequate care efforts in home Too early Increased variability, possibility of miss‑ ing actual care needs On time Dampening of variability, promot‑ ing a safe and secure care adapted to the patient’s care needs Too late Increased variability Omission Increased variability Precise Dampening of variability Acceptable No effect Imprecise Timing of and variability in upstream functions determines the avail‑ ability of resources for the patient. Depending on the day of the week and the time of day, the resulting needs can be handled either at short notice, with a large amount of effort or not at all. May result in a readmis‑ sion in hospital when all options are exhausted at home apparent in functions related to the planning and prepa- ration for the care transition (during the ‘Plan and Agree’ and ‘Facilitate’ phases) and the evaluation at home (the ‘Re-evaluate’ phase) based on timing and precision of performance. The potential for internal variability relates to bias in assessments of what care aids a patient may need on arrival at home (originating from the func- tions ‘Assess ADL and mobility’ and ‘Plan and order care aids’), or whether a patient is truly ready for dis- charge (‘Assess patient ready for discharge’). The quality of communication via digital systems can also be a fac- tor complicating the assessment of care needs. External variability is exemplified by the legal constraints limiting the transfer and sharing of patient information between the stakeholders involved in a care transition or by the limited personnel resources available when a patient is discharged late in the afternoon. Examples of potential variability related to timing and precision in a selection of functions are presented in Table 5. Timing Timing is essential within the WAD process as it may impact on both the prerequisites for and the outcomes of a care transition. If a patient is discharged from hos- pital while still in recovery, the course of the next few Hedqvist et al. BMC Health Services Research (2023) 23:851 Page 21 of 28 days will be dynamic. If care planning is performed too early, the patient’s status may change and no longer match the decisions made, resulting in inadequate care or a need to review these decisions: in discharges. During evenings and weekends, resources are reduced, with limited access to decision-makers and personnel with knowledge, existing relationships with patients and insight into the care planning processes, which were performed during weekdays. At best, the per- sonnel might have access to written documentation, but this could also be limited during weekends if secretar- ies had not yet had the time to transcribe the physicians’ notes into the electronic patient records. It all depends on when in time you have the dis- charge or care planning. Sometimes it happens quickly and then things happen, and nothing matches. Then you might even get a coordinated care plan. One of our coordinators is doing a care planning for this one gentleman today, where there has already been a care planning meeting and he wasn’t supposed to get any home care. Now he’s gotten worse and will definitely need home care. […] So, it depends a bit on when in time it’s done. (Participant 25, registered nurse, municipal home care) If someone is discharged on a Friday afternoon and then there’s just temporary staff all weekend and then maybe, you’ll get a message about that on Mon- day or something and then maybe you’ll talk it over with the regular municipal personnel. But of course, things can happen over the weekend and probably do sometimes. And sometimes it’s not … sometimes it takes a long time before the patient discharge sum- mary is transcribed and sometimes things there are missed. I think that’s a potential problem. (Participant 22, physician in primary care) However, if care planning is performed too late, the personnel at the receiving end will have limited time to arrange for the arrival at home. The timing of the functions also relates to the concept of synchronisa- tion, i.e., agreement between the sending unit and the receiving unit. Precisionh The precision of the performed functions relates to the quantity and quality of patient care. During a hospital stay, many units and professions are involved in the pro- cess of planning for sustainable continued care at home. The findings of the study suggest that gaps occur in this process, obscuring possible problems which can surface when the patient comes home. One such gap is the fact that the social care personnel with the closest relation- ships to the patient are neither invited nor informed first- hand regarding care needs during the discharge process. Another illustrative example relates to information trans- fer: too little information about a patient leads to knowl- edge gaps, creating extra work to gather the missing information. This may lead to unnecessary examinations or tests or deficient care. However, too much or irrele- vant information can also create problems, as important information may be obscured. Yeah, things move quickly once they’re … when you think that now such-and-such is done with treat- ment and should go home and then it’s just wham- bam… (Participant 4, occupational therapist, municipal rehabilitation services) Yeah, things move quickly once they’re … when you think that now such-and-such is done with treat- ment and should go home and then it’s just wham- bam… (Participant 4, occupational therapist, municipal rehabilitation services) Additionally, the time of day when functions are per- formed can have major consequences for any worka- rounds that must be made to gain control over a situation and resolve it in the patient’s best interest. In particular, the weekend is a vulnerable time where the personnel must use ‘firefighting’ methods to meet care needs, until more permanent solutions can be put in place: We had one person come home on a Friday, who couldn’t get out of their wheelchair and did not have a wheelchair lift at home. And then we got a hold of … an emergency occupational therapist and physi- cal therapist and had them bring a lift that they could borrow and use over the weekend. And then our occupational therapist and physical therapist had to come over on the Monday. (Participant 37, assistant nurse, municipal social care) That the information is given, that it is relevant, a bit brief. If there’s too much text, you realise that you kind of lose track of what’s important. Straightfor- ward, clear communication, with short pathways, that they aren’t too long. Timing The analysis revealed the importance of having time and opportunity for preparation, so that everyone is ready when the patient is discharged. This includes both social care personnel, the patient and their family: Precisionh (Participant 13, registered nurse, municipal home care) And then I’ll come back a day or two later and talk a bit more once they’ve settled in at home and when there’s hopefully a bit more peace and quiet […] get an idea of how things have actually gone during the first few days at home. That follow-up is valuable, I think, because then I can see quite a lot. (Participant 1, registered nurse, municipal home care) There is an apparent gap in information transfer in the system, which can be illustrated by the case of ambulance personnel arriving at an older person’s home and try- ing to get the bigger picture of what the patient’s usual status was and what had been decided at hospital dis- charge. Access to written information was limited or non-existent, and there was difficulty getting in contact with municipal healthcare personnel with knowledge of the patient. The result was that the patient was returned to hospital and readmitted, but the solution could have been different: Precisionh (Participant 14, registered nurse, municipal home care) As emphasised in the quotation above, effective, clear and concise communication is of utmost importance for successful care transitions. The precision of information and communication regarding relevant issues is equally Friday night was a time that was frequently mentioned in the narratives of the participants as a known risk factor Hedqvist et al. BMC Health Services Research (2023) 23:851 Page 22 of 28 important for social care personnel and when informing the patient: important for social care personnel and when informing the patient: The precision of the care planning is also a central issue. Multiple stakeholders in various professions pre- pare a plan of action, together with the patient and fam- ily, regarding the care efforts needed when the patient gets home. However, such planning is hypothetical, since there are limited possibilities to know what everyday life will be like for the patient after discharge: The patient is just buried in information and paper- work when they are discharged from hospital, so they can’t remember what’s important. They get confused … it’s better if the important stuff is written down and then they can read it afterward. (Participant 118, coordinator, municipal home care) The environment is different when you get home. The conditions are completely different. (Participant 4, occupational therapist, municipal rehabilitation services) The environment is different when you get home. The conditions are completely different. (Participant 4, occupational therapist, municipal rehabilitation services) The core issue of precision in care transitions may thus emerge in the quality of information transfer and the availability, procedure and use of systems where informa- tion is stored. Availability of information in multiple sys- tems can be considered a strength, but can also generate extra work: To counteract this, municipal care personnel would wait a few days to let the patient get acquainted with the care efforts and aid tools and have time to recover from the hospital visit: Yeah, the systems are … I kind of think that you should have that information more available. There are so many different systems that you have to access to see it. Management of possible occurrences of uncontrolled performance variability In the final step of the FRAM analysis, we propose ways to manage possible occurrences of uncontrolled performance variability as presented above. The preci- sion of functions related to the planning of resources was shown to vary greatly. This indicates that for healthcare providers to have the ability to act even in uncertain conditions, their possibilities for adap- tation need to be increased. Functions like ‘Execute coordinated care at home’ have several prerequisites and controls originating from the output of the func- tion ‘Execute care transition’. However, the analy- sis indicates that variability in precision can also be dealt with by increasing the flexibility in a patient’s home after discharge. Thus, one way to dampen the effects of functional resonance could be to redistribute resources and increase them in a patient’s home the first few days after homecoming and then perform reg- ular and frequent reassessments, to adjust care flexibly and accurately. This would enable adaption to the care needs that emerge during the first few days as a way to manage uncontrolled performance variability.hf It’s so hard to get information about the patient … what has been decided … I’ll look through binders and call, but I feel like I never get any information, you just stand there with your papers and wonder … So that’s what’s missing, from the side of the munici- pality. I mean, if you’re supposed to call one of the municipal nurses, then the person in charge will have gone home or is off or it’s in the evening or there’s no binder and there aren’t any papers and I don’t have the medical records and I don’t have anything … a care plan or whatever it is that you’re supposed to … that’s been decided, and when I’m standing there and I don’t have any idea, then maybe we’ll load up that patient and head to the emergency room. And then they’ll do something … small or, yeah, they’ll admit the patient for a night or something and then it all starts over. So, you get unnecessary care and unneces- sary trips for these kinds of patients. (Participant 40, ambulance nurse) y The timing of the care transition also affects the pos- sibility of responding to unexpected events, as the Hedqvist et al. Management of possible occurrences of uncontrolled performance variability BMC Health Services Research (2023) 23:851 Page 23 of 28 Page 23 of 28 planning, suggesting that detailed planning for care at home is almost impossible. If viewed using the frame- work of WAx [17], WAI in this study would rely on work- as-prescribed and work-as-normative as presented in the guidelines and policy documents by blunt-end operators. While the policy documents presented hospital discharge as a linear process in eight consecutive steps, the FRAM model revealed a total of 52 dynamic functions to realise a transfer from hospital to home. In practice, the func- tions are often performed in parallel, are intertwined, or are performed in a back-and-forth, dynamic cooperation between a multitude of professions and stakeholders. These people work in separate documentation systems, with different technical solutions and access, regulated by different laws and regulations, which further increases complexity. Although the descriptions in WAI from ‘the blunt-end operators’ (i.e., managers and policymakers) [17] seek to give a direction and goal for the discharge process, following a standardised and normative protocol would be difficult – if at all desirable, given the diverse needs of and requirements for the patients being dis- charged from hospital to home. The findings reveal WAD to be inherently different from WAI, despite the best efforts of HSCPs. The differences in professional back- grounds, values, and cultures, along with the patients’ varying recovery processes, home environments, and supportive resources at home, mean that the reality is almost always different from the plan [53–55]. This study suggests that resources could be more efficiently utilised to manage possible occurrences of uncontrolled perfor- mance variability. Primarily, resources could be redis- tributed to allow flexible adjustments during the first few days after the return home, as some patients need less resources than planned while others need more. system appears to be more vulnerable during evenings and weekends. Variability in timing can mean that the patient is discharged too early or too late, causing a ripple effect and functional resonance with consequences for downstream functions. One way of managing this could be adding prerequisites and controls for the function ‘Execute care transition’ so that the time of discharge will not be during the vulnerable periods. Discussionh The study aimed to map coordination of care across healthcare and social care organisations, describe inter- dependencies and system variability in the discharge process for older people with complex care needs and evaluate the alignment of the discharge planning with the needs that arise at home. The modelling of the discharge process reveals, in line with former studies [27, 29, 30], that discharge planning is complex. There are multiple task interactions and numerous functional dependen- cies across organisations, with countless occasions where things can go wrong. As shown in a study of complex sociotechnical systems, a reductionist approach cannot provide a sufficiently detailed and holistic picture of the interacting components of a system to guide improve- ment [52]. The results of the current study demonstrated that the FRAM could help create an accurate and com- prehensive view of the cross-organisational collaboration, making it possible to identify areas of improvement and changes that might lead to a more efficient and effective discharge process. The FRAM provided insight into the complexities of interdependencies and variability in the components involved, such as how individuals, teams, and organisational structures – as well as the technical aspects of the discharge process – interact and influence each other throughout the discharge process. This could be used to identify potential barriers and to set strategies to achieve better outcomes for patients.h Discrepancies between daily practice (WAD) and how the process is described in protocols (WAI) have also been found in other studies [41, 53, 54, 56–59]. A large discrepancy between WAI and WAD may be an indica- tor of brittleness in the system [60]. Thus, understand- ing why deviations occur is fundamental to improving patient safety [60, 61].i However, there is a fine line between adapting to vary- ing conditions (i.e., being resilient) and going beyond the realm of safety due to the pressure of a high workload, limited resources, or the system’s expectations of high efficiency [62]. The FRAM model highlights the limita- tions in the system design that lead to ‘workarounds’ in order to manage certain tasks in time and align WAD with WAI [59]. This study reveals several gaps aris- ing during the discharge process, though these are not always visible until the patient faces reality at home, fur- ther emphasising the importance of flexibility and adapt- ability. Management of possible occurrences of uncontrolled performance variability The downstream function ‘Execute coordinated care at home’ requires some components supplied from the hospital – such as medical information regarding the care given, medica- tions needed during the first few days, and an updated medication list – and some components from the munic- ipal organisation, such as availability of adapted care aids for basic mobility and ergonomic work conditions for personnel. The most critical functions in the system for monitoring and directing efforts to manage variability are ‘Co-create plan of coordinated care’, ‘Prepare care transi- tion to home’ and ‘Execute care transition’. These three functions together constitute the grounds for the care performed at home. Discussionh The process mapped in WAD works because This study adds to the knowledge from other studies using the FRAM, by increasing our understanding of the uncontrolled variability in the outcome of the discharge Hedqvist et al. BMC Health Services Research (2023) 23:851 Page 24 of 28 Page 24 of 28 Other studies indicate that the time between the deci- sion on discharge and the care transition is a poten- tial barrier to information sharing, as time constraints lead to greater time pressure, increased performance demands and decreased flexibility [74]. Complex systems are resilient when they have diversity, as different profes- sionals deploy different behaviours to respond to actions and situations based on evidence and their own experi- ences [75]. However, according to complexity theory, a system can never be fully described or fully controlled [76]. This creates challenges in care transitions since the findings suggest many interdependencies between the actors involved, meaning that they depend on each other and on collaboration. Rasmussen [77] emphasises how circumstances may not be under the control of any one person, and that there may be systematic but unseen traps set by upstream decisions. individuals go beyond their responsibilities and do more than expected to manage the negative effects of variabil- ity. The findings thus indicate that the discharge process as described in WAI is not adapted to the current health- care system design, given existing staffing levels, struc- tures, and information systems. Furthermore, gaps occur in the information exchange between HSCPs in the planning and performance of care transitions. In the findings, the most prominent gap is that home care personnel, who often have the most up- to-date information about a patient, are not included in the information transfer to the same extent as other parties. Instead, they are supplied with certain informa- tion fragments after the patient has returned home [63]. Experiences of gaps in care are common, often arising from failures in communication and coordination of care [64] and interoperability of systems [65]. Cross-organi- sational bridges may mitigate such gaps in care [66]. The findings suggest that there are possibilities of creating shared documentation of a patient’s current care needs and plans. Strengths and limitations Care transitions for older people with complex care needs involve multiple stakeholders and a diversity of professionals and organisations. Furthermore, the sys- tem’s adaptive complexity makes it difficult to over- view, study, and evaluate. The FRAM showed potential to uncover the complexity in collaborative work in the dynamic discharge process, which can unfold in a vari- ety of ways. This might have been difficult to do with other methods. The FRAM allowed for a visualisation of the everyday activities in the interprofessional team col- laboration across organisational borders and the roles of the different professionals during the discharge process. In this study, the FRAM offered a thick description of a process that normally remains hidden and thus enabled potential development of new guidelines. Further, it also provided a detailed model of WAD and performance variability of the complex coordination of transitional care across care providers, which can be used as a guide for improvements. However, using the FRAM requires expert knowledge and experience along with access to the research field. Therefore, the method cannot be used for all types of processes [39] and may be too resource- intensive and demanding for use in healthcare quality improvement generally. Collaboration involving multiple individuals and teams within and across organisational boundaries is expected during the transition process, even though not all indi- viduals and teams communicate. If the healthcare team is seen as a CAS [72], healthcare professionals need to expand the system boundaries to understand how any action taken (or not taken) in their part of the system has a ripple effect throughout the entire system. Health- care professionals in hospital care must anticipate what problems HSCPs at the receiving end will have when the patient is discharged. In their turn, professionals in home healthcare need to manage adaptations and moderation of goals when care plans ‘as imagined’ do not fit with the needs in the patient’s home. Systems thinking and antici- pation of the consequences of processes across depart- ments remain major challenges [73]. As regards the issues of timing in the discharge pro- cess, the study shows that the time of day when the patient is discharged strongly affects the ability of per- sonnel to deal with unexpected situations. Friday after- noons are mentioned as a particularly vulnerable time for discharge of people with complex care needs. Discussionh Previous research indicates that sharing of patient data across systems, if performed effectively, can create the basis for safe care [67, 68], while also reduc- ing the workload for healthcare providers [69], limiting treatment delays [70] and decreasing overall costs for the healthcare system [71]. Strengths and limitations Pre- vious studies have shown that the time of day when a patient is considered medically fit sets temporal condi- tions and time pressures for subsequent actions [29]. Decisions later in the day were associated with increased time pressure and variability, thus affecting precision. A key strength of this study was inclusion of multi- ple perspectives across a variety of healthcare settings to explore how different clinical teams successfully sup- port care transitions. However, the heterogeneous sam- ple may have limited the ability to achieve theoretical saturation. On the other hand, we were not looking for a representative sample, but one that encompassed par- ticipants with as broad knowledge, skills, and expertise as needed to answer the research question [78, 79]. The number of participants is related to concerns about the Hedqvist et al. BMC Health Services Research (2023) 23:851 Page 25 of 28 Page 25 of 28 always surface, making the planning of care at home a hypothetical reality, left for HSCPs in the patient’s home to deal with after discharge. To achieve a safe and secure discharge and subsequent care at home, proactive work during the discharge planning process needs to be facili- tated. This may be done by increasing precision in the assessment and planning of a patient’s care. Since this is a time-sensitive process, considerations need to be made as to at what time and on what day of the week the dis- charge will be performed, so the variable needs can be dealt with accordingly. It could be useful to have a clear definition of what a secure discharge for patients with complex care needs should be, and what parameters need to be fulfilled. If security is intentionally created, the dis- charge process can proceed and perform successfully. Furthermore, to ensure the ability to respond to unex- pected events and variations by adapting care efforts at home, a redistribution of resources during the first few days after the return home would increase flexibility and patient safety. richness and thickness of the data [79]. Thus, the large amount of material represents a strength in this study. Multiple data collection methods were used as triangu- lation enables qualitative researchers to study relatively complex entities or phenomena in a holistic way [80]. The study was performed over the course of more than a year. Strengths and limitations To reduce a possible ‘Hawthorne effect’ in obser- vations [35], the researchers emphasised that it was not the performance of individuals that was being observed and that the participants were not in any way being assessed in their work. Furthermore, the observer was dressed in hospital scrubs, like other healthcare person- nel, to blend in.f Constructing the FRAM model was a trade-off between showing all the related functions and achieving a usable and understandable model. Although in-depth informa- tion was obtained on each function, some perspectives to create a complete picture of the discharge process may be lacking. To mitigate this, the model was checked for sense by professionals with experience of working with older people with complex care needs and the care transitions involved. This expert audit was performed to increase ecological validity.h By promoting insight and knowledge of other HSCPs’ areas and creating the basis for a resilient organisation with HSCPs that anticipate and adapt by going outside their boundaries, we could increase the possibility of safely bridging the gaps in the system. This study was performed in a single region of south- ern Sweden and the sampling in hospital was limited to medical and geriatric wards, to gather data that were gen- eral across the different geographical locations. Future studies should be performed in other settings to elucidate whether the results may be transferred. The study aimed at mapping and describing intra- and inter-organisational processes with a focus on coordination and collaboration between different professionals and stakeholders. This study contributed to increased understanding of the sys- tem of hospital discharge and care transitions from the perspective of healthcare professionals. It did not include patients, family, or other informal caregivers. We want to emphasise the importance of including patients and fam- ilies in future studies [15, 81]. Conclusionsh The transition process from hospital to home for older people is complex, encompassing couplings and inter- dependencies in the exchange of information between various individuals, teams, and organisations. The FRAM- based approach revealed barriers and cracks in team col- laborations. Discharge planning was carried out within a system that lacked control because of limited access to patient information and complexity in the communica- tion channels between different organizations. Due to the varying health conditions of patients, and as their com- plex needs and requirements are difficult to predict and plan for, a mismatch between discharge planning and the needs at home may occur. This makes the arrival at home the most vulnerable point of the transition process. To optimise the outcome of the discharge process for patients with complex needs, the system’s variability and adapta- tions should not be stifled. Instead, the study suggests, the system might be kept stable and resilient through flexibil- ity, adjustments, and allocation of adequate resources at the time of the patient’s return home.hi Implications for practiceh This study provides a comprehensive visualisation of the most prominent gaps and vulnerabilities in the care transition process indicating where special attention to communication and coordination is needed. The findings show the importance of consensus in the care planning process and that all relevant information is collected, dis- tributed and made available to the parties involved in the decision process and particularly in patient care at home. Reinforcing the ability to bridge gaps through collabora- tion may improve safety [82]. However, the possibility of bridging the gaps depends on both access to informa- tion and inclusion in the process. During the hospital stay, possible problems or issues waiting at home do not The findings of this study can inform managers and other decision-makers on how coordinated care is designed and managed in care transitions to align with the varying needs in patients’ homes after discharge. It can serve as a roadmap to future studies regarding the development of guidelines and training, as well as Page 26 of 28 Page 26 of 28 Hedqvist et al. BMC Health Services Research (2023) 23:851 multidisciplinary simulations to improve intra- and inter- organisational collaboration and ultimately improve care for people with complex care needs. multidisciplinary simulations to improve intra- and inter- organisational collaboration and ultimately improve care for people with complex care needs. Received: 28 October 2022 Accepted: 18 July 2023 Acknowledgements g We thank Emelie Ingvarsson, PhD student, for her assistance in data collec‑ tion. We would also like to thank the participating healthcare and social care professionals, who made this study possible. 7. Tinetti ME, Fried TR, Boyd CM. Designing health care for the most com‑ mon chronic condition—multimorbidity. JAMA. 2012;307(23):2493–4. 8. Kannampallil TG, Schauer GF, Cohen T, Patel VL. Considering complexity healthcare systems. J Biomed Inform. 2011;44(6):943–7. 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COVA1-18 neutralizing antibody protects against SARS-CoV-2 in three preclinical models
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ARTICLE ARTICLE COVA1-18 neutralizing antibody protects against SARS-CoV-2 in three preclinical models COVA1-18 neutralizing antibody protects against SARS-CoV-2 in three preclinical models NATURE COMMUNICATIONS | (2021) 12:6097 | https://doi.org/10.1038/s41467-021-26354-0 | www.nature.com/naturecommunications A full list of author affiliations appears at the end of the paper. ARTICLE ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26354-0 A A cross the world, the Coronavirus Disease 19 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to escalate1. Despite the progressive rollout of vaccines, there remains an urgent need for both curative and preventive measures, especially in individuals with high risk. Monoclonal neutralizing antibodies (NAbs), isolated from convalescent COVID-19 patients, are one of the most promising approaches and two NAb-based products have already received emergency use authorizations by regulatory agencies in both the US2,3 and Europe4,5. Although their clinical efficacy in hospitalized patients remains to be fully assessed, their capability to reduce viral loads and hospitalization in high risk individuals shows that NAbs constitute an effective treatment when administered early enough after symptom onset6–8. weaker binding to RBD compared to IgG (84 nM), with the difference mainly caused by a faster Fab off-rate (Fig. 1a, Table 1), as also observed in a different assay setting (Supplementary Fig. 1d). With an IC50 of 199 ng ml−1, the COVA1-18 Fab was 237-fold less potent at neutralizing SARS-CoV-2 pseudovirus, showing that the IgG avidity effect is important for COVA1-18 neutralization potency (Supplementary Fig. 1a, Table 1). COVA1-18 inhibits viral replication in rodents. We sought to evaluate whether COVA1-18 could control SARS-CoV-2 viral infection in a previously described Ad5-hACE2 mouse model22,23 using a 10 mg kg−1 dose. COVA1-18 administered intraper- itoneally 24 h either prior to or after a SARS-CoV-2 challenge with 104 plaque forming units (PFU) (n = 5 for treated groups, n = 3 for control group) was fully protective with no detectable viral replication in the lungs (Fig. 1b, c). We then tested the efficacy of COVA1-18 in the golden Syrian hamster model (n = 5 per group), which is naturally susceptible to SARS-CoV-2 and develops severe pneumonia upon infection24. We evaluated the effect on lung viral loads of 10 mg kg−1 of COVA1-18 given 24 h after a 105 PFU intranasal challenge (Fig. 1b, d). At 3 days post- infection (d.p.i.), 3/5 animals had high serum neutralization, while for 2/5 animals, low neutralization activity was observed (Supplementary Fig. 1e). On day 3, the COVA1-18 treated group had significantly lower median lung viral titers compared to the control group (3.5 vs 6.7 log10 PFU g−1, respectively, p < 0.01) with lowest viral titers corresponding to the higher neutralizing serum activity (Fig. 1d). Results COVA1-18 in vitro potency is dependent on avidity. To advance our earlier in vitro results9 on COVA1-18 and allow for better comparability with other studies, we used two pseudovirus assays, one using lentiviral pseudotypes with an ACE2-expressing 293 T cell line13, and one using VSV-pseudotypes with Vero E6 cells14, to confirm the potency of COVA1-18. With these assays, we found that COVA1-18 IgG inhibited lentiviral SARS-CoV-2 pseudovirus with an IC50 of 1.7 ng ml−1 (11.3 pM) and VSV- based pseudovirus with an IC50 of 9 ng ml−1 (60 pM), confirming the remarkable potency previously observed against authentic virus9 (Supplementary Fig. 1a, Table 1). These results were cor- roborated in multiple independent labs and COVA1-18 was also equipotent against the D614G variant (Table 1) that now dom- inates worldwide15–19 as well as the recently emerged B.1.1.7 variant that includes the N501Y mutation in the RBD20,21 (Table 2). COVA1-18 PrEP prevents infection in NHP. We evaluated the potential of COVA1-18 to prevent SARS-CoV-2 infection in cynomolgus macaques in a pre-exposure prophylaxis (PrEP) study. The animals were treated intravenously 24 h prior to viral challenge with a dose of 10 mg kg−1 of COVA1-18 (Fig. 2a). Treated and control animals (n = 5 per group) were challenged on day 0 with 106 PFU of SARS-CoV-2 via combined intranasal and intratracheal routes using an experimental protocol developed previously25,26. On the day of challenge, the mean COVA1-18 serum concentration was 109 ± 2.7 μg ml−1 (Fig. 2b, Supplementary Fig. 2a). COVA-18 was also detected in all respiratory tract samples and rectal samples (Fig. 2c–e, Supple- mentary Fig. 2b–d), and represented on average 1.5% and 1.2% of the total IgG in heat-inactivated content in the nasopharyngeal and tracheal mucosae, respectively. These levels remained con- stant throughout the study period and similar levels were detected at 3 d.p.i. in bronchoalveolar lavages (BAL) and saliva (Fig. 2e, f). As SARS-CoV-2 can cause damage to non-respiratory organs, we performed a pharmacokinetic study on two additional macaques to characterize the COVA1-18 distribution within the first 24 h using non heat-inactivated samples (Fig. 2g and Supplementary COVA1-18 bound strongly to SARS-CoV-2 S protein and showed no cross-reactivity with S proteins of SARS-CoV, MERS- CoV and common cold coronaviruses HKU1-CoV, 229E-CoV and NL63-CoV (Supplementary Fig. 1b)9. ARTICLE The time of treatment (24 h post-infec- tion) and 3-day study period did not allow for prevention of lung damage and recovery monitoring in this model (Supplementary Fig. 1f, g and Supplementary Table 1). We and others have previously isolated and characterized several highly potent monoclonal NAbs with half-maximum inhibitory concentration (IC50) values in the picomolar range9–12, with the majority of these targeting the receptor binding domain (RBD) on the S1 subunit of the S protein. We previously iden- tified COVA1-18, an RBD-specific monoclonal Ab, as one of the most potent NAb in vitro9. In this work, we use three different experimental models as well as mathematical modeling to demonstrate that COVA1-18 rapid and extensive biodistribution is associated with a very potent antiviral effect, and make it a promising candidate for clinical evaluation, both as a prophylactic or therapeutic treatment of COVID-19. COVA1-18 neutralizing antibody protects against SARS-CoV-2 in three preclinical models Pauline Maisonnasse 1,21, Yoann Aldon 2,21, Aurélien Marc3, Romain Marlin 1, Nathalie Dereuddre-Bosquet 1, Natalia A. Kuzmina 4,5, Alec W. Freyn 6, Jonne L. Snitselaar2, Antonio Gonçalves 3, Tom G. Caniels 2, Judith A. Burger2, Meliawati Poniman2, Ilja Bontjer 2, Virginie Chesnais7, Ségolène Diry7, Anton Iershov7, Adam J. Ronk 4,5, Sonia Jangra6, Raveen Rathnasinghe6,8, Philip J. M. Brouwer2, Tom P. L. Bijl2, Jelle van Schooten 2, Mitch Brinkkemper2, Hejun Liu9, Meng Yuan 9, Chad E. Mire 5,10, Mariëlle J. van Breemen2, Vanessa Contreras1, Thibaut Naninck 1, Julien Lemaître1, Nidhal Kahlaoui1, Francis Relouzat1, Catherine Chapon1, Raphaël Ho Tsong Fang1, Charlene McDanal11, Mary Osei-Twum12, Natalie St-Amant12, Luc Gagnon12, David C. Montefiori11, Ian A. Wilson 9, Eric Ginoux7, Godelieve J. de Bree13, Adolfo García-Sastre 6,14,15,16, Michael Schotsaert 6,16, Lynda Coughlan 6,17, Alexander Bukreyev 4,5,10, Sylvie van der Werf 18,19, Jérémie Guedj 3, Rogier W. Sanders2,20✉, Marit J. van Gils2✉& Roger Le Grand 1✉ Effective treatments against Severe Acute Respiratory Syndrome coronavirus 2 (SARS- CoV-2) are urgently needed. Monoclonal antibodies have shown promising results in patients. Here, we evaluate the in vivo prophylactic and therapeutic effect of COVA1-18, a neutralizing antibody highly potent against the B.1.1.7 isolate. In both prophylactic and therapeutic settings, SARS-CoV-2 remains undetectable in the lungs of treated hACE2 mice. Therapeutic treatment also causes a reduction in viral loads in the lungs of Syrian hamsters. When administered at 10 mg kg-1 one day prior to a high dose SARS-CoV-2 challenge in cynomolgus macaques, COVA1-18 shows very strong antiviral activity in the upper respira- tory compartments. Using a mathematical model, we estimate that COVA1-18 reduces viral infectivity by more than 95% in these compartments, preventing lymphopenia and extensive lung lesions. Our findings demonstrate that COVA1-18 has a strong antiviral activity in three preclinical models and could be a valuable candidate for further clinical evaluation. A full list of author affiliations appears at the end of the paper. 1 NATURE COMMUNICATIONS | (2021) 12:6097 | https://doi.org/10.1038/s41467-021-26354-0 | www.nature.com/naturecommunicatio Results b Study design with n = 5 per group, except mouse control group (n = 3). Hamsters were infected with 105 PFU on day 0 and treated on day 1. Mice received COVA1-18 24 h prior to or after exposure to 104 PFU. Lung viral titers at 3 days post-infection are shown for mice (c) and hamsters (d). Bars indicate medians. Mann-Whitney unpaired two-tailed t-test, p values: *:0.0179, **:0.0079. Ctl. control group (black), KD dissociation constant, PFU Plaque forming unit, PrEP pre-exposure prophylaxis (dark blue), Ther. therapeutic (light blue). 0.01 0.1 1 10 BAL 3 Saliva SARS-CoV-2 106 PFU D-1 D0 Mucosal & serum sampling D28 a b c d e COVA1-18 10 mg kg-1 Baseline sampling n = 5 0.01 0.1 1 10 -30 -15 0 5 15 20 25 0.01 0.1 1 10 10 -30-15 0 5 10 15 20 25 0.01 0.1 1 10 3 COVA1-18 (µg ml-1) Serum f Control COVA1-18 Days post-infection COVA1-18 (% of total IgG) Nasopharyngeal fluid Tracheal fluid -30 -15 0 5 10 15 20 25 0.1 1 10 100 Days post-infection COVA1-18 (% of total IgG) g Accessory Caudal L Caudal R Cranial L Cranial R Middle L Middle R Heart Liver Spleen Kidney Brain Trachea 0.1 1 10 100 COVA1-18 (ng per mg of tissue) Lungs Biodistribution - PK Fig. 2 COVA1-18 serum and mucosal pharmacokinetic in infected cynomolgus macaques. a Study design. Two groups of n = 5 were exposed to 106 PFU of SARS-CoV-2 (intranasal and intratracheal routes). Treated animals received 10 mg kg−1of COVA1-18 1 day before challenge. b COVA1-18 serum concentration (mean with range). COVA1-18 concentration reported as percent of total cynomolgus IgG in heat-inactivated (c) nasopharyngeal fluid, d tracheal fluid (means with range), e bronchoalveolar lavage (BAL) and f saliva (means ± SEMs) with n = 5, except for the control group in (f) where n = 1. g The two macaques from the pharmacokinetic study were euthanized at 24 h post-treatment and their organs analyzed to assess the biodistribution of COVA1-18. The concentration of COVA1-18 was normalized to the weight of each sample for every organ. Bars represent means. The red dashed line indicates challenge day. L left, PFU Plaque forming unit, PK pharmacokinetic, R right. Results Active viral replication, as assessed by sgRNA levels, peaked at 1-2 d.p.i. in nasopharyngeal 50 nM 100 nM 200 nM 400 nM ctl. 100 nM 200 nM 400 nM 800 nM IgG Fab a Response (nm) 0 1 2 3 4 5 KD = 7 nM KD = 84 nM 0 100 200 0 100 200 Time (s) Time (s) d b D0 D1 D3 SARS- CoV-2 Lung viral titer ctl. PrEP Ther. 0 1 2 3 4 5 D-1 PrEP Therapeutic Hamsters hACE2 mice c ctl. Ther. 2 3 4 5 6 7 8 Viral titer (log10 PFU g-1) ** * * Viral titer (log10 PFU ml-1) 50 nM 100 nM 200 nM 400 nM ctl. 100 nM 200 nM 400 nM 800 nM IgG Fab a d Response (nm) b D0 D1 D3 SARS- CoV-2 Lung viral titer 0 1 2 3 4 5 ctl. PrEP Ther. 0 1 2 3 4 5 D-1 PrEP Therapeutic Hamsters hACE2 mice c KD = 7 nM KD = 84 nM ctl. Ther. 2 3 4 5 6 7 8 Viral titer (log10 PFU g-1) ** * * Viral titer (log10 PFU ml-1) 0 100 200 0 100 200 Time (s) Time (s) Fig. 1 COVA1-18 avidity and SARS-CoV-2 protection in rodents. a Biolayer interferometry sensorgrams comparing COVA1-18 IgG and Fab binding to RBD. KDs are indicated. Representative of 3 independent experiments. b Study design with n = 5 per group, except mouse control group (n = 3). Hamsters were infected with 105 PFU on day 0 and treated on day 1. Mice received COVA1-18 24 h prior to or after exposure to 104 PFU. Lung viral titers at 3 days post-infection are shown for mice (c) and hamsters (d). Bars indicate medians. Mann-Whitney unpaired two-tailed t-test, p values: *:0.0179, **:0.0079. Ctl. control group (black), KD dissociation constant, PFU Plaque forming unit, PrEP pre-exposure prophylaxis (dark blue), Ther. therapeutic (light blue). d ctl. PrEP Ther. 0 1 2 3 4 5 Hamsters hACE2 mice c ctl. Ther. 2 3 4 5 6 7 8 Viral titer (log10 PFU g-1) ** * * Viral titer (log10 PFU ml-1) b Fig. 1 COVA1-18 avidity and SARS-CoV-2 protection in rodents. a Biolayer interferometry sensorgrams comparing COVA1-18 IgG and Fab binding to RBD. KDs are indicated. Representative of 3 independent experiments. Results 2 3 4 5 6 7 8 Viral titer (log10 PFU g-1) ** * * Viral titer (log10 PFU ml-1) 0 100 200 0 100 200 Time (s) Time (s) Fig. 1 COVA1-18 avidity and SARS-CoV-2 protection in rodents. a Biolayer interferometry sensorgrams comparing COVA1-18 IgG and Fab binding to RBD. KDs are indicated. Representative of 3 independent experiments. b Study design with n = 5 per group, except mouse control group (n = 3). Hamsters were infected with 105 PFU on day 0 and treated on day 1. Mice received COVA1-18 24 h prior to or after exposure to 104 PFU. Lung viral titers at 3 days post-infection are shown for mice (c) and hamsters (d). Bars indicate medians. Mann-Whitney unpaired two-tailed t-test, p values: *:0.0179, **:0.0079. Ctl. control group (black), KD dissociation constant, PFU Plaque forming unit, PrEP pre-exposure prophylaxis (dark blue), Ther. therapeutic (light blue). Table 2 COVA1-18 and COVA1-16 neutralization potency against variants in HEK293T hACE2 cells. IC50 (ng ml−1) COVA1-18 COVA1-16 COVA1-18 + COVA1-16 (1:1) Wild type 1.7 80.7 N/A D614G 0.7 109.2 1.0 B.1.1.7 1.4 90.8 2.2 E484K >12500 94.3 N/A B.1.351 >50000 50.6 97.6 Mean IC50 values with n ≥3, except for the cocktail with n = 2. N/A, not assessed. Fig. 2e, f). COVA1-18 was found in all organs studied, including the lungs, at concentrations of 4 to 22 ng mg−1 of tissue, except for the brain where concentrations were substantially lower (250 pg mg−1 of tissue) (Fig. 2g). Altogether, these data showed that COVA1-18 administered intravenously was rapidly and efficiently distributed to the natural sites of infection as well as to organs affected by COVID-19 pathology. Table 2 COVA1-18 and COVA1-16 neutralization potency against variants in HEK293T hACE2 cells. IC50 (ng ml−1) COVA1-18 COVA1-16 COVA1-18 + COVA1-16 (1:1) Wild type 1.7 80.7 N/A D614G 0.7 109.2 1.0 B.1.1.7 1.4 90.8 2.2 E484K >12500 94.3 N/A B.1.351 >50000 50.6 97.6 Mean IC50 values with n ≥3, except for the cocktail with n = 2. N/A, not assessed. Table 2 COVA1-18 and COVA1-16 neutralization potency against variants in HEK293T hACE2 cells. g y gy Following viral challenge, control animals showed similar genomic (g)RNA and subgenomic (sg)RNA levels and kinetics as previously described25,26 with median peak viral loads (VL) of 6.4 and 6.2 log10 copies per ml at 1-2 d.p.i. in the nasopharyngeal and tracheal swabs, respectively (Fig. 3a). Results Biolayer interferometry experiments showed that COVA1-18 IgG bound to soluble SARS- CoV-2 S protein with an apparent dissociation constant (KD) of 5 nM, and its affinity for RBD was similar at 7 nM (Fig. 1a, Supplementary Fig. 1c, d, Table 1). Its Fab displayed a 12-fold Table 1 BLI and neutralization potency of COVA1-18 IgG vs Fab in HEK293T hACE2 cells. IC50 BLI AMC (n ≥4) Duke (n = 1) Duke D614G (n = 1) Nexelis (n = 1) RBD loaded (n = 3) Soluble S loaded (n = 3) ng ml−1 pM ng ml−1 KD (nM) Ka (M−1s−1) Kd (s−1) KD (nM) Ka (M−1s−1) Kd (s−1) 1–18 IgG 1.7 11.3 9.0 7.0 9.0 7.0 1.7E+05 1.3E-03 5.0 3.7E+05 1.9E-03 Fab 199.0 3968.0 N/A N/A N/A 84.1 5.0E+04 4.1E-03 N/A N/A N/A AMC and Duke neutralization assays use lentiviral pseudotyped particles and HEK293T hACE2 cells. Nexelis neutralization assay uses VSVΔG pseudotyped particles and Vero E6 cells. BLI biolayer interferometry, RBD receptor binding domain. Table 1 BLI and neutralization potency of COVA1-18 IgG vs Fab in HEK293T hACE2 cells. 2 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26354-0 Fig. 2e, f). COVA1-18 was found in all organs studied, including the lungs, at concentrations of 4 to 22 ng mg−1 of tissue, except for the brain where concentrations were substantially lower (250 pg mg−1 of tissue) (Fig. 2g). Altogether, these data showed that COVA1-18 administered intravenously was rapidly and efficiently distributed to the natural sites of infection as well as to organs affected by COVID-19 pathology. Following viral challenge, control animals showed similar genomic (g)RNA and subgenomic (sg)RNA levels and kinetics as previously described25,26 with median peak viral loads (VL) of 6.4 and 6.2 log10 copies per ml at 1-2 d.p.i. in the nasopharyngeal and tracheal swabs, respectively (Fig. 3a). Active viral replication, as assessed by sgRNA levels, peaked at 1-2 d.p.i. in nasopharyngeal 50 nM 100 nM 200 nM 400 nM ctl. 100 nM 200 nM 400 nM 800 nM IgG Fab a d Response (nm) b D0 D1 D3 SARS- CoV-2 Lung viral titer 0 1 2 3 4 5 ctl. PrEP Ther. 0 1 2 3 4 5 D-1 PrEP Therapeutic Hamsters hACE2 mice c KD = 7 nM KD = 84 nM ctl. Ther. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26354-0 Nasopharyngeal Tracheal a b -2 0 2 4 6 8 3 4 5 6 7 8 9 Viral load (log10 copies per ml) ** ** sgRNA (log10 copies per ml) ** ** * 0 1 2 3 2 3 4 5 6 7 8 Days post-infection Days post-infection -2 0 2 4 6 8 0 1 2 3 ** ** ** ** ** ** ** ** ** ** 3 COVA1-18 LoD MF1 MF2 MF3 MF4 MF5 MF6 MF7 MF8 MF9 MF10 BAL Control LoQ 3 Nasopharyngeal Tracheal a -2 0 2 4 6 8 3 4 5 6 7 8 9 Viral load (log10 copies per ml) Days post-infection -2 0 2 4 6 8 ** ** ** ** ** ** ** ** ** ** BA 3 b ** ** sgRNA (log10 copies per ml) ** ** * 0 1 2 3 2 3 4 5 6 7 8 Days post-infection Days post-infection 0 1 2 3 3 COVA1-18 LoD MF1 MF2 MF3 MF4 MF5 MF6 MF7 MF8 MF9 MF10 Control d 0 2 4 6 8 10 Lymphocytes (x103 cells per µl) -4 2 0 4 6 7 ** Days post-infection Control COVA1-18 c Lung lesions D2 D5 ctl. 1-18 Historical ctl. D3 0 5 10 15 20 CT score d c Days post-infection D3 Historical ctl. Fig. 3 COVA1-18 pre-exposure prophylaxis protects cynomolgus monkeys against SARS-CoV-2 challenge and clinical symptoms. a Genomic (g)RNA and b subgenomic (sg)RNA loads determined by PCR in nasopharyngeal fluids (left), tracheal fluids (middle) and bronchoalveolar lavages (BAL) (right). Individual values are plotted for nasopharyngeal and tracheal samples and bars represent medians for BAL. For b, boxes and whiskers representation with min-max., median, 25th–75th percentile for n = 5 per group. c Chest CT scores were determined at 3 d.p.i. and at 2 or 5 d.p.i for historical controls (n = 8). d Absolute lymphocyte count in the blood (mean with range). Mann-Whitney unpaired two-tailed t-test, p values: * < 0.05, ** < 0.01. 1–18, COVA1-18; CT Computed Tomography, Ctl. control group, LoD limit of detection, LoQ limit of quantification. the progressive elimination of the challenge inoculum, and does not result from active replication. The gRNA and sgRNA loads in BAL were also lower in COVA1-18 recipients compared to controls but the difference did not reach statistical significance (Fig. 3a, b). Cynomolgus anti-S IgM was detected as early as 6 d.p.i. Results SARS-CoV-2 106 PFU D-1 D0 Mucosal & serum sampling D28 a b c COVA1-18 10 mg kg-1 Baseline sampling n = 5 -30 -15 0 5 15 20 25 0.01 0.1 1 10 10 COVA1-18 (µg ml-1) Serum Control COVA1-18 Nasopharyngeal fluid -30 -15 0 5 10 15 20 25 0.1 1 10 100 Days post-infection COVA1-18 (% of total IgG) 0.01 0.1 1 10 BAL d e -30-15 0 5 10 15 20 25 0.01 0.1 1 10 3 Days post-infection COVA1-18 (% of total IgG) Tracheal fluid Days post infec f f 3 Saliva 0.01 0.1 1 10 f g Accessory Caudal L Caudal R Cranial L Cranial R Middle L Middle R Heart Liver Spleen Kidney Brain Trachea 0.1 1 10 100 COVA1-18 (ng per mg of tissue) Lungs Biodistribution - PK d g Biodistribution - PK Lungs Fig. 2 COVA1-18 serum and mucosal pharmacokinetic in infected cynomolgus macaques. a Study design. Two groups of n = 5 were exposed to 106 PFU of SARS-CoV-2 (intranasal and intratracheal routes). Treated animals received 10 mg kg−1of COVA1-18 1 day before challenge. b COVA1-18 serum concentration (mean with range). COVA1-18 concentration reported as percent of total cynomolgus IgG in heat-inactivated (c) nasopharyngeal fluid, d tracheal fluid (means with range), e bronchoalveolar lavage (BAL) and f saliva (means ± SEMs) with n = 5, except for the control group in (f) where n = 1. g The two macaques from the pharmacokinetic study were euthanized at 24 h post-treatment and their organs analyzed to assess the biodistribution of COVA1-18. The concentration of COVA1-18 was normalized to the weight of each sample for every organ. Bars represent means. The red dashed line indicates challenge day. L left, PFU Plaque forming unit, PK pharmacokinetic, R right. NATURE COMMUNICATIONS | (2021) 12:6097 | https://doi.org/10.1038/s41467-021-26354-0 | www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26354-0 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26354-0 Nasopharyngeal Tracheal a b -2 0 2 4 6 8 3 4 5 6 7 8 9 Viral load (log10 copies per ml) ** ** sgRNA (log10 copies per ml) ** ** * 0 1 2 3 2 3 4 5 6 7 8 Days post-infection Days post-infection d c 0 2 4 6 8 10 Lymphocytes (x103 cells per µl) Lung lesions -4 2 0 4 6 7 ** -2 0 2 4 6 8 0 1 2 3 D2 D5 ctl. 1-18 Historical ctl. D3 0 5 10 15 20 Days post-infection CT score ** ** ** ** ** ** ** ** ** ** Control COVA1-18 3 COVA1-18 LoD MF1 MF2 MF3 MF4 MF5 MF6 MF7 MF8 MF9 MF10 BAL Control LoQ 3 Fig. 3 COVA1-18 pre-exposure prophylaxis protects cynomolgus monkeys against SARS-CoV-2 challenge and clinical symptoms. a Genomic (g)RNA and b subgenomic (sg)RNA loads determined by PCR in nasopharyngeal fluids (left), tracheal fluids (middle) and bronchoalveolar lavages (BAL) (right). Individual values are plotted for nasopharyngeal and tracheal samples and bars represent medians for BAL. For b, boxes and whiskers representation with min-max., median, 25th–75th percentile for n = 5 per group. c Chest CT scores were determined at 3 d.p.i. and at 2 or 5 d.p.i for historical controls (n = 8). d Absolute lymphocyte count in the blood (mean with range). Mann-Whitney unpaired two-tailed t-test, p values: * < 0.05, ** < 0.01. 1–18, COVA1-18; CT Computed Tomography, Ctl. control group, LoD limit of detection, LoQ limit of quantification. NATURE COMMUNICATIONS | (2021) 12:6097 | https://doi.org/10.1038/s41467-021-26354-0 | www.nature.com/naturecommunications Discussion Despite the recent approval of several SARS-CoV-2 vaccines by health authorities, the slow roll-out of vaccination campaigns will not result in resolution of the pandemic in the immediate future. Furthermore, the emergence of viral escape mutants may lead to reduced vaccine efficacy, and some individuals, such as immu- nocompromised patients or the elderly, may not mount adequate protective immune responses to vaccination. Thus, there is an urgent need to develop effective therapeutics, in particular for individuals with high risk of severe disease. Prediction models refine COVA1-18 dosage. Next, we used a viral dynamic model previously developed in the same SARS- CoV-2 NHP experimental model29 to evaluate the level of pro- tection conferred by COVA1-18, and guide potential subsequent studies on SARS-CoV-2 MAbs. The model considers a target cell limited infection in both nasopharyngeal and tracheal compart- ments. In addition to the previously developed model, we assumed that sgRNA was a proxy for the total number of non- productively and productively infected cells (see supplementary methods) and we further assumed that COVA1-18 plasma drug concentrations over time, noted C(t), was the driver of drug efficacy. We modeled the changes in C(t) using a standard first order absorption and elimination model, and we estimated the half-life of COVA1-18 in plasma to be 12.6 days (Supplementary Fig. 4a). We assumed that COVA1-18 reduces infectivity rate in both tracheal and nasopharyngeal compartments with an efficacy, noted η(t), determined by the following model ηðtÞ ¼ CðtÞ CðtÞþEC50, where EC50 is the plasma COVA1-18 concentrations corre- sponding to a 50% reduction of viral infectivity. The model fitted the viral kinetics well in all animals (Fig. 4a, Supplementary Fig. 4b, Supplementary Table 2). The EC50 was estimated to be 2.2 and 0.053 µg ml−1 in the nasopharynx and trachea, respectively, which is roughly 50 and 2000 times lower than the plasma drug concentrations of 109 µg ml−1 observed on the day of infection (see above). Thus, these results confirm that the efficacy of COVA1-18 was very high, with efficacies above 95% and 99.9% in nasopharyngeal and tracheal compartments on the day of infec- tion, respectively (Fig. 4a, Supplementary Fig. 4b). Discussion Given the long half-life of the drug, this efficacy was maintained over time, and we estimated that the mean individual efficacy of the COVA1-18 in the first 10 days following infection ranged between 96.67% and 97.50% in the nasopharynx and between 99.91% and 99.94% in the trachea (Supplementary Fig. 4c). g In hACE2-expressing mice and golden Syrian hamsters, COVA1-18 showed remarkable control of SARS-CoV-2 infection. These promising results were confirmed in NHPs, with COVA1- 18 given one day prior to infection achieving nearly complete protection in the upper respiratory tract in cynomolgus maca- ques. Using a viral dynamic model, we estimated that COVA1-18 reduced viral infectivity by >95% and 99.9% in nasopharyngeal and tracheal compartments, respectively. The robustness of these results are reinforced by the high challenge dose that we used, which was 10 to 100-fold higher than in other NHP studies evaluating NAbs for PrEP against SARS-CoV-232–38. In fact, the model allowed us to predict, without using additional animals, that a high level of protection could be achieved with lower doses of 5 mg kg−1 and 1 mg kg−1 with lower inoculum doses of 105 or 104 PFU, both in prophylactic and therapeutic settings (Supple- mentary Fig. 4, Supplementary Fig. 5). How do these levels of efficacy greater than 95% translate into clinical efficacy? In previous work, we estimated that achieving 90% efficacy would be sufficient to confer a high level of pro- tection against infection acquisition if treatment can be admi- nistered prophylactically or just after a high-risk contact38. In hospitalized patients, where viral load kinetics after admission are associated with the risk of death, we estimated that administra- tion of treatment with an efficacy higher than 90% could reduce the time to viral clearance by more than 3 days in patients over 65 years of age, which could translate into significantly lower rates of mortality in this population39. y p p Several NAbs are being developed and some have achieved clinical endpoints, such as the reduction of the risk of hospitaliza- tion in patients that initiate treatment within 5 days of symptom onset6–8, leading to their approval for emergency use22,32–37,40. However, the narrow efficacy range of FDA-approved NAbs41–43, together with rapidly spreading new variants complicate treatment strategies30,31,44,45, highlighting the need for additional treatment options, including potent NAbs, such as COVA1-18, that could be used in combination with other NAbs. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26354-0 One mutation (E725G) was detected in the S gene in the MF7 BAL sample when applying standard quality filters, but this mutation has not been previously implicated in immune escape and located outside the epitope of COVA1-18 (Supplementary Fig. 3 and Supplementary Informa- tion). The high efficacy of COVA1-18 treatment prevented recovery of viral genetic information past 3 d.p.i. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26354-0 in control animals, while no IgM was detected in treated animals at early timepoints (Supplementary Fig. 2g). Some IgM was detected at 28 d.p.i. in 3 treated animals (MF6, MF7, MF9), although levels remained lower than controls at 6 d.p.i. No anti-S specific cynomolgus IgG was detected up to the day of euthanasia in control animals (7 d.p.i.) or in treated animals up to 28 d.p.i. (Supplementary Fig. 2h). Overall, these results demonstrate that a 10 mg kg−1 dose of COVA1-18 PrEP dramatically reduced the acquisition and/or early spread of SARS-CoV-2 in the different respiratory compartments. and tracheal swabs with median values of 4.6 and 4.0 log10 copies per ml, respectively (Fig. 3b). At 3 d.p.i., viral loads were detected in the BAL with a median value of 4.9 log10 copies per ml of gRNA and 3.2 log10 copies per ml of sgRNA, including 3 animals with no detectable sgRNA. In comparison, treated animals had a reduction of 2.2 and 3.4 log10 median gRNA VL in tracheal swabs on days 1 and 2 (both p < 0.01 to controls), and had undetectable VL after day 4 (Fig. 3a). The difference was also evident in nasopharyngeal swabs, with treated animals having a reduction of 1.5 and 2.2 log10 gRNA VL on days 1 and 2 (both p < 0.01 to controls). By day 4, 4/5 treated animals had undetectable gRNA in the nasopharyngeal swabs while one animal (MF7) remained positive with a low residual gRNA signal up to 7 d.p.i. COVA1-18 treatment dramatically hindered viral replication in the upper respiratory tract as evidenced by the absence of detectable sgRNA in the nasopharyngeal and tracheal swabs for all treated animals with the exception of animal (MF9) that showed a low signal at 1 d.p.i. only in the tracheal swabs (Fig. 3b). Therefore, in the treated group, most upper respiratory tract gRNA VL likely represents p y p Analysis of lung lesions by chest computed tomography (CT) showed that all treated animals had few and small lung lesions as recorded by low CT scores at 3 d.p.i. while 2/5 controls showed mild pulmonary lesions characterized by non-extended ground- glass opacities (GGOs) with scores superior to 5, consistent with 4 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26354-0 emerging variants-of-concern (VOC), in particular E484K30,31. We evaluated the ability of COVA1-18 to neutralize VOCs B.1.1.7 and B.1.351 as well as a E484K single mutant virus. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26354-0 While COVA1-18 retains its high potency against the B.1.1.7 strain, it lost its capacity to neutralize the B.1.351 strain due primarily to the RBD E484K mutation in the spike (Supplementary Fig. 6). Therefore, we also evaluated the in vitro potency of COVA1-18 in a cocktail with COVA1-16, an antibody that neutralizes B.1.351 as well as SARS-CoV-1, but is less potent than COVA1-189 (Supplementary Fig. 6 and Table 2). This mAb cocktail retained the high potency of COVA1-18 against wild-type, D614G and B.1.1.7 and also efficiently neutralized B.1.351, providing an avenue for broad mAb prophylaxis and treatment against VOCs. what was observed in historic controls25 and mirroring the heterogeneity of COVID-19 infection in humans27 (Fig. 3c). In addition, we observed that all control animals were lymphopenic at 2 d.p.i., consistent with previous studies25,26, while all treated animals had normal lymphocyte counts throughout the study (p < 0.01 for the comparison) (Fig. 3d and Supplementary Fig. 2i). One concern about SARS-CoV-2 vaccines and NAb treatments is the possible generation of suboptimal concentrations of NAb in individuals, which could foster viral escape28. Sequencing analysis of nasopharyngeal, tracheal and BAL samples at 3 d.p.i. showed that COVA1-18 treatment resulted in enrichment of subclonal variations in N and ORF1ab. One mutation (E725G) was detected in the S gene in the MF7 BAL sample when applying standard quality filters, but this mutation has not been previously implicated in immune escape and located outside the epitope of COVA1-18 (Supplementary Fig. 3 and Supplementary Informa- tion). The high efficacy of COVA1-18 treatment prevented recovery of viral genetic information past 3 d.p.i. what was observed in historic controls25 and mirroring the heterogeneity of COVID-19 infection in humans27 (Fig. 3c). In addition, we observed that all control animals were lymphopenic at 2 d.p.i., consistent with previous studies25,26, while all treated animals had normal lymphocyte counts throughout the study (p < 0.01 for the comparison) (Fig. 3d and Supplementary Fig. 2i). One concern about SARS-CoV-2 vaccines and NAb treatments is the possible generation of suboptimal concentrations of NAb in individuals, which could foster viral escape28. Sequencing analysis of nasopharyngeal, tracheal and BAL samples at 3 d.p.i. showed that COVA1-18 treatment resulted in enrichment of subclonal variations in N and ORF1ab. NATURE COMMUNICATIONS | (2021) 12:6097 | https://doi.org/10.1038/s41467-021-26354-0 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26354-0 LoQ LoQ y p Days post-infection b c 0 -2 4 6 2 8 0 -2 4 6 2 8 Therapeutic Inoculum: 106 PFU Nasopharynx Trachea 3 4 5 6 7 8 9 2 3 4 5 6 7 8 9 2 3 4 5 6 7 8 9 2 3 4 5 6 7 8 9 2 0 -2 4 6 2 8 0 -2 4 6 2 8 PrEP Inoculum: 106 PFU Nasopharynx Trachea gRNA sgRNA Viral load (log10 copies per ml) Control 10 mg kg-1 5 mg kg-1 1 mg kg-1 0.1 mg kg-1 COVA1-18 dose Injection i.v. Days post-infection b 3 4 5 6 7 8 9 2 3 4 5 6 7 8 9 2 0 -2 4 6 2 8 0 -2 4 6 2 8 PrEP Inoculum: 106 PFU Nasopharynx Trachea LoQ LoQ c 0 -2 4 6 2 8 0 -2 4 6 2 8 Therapeutic Inoculum: 106 PFU Nasopharynx Trachea 3 4 5 6 7 8 9 2 3 4 5 6 7 8 9 2 b c Days post-infection 0 2 4 6 2 Fig. 4 Modeling of viral dynamics and treatment efficacy. a Individual prediction of the nasopharyngeal genomic (g)RNA and subgenomic (sg)RNA in control (top) and treated animals (bottom) with individual efficacy prediction indicated (green line). The dashed red line indicates the time of infection. gRNA (squares) and sgRNA (circles) data are indicated as plain (above LoQ) or open (below LoQ). b Model predictions of gRNA and sgRNA dynamics with 4 doses of COVA1-18 given 24 h prior to challenge (arrow). c Simulation as in (b) with COVA1-18 given 24 h post-infection. Black dotted lines indicate LoQ (limit of quantification), i.v. intravenous, PFU plaque forming units, PrEP Pre-exposure prophylaxis. lower inoculum doses of 104 or 105 PFU, as used in other studies32,33,37 (Supplementary Table 3), a dose of 10 mg kg−1 COVA1-18 could reduce the viral load even more dramatically (Supplementary Fig. 4d and Supplementary Fig. 5a, b). Although it is difficult to compare results obtained with different experimental and virological models, this model shows that the in vivo efficacy of COVA1-18 is comparable with what has been obtained for other advanced NAbs in clinical development. found in the B.1.1.28 lineage, similar to what has been found with first wave convalescent plasma and many NAbs30,31. Discussion The plasma half-life was 12.6 days, albeit lower to what is found typically for human NAbs in humans37, ranging from 15 to 25 days, and consistent with values reported for other human NAbs in the macaque model (Supple- mentary Table 3). The efficacy in this model was high, despite the high challenge dose (106 PFU) used here. We estimated that with Next, we used our model to investigate changes in experimental conditions, such as COVA1-18 dose being administered at a lower dose and/or after the viral challenge (see methods). In all scenarios considered, a dose of 5 mg kg−1 was determined to provide nearly similar results than 10 mg kg−1 (Fig. 4b, c, Supplementary Fig. 5a, b). A dose of 1 mg kg−1 could be sufficient to prevent active viral replication as long as treatment is given prior to infection, but might be insufficient in a therapeutic setting. However, this dose could be relevant if lower doses of virus were used for infection, such as 104 or 105 PFU (Supplementary Fig. 4d–g). COVA1-18/1-16 cocktail neutralizes B.1.351. Many highly potent RBD-targeting mAbs are affected by mutations in 5 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26354-0 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26354-0 gRNA sgRNA Viral load (log10 copies per ml) Control 10 mg kg-1 5 mg kg-1 1 mg kg-1 0.1 mg kg-1 COVA1-18 dose Injection i.v. LoQ LoQ MF1 MF2 MF3 MF4 MF5 gRNA sgRNA MF6 MF7 MF8 MF9 3 4 5 6 7 8 9 2 3 4 5 6 7 8 9 2 99.99 99.9 99 90 99.99 99.9 99 90 Viral load (log10 copies per ml) % Efficacy Control COVA1-18 0 2 4 6 8 Days post-infection 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 a MF10 gRNA sgRNA Efficacy Days post-infection b c 0 -2 4 6 2 8 0 -2 4 6 2 8 Therapeutic Inoculum: 106 PFU Nasopharynx Trachea 3 4 5 6 7 8 9 2 3 4 5 6 7 8 9 2 3 4 5 6 7 8 9 2 3 4 5 6 7 8 9 2 0 -2 4 6 2 8 0 -2 4 6 2 8 PrEP Inoculum: 106 PFU Nasopharynx Trachea Fig. 4 Modeling of viral dynamics and treatment efficacy. a Individual prediction of the nasopharyngeal genomic (g)RNA and subgenomic (sg)RNA in control (top) and treated animals (bottom) with individual efficacy prediction indicated (green line). The dashed red line indicates the time of infection. gRNA (squares) and sgRNA (circles) data are indicated as plain (above LoQ) or open (below LoQ). b Model predictions of gRNA and sgRNA dynamics with 4 doses of COVA1-18 given 24 h prior to challenge (arrow). c Simulation as in (b) with COVA1-18 given 24 h post-infection. Black dotted lines indicate LoQ (limit of quantification), i.v. intravenous, PFU plaque forming units, PrEP Pre-exposure prophylaxis. MF1 MF2 MF3 MF4 MF5 gRNA sgRNA MF6 MF7 MF8 MF9 3 4 5 6 7 8 9 2 3 4 5 6 7 8 9 2 99.99 99.9 99 90 99.99 99.9 99 90 Viral load (log10 copies per ml) % Efficacy Control COVA1-18 0 2 4 6 8 Days post-infection 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 a MF10 gRNA sgRNA Efficacy % Efficacy gRNA sgRNA Viral load (log10 copies per ml) Control 10 mg kg-1 5 mg kg-1 1 mg kg-1 0.1 mg kg-1 COVA1-18 dose Injection i.v. Methods I G F b All mice were housed in a temperature con- trolled environment (68–72 degrees Fahreheit, 50–60% humidity) with twelve hours of light per day at the Center for Comparative Medicine and Surgery (CCMS) at Icahn School of Medicine at Mount Sinai (New York, NY, USA). All experiments involving viral infections were carried out in a CDC/ USDA-approved BSL-3 facility at CCMS and animals were transferred into the facility four days prior to onset of experiments. Mice were housed in Allentown individually ven- tilated cages with ad libitum access to food and water. The mouse experimental study was approved by the Icahn School of Medicine at Mount Sinai Institutional Animal Care and Use Committee (IACUC-2017-0170 and IACUC-2017-0330). Hamsters were housed in the ABSL-4 facility of the Galveston National Laboratory. The animal protocol # 2004049 was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Texas Medical Branch at Galveston (UTMB). Ni-NTA-capture ELISA. SARS-CoV-2, SARS-CoV, MERS, HKU1, 229E and NL63 S His-tagged proteins were loaded at 2 µg ml−1 in TBS/2% skimmed milk (100 µl/ well) on 96-well Ni-NTA plates (Qiagen) for 2 h at room temperature (RT). Three- fold serially diluted COVA NAb were then added onto the plates for 2 h at RT followed by the addition goat anti-human IgG-HRP (Jackson Immunoresearch) secondary Ab (1:3000) for 1 h at RT. The plates were developed for 3 min using TMB solution and then stopped. Optical densities were measured at 450 nm on a spectrophotometer and data graphed using GraphPad Prism software (v8.3.0). Hamsters were housed in the ABSL-4 facility of the Galveston National Laboratory. The animal protocol # 2004049 was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Texas Medical Branch at Galveston (UTMB). Cynomolgus macaques (Macaca fascicularis) originating from Mauritian AAALAC certified breeding centers were used in this study. All animals were housed in IDMIT infrastructure facilities (CEA, Fontenay-aux-roses), under BSL-2 and BSL-3 containment when necessary (Animal facility authorization #D92-032- 02, Préfecture des Hauts de Seine, France) and in compliance with European Directive 2010/63/EU, the French regulations and the Standards for Human Care and Use of Laboratory Animals, of the Office for Laboratory Animal Welfare (OLAW, assurance number #A5826-01, US). The protocols were approved by the institutional ethical committee “Comité d’Ethique en Expérimentation Animale du Commissariat à l’Energie Atomique et aux Energies Alternatives” (CEtEA #44) under statement number A20-011. Methods I G F b IgG, Fab, and soluble viral protein expression. COVA1-18 was isolated from a participant in the “COVID-19 Specific Antibodies” (COSCA) study as described9. The COSCA study was conducted at the Amsterdam University Medical Centre, location AMC, the Netherlands, and approved by the local ethical committee of the AMC (NL 73281.018.20). COVA1-18 IgG was produced in HEK293F suspension cells as previously described9. COVA1-18 His-tagged Fab was produced in Expi- CHO cells as previously described50. Spike and RBD proteins were produced and purified as previously described9. Briefly, cells were transfected at a density of 0.8–1.2 million cells per mL by addition of a mix of PEImax (1 μg μl−1) with expression plasmids (312.5 μg l−1) in a 3:1 ratio in OptiMEM. Supernatants of glycoproteins were harvested six days post transfection, centrifuged for 30 min at 4000 rpm and filtered). Constructs with a his-tag were purified by affinity pur- ification using Ni-NTA agarose beads. Protein eluates were concentrated and buffer exchanged to PBS using Vivaspin filters with a 100 kDa molecular weight cutoff (GE Healthcare) for Spike proteins or 10 kDa for RBD. Protein concentrations were determined by the Nanodrop method using the proteins peptidic molecular weight and extinction coefficient as determined by the online ExPASy software (ProtParam). Pseudovirus neutralization assay. Neutralization assays were performed using SARS-CoV-2 S-pseudotyped HIV-1 virus and HEK293T hACE2 cells as described previously13. In brief, pseudotyped virus was produced by co-transfecting expression plasmids of SARS-CoV-2Δ19 S proteins (GenBank MT449663.1) with an HIV backbone expressing NanoLuc luciferase (pHIV-1NL4-3 ΔEnv-NanoLuc) in HEK293T cells (ATCC, CRL-11268). After 2 days, the cell culture supernatants containing SARS-CoV-2 S-pseudotyped HIV-1 viruses were harvested and stored at −80 °C. HEK293T hACE2 cells were seeded 20,000 cells/well in a flat-bottom 96-well plates one day prior to the start of the neutralization assay. COVA1-18 IgG and His6- tagged Fab as well as heat-inactivated serum samples were serially diluted in 3-fold steps using cell culture medium and then mixed with pseudotyped virus in a 1:1 ratio and incubated for 1 h at 37 °C. The mixtures were then added to the HEK293T hACE2 cells in a 1:1 medium to mixture ratio. The final starting concentration for IgGs was 20 µg ml−1 and 13.33 µg ml−1 for Fab. The cells were then incubated at 37 °C for 48 h followed by one PBS wash and lysis buffer addition. Methods I G F b The luciferase activity in the cell lysates was measured using the Nano-Glo Luciferase Assay System (Promega) and GloMax Discover microplate reader. Relative luminescence units (RLU) were normalized to those from positive control wells where cells were infected with SARS-CoV-2 pseudovirus without IgG, Fab or serum. The inhibitory con- centration (IC50) and neutralization titers (ID50) were determined as the IgG/Fab concentration or serum dilution at which infectivity was inhibited by 50%. d d l ( ) l d l Bio-layer interferometry. The affinity of COVA1-18 IgG and His-tagged Fab versions were determined using Ni-NTA biosensors (ForteBio) onto which 20 µg ml−1 of SARS-CoV-2 RBD was in running buffer (PBS, 0.02% Tween-20, 0.1% BSA) was loaded for 300 s as previously described50. The association rate and dissociation step were assessed over a 120 s step each. Serially diluted IgG (50, 100, 200, and 400 nM) and Fab (100, 200, 400, and 800 nM) were tested and an anti- HIV-1 His-tagged Fab at 800 nM in running buffer was included as negative control. KDs were determined using ForteBio Octet CFR software using a 1:2 fitting model for IgGs and a 1:1 fitting model for Fabs. The apparent affinity of COVA1- 18 IgG to the SARS-CoV-2 S trimer was determined as described above except that 20 µg ml−1 SARS-CoV-2 S 2 P Fld His protein was loaded instead of RBD. The COVA1-18 IgG avidity effect was further evaluated by titrating the loaded SARS- CoV-2 RBD (5, 1, 0.2, and 0.04 μg ml−1). An additional loading step using His- tagged HIV-1 gp41 was performed to minimize background binding of His-tagged Fabs to the biosensor and both the COVA1-18 IgG and Fab concentrations were set at 250 nM. All other steps were performed as described above. Data were acquired with Octet Data Acquisition 10.0.03.12 and analyzed with Octet Analysis HT 10.0.3.7 (ForteBio). Pseudotyped Vesicular Stomatitis Virus (VSVΔG) particles displaying SARS- CoV-2Δ19 S and containing a luciferase reporter were used as previously described14. Two-fold dilution series of COVA1-18 were prepared in complete medium, pseudotyped virus added and the mixture incubated for 1 h at 37 °C. The virus-antibody mixtures were then loaded onto plates seeded with Vero E6 cells 24 h prior this step. Following a 20 h incubation at 37 °C, the luciferase substrate was added to lysed cells and RLU determined and analyzed as described above. Ethics and biosafety statement. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26354-0 ARTICLE In conclusion, our COVA1-18 in vitro data translated into a powerful protective drug in three preclinical models to prevent SARS-CoV-2 replication. Together with our prediction model, these data showed that COVA1-18 could be used in patients at low doses either to prevent infection or to reduce viral loads in a therapeutic setting, with a potential greater impact in high-risk patients. The high in vivo efficacy of COVA1-18 and its demonstrated potency against the B.1.1.7. isolate also suggests that it is a promising candidate for a NAb cocktail. Cynomolgus monkey IgG ELISA. Half area high binding 96-well plates were coated overnight (4 °C) with goat anti-Human IgG λ and goat anti-Human IgG κ (Southern Biotech), 1:2000 (each) in PBS, 50 µl/well. The plates were washed (1X TBS – 0,05% Tween20) and blocked for 2 h at RT with 50 µl/well casein buffer. Serially diluted mucosal and serum samples were loaded onto the plates. Serially diluted polyclonal cynomolgus IgG (Molecular Innovations) was used as standard. Following a 1 h incubation at RT, mouse anti-Monkey IgG Fc-BIOT (Southern Biotech) was loaded onto the plate (1:50000). After 1 h at RT, poly-HRP40 was added (1:10000) and the plates incubated for 1 h. Finally, the plates were washed 5 times, developed for 5 min, and analyzed as described above. Cynomolgus anti-S IgG and IgM ELISA were performed as described above except that 2 µg ml−1 SARS-CoV-2 S Fld His-tagged protein were coated in the sample wells instead of goat anti-Human IgG λ and goat anti-Human IgG κ. For the IgM ELISA, the standard was obtained from Molecular Innovations and the detection goat anti-Monkey IgM (μ-chain specific)-Biotin antibody from Sigma Aldrich and used at 1:20000 dilution. Strep-HRP (R&D systems) at 1:500 was used for IgG and poly-HRP40 for IgM. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26354-0 This finding highlights the necessity of using NAbs cocktails targeting distinct epitopes and we propose the use of the SARS-CoV-1 cross- neutralizing antibody COVA1-16, which can effectively neutralize B.1.351, in a 1:1 cocktail with COVA1-18. In addition, the half- life of COVA1-18 can be extended by incorporating the LS or YTE47 mutations, which can further reduce the protective dose required and reduce the cost of treatment. An optimal cocktail should not only be based on intrinsic efficacy against wild-type virus of each NAbs, but rather whether synergy could be achieved in terms of binding domain and/or spectrum of efficacy. Indeed, the increasing prevalence of mutant strains has reduced the sensitivity to pre-existing NAbs, including those given in combination30. Escape mutations can arise fol- lowing single NAb treatment as recently demonstrated37,46 and the one S mutation found in a unique sample from one animal treated with COVA1-18 is not in the epitope of COVA-18. Importantly, we and others have determined that COVA1-18 retains high potency against the B.1.1.7 variant, which includes the N501Y mutation20,21. However, COVA1-18 lost its potency against B.1.351 which harbors the E484K mutation that is also q While approved SARS-CoV-2 mAbs are given intravenously, other therapeutic mAbs are given intramuscularly or by sub- cutaneous injection48. SARS-CoV-2 mAbs could potentially also be administered intranasally or delivered via gene therapy to the airways49, to provide protection where it is most needed, i.e. the respiratory tract. The biodistribution of COVA1-18 by different routes of administration would also have to be investigated. In addition, we note that COVA1-18 and numerous potent neu- tralizing Abs isolated to date against SARS-CoV-2 have very low levels of somatic hypermutation. Thus, these antibodies are very close to the germline precursor and unlikely to trigger anti- idiotypic response in patients. NATURE COMMUNICATIONS | (2021) 12:6097 | https://doi.org/10.1038/s41467-021-26354-0 | www.nature.com/naturecommunications 6 ARTICLE Lesions and scoring were assessed in each lung lobe blindly and independently by two persons and the final results were established by consensus. Overall CT scores include the lesion type (scored from 0 to 3) and lesion volume (scored from 0 to 4) summed for each lobe as previously described25,26. Animals and study design. Seven week old female Balb/cJ mice (Jackson Laboratories Bar Harbor, ME) were anesthetized before being administered with 2.5 × 108 PFU of human adenovirus type 5 encoding the human angiotensin converting enzyme-2 receptor (Ad5-hACE2) 5-days prior to challenge with SARS- CoV-2, as previously described29,30. Animals were transferred to the BSL-3 facility where two groups of n = 5 mice per group received 10 mg kg−1 of COVA1-18 intraperitoneally 24 h prior to, or post-infection with 104 PFU SARS-CoV-2 in 50 μl PBS. A control group of n = 3 mice received 50 μl PBS. Mice were euthanized 3 d.p.i. and lungs harvested to quantify viral lung titers. Lungs were homogenized in PBS using a Beadblaster Microtube homogenizer (Benchmark Scientific). SARS- CoV-2 plaque assay was performed on 10-fold serial dilutions of lung homogenates prepared in 0.2% bovine serum albumin (BSA) in PBS that were plated onto a Vero E6 cells monolayer and incubated with shaking for 1 h. Inoculum was removed and plates were overlaid with Minimal Essential Media (MEM) containing 2% FBS/ 0.05% oxoid agar and incubated for 72 h at 37 °C. Plates were fixed with 4% formaldehyde overnight, stained with a mAb cocktail composed of SARS-CoV-2 spike and SARS-CoV-2 nucleoprotein (Center for Therapeutic Antibody Dis- covery; NP1C7C7) followed by anti-Mouse IgG-HRP (Abcam ab6823) and developed using KPL TrueBlue peroxidase substrate (Seracare; 5510-0030). Viral sequencing. 30 RNA samples from nasopharyngeal and tracheal swabs as well as BAL fluids at 3 d.p.i. were selected for sequencing along with the inoculum. cDNA and multiplex PCR reactions were prepared following the ARTIC SARS- CoV-2 sequencing protocol v256. V3 primer scheme (https://github.com/artic- network/primer-schemes/tree/master/nCoV-2019/V3) was used to perform the multiplex PCR for SARS-CoV-2. All samples were run for 35 cycles in the two multiplex PCRs. Pooled and cleaned PCR reactions were quantified using QubitTM fluorometer (Invitrogen). The Ligation Sequencing kit (SQK-LSK109; Oxford Nanopore Technologies) was used to prepare the library following the manu- facturer’s protocol (“PCR tiling of COVID-19 virus”, release F; Oxford Nanopore Technologies). ARTICLE Twenty-four samples were multiplexed using Native Barcoding Expansion 1–12 and Native Barcoding Expansion 13–24 kits (EXP-NBD104 and EXP-NBD114; Oxford Nanopore Technologies). Two libraries of 24 samples were prepared independently and quantified by QubitTM fluorometer (Invitrogen). After the quality control, two R9.4 flowcells (FLO-MIN106; Oxford Nanopore Technologies) were primed as described in the manufacturer’s protocol and loaded with 45 and 32 ng of library. Sequencing was performed on a GridION (Oxford Nanopore Technologies) for 72 h with high-accuracy Guppy basecalling (v3.2.10). After sequencing, demultiplexing was performed using Guppy v4.0.14 with the option -require_barcodes_both_ends to ensure high quality demultiplexing. Reads were then filtered by Nanoplot v1.28.1 based on length and quality to select high quality reads. Then, reads were aligned on the SARS-CoV-2 reference genome NC_045512.2 using minimap2 v2.17. Primary alignments were filtered based on reads length alignment and reads identity. Reads were basecalled and demulti- plexed with Guppy 4.0.14. The potential clonal and subclonal variants were detected with a custom pipeline based on ARTIC network workflow. Longshot v0.4.1 was used for variant detection. The potential subclonal variants were manually curated by comparing the generated VCF files and visual inspection of the alignments in IGV browser. p g p Female golden Syrian hamsters, aged 6–7 weeks, were randomly assigned to two groups of n = 5 and microchipped 24 h before SARS-CoV-2 challenge. On the day of challenge, hamsters were anesthetized with ketamine/xylazine and challenged by the intranasal route with 105 PFU of SARS-CoV-2 diluted in sterile PBS in the total volume 100 µl. Body weight and body temperature were measured each day, starting at day 0. Twenty four hours post-challenge, hamsters were treated with 10 mg kg−1 of COVA1-18 diluted in 0.5 ml of sterile PBS via the intraperitoneal route. The control group of animals received an equal volume of sterile PBS via the intraperitoneal route. All animals were euthanized 72 h post-infection with an overdose of anesthetic (isoflurane or ketamine/xylazine) followed by bilateral thoracotomy, and terminal blood and lungs were collected at necropsy. Right lungs were frozen in 5 ml L-15 Leibowitz medium (Gibco) with 10% FBS. Tissue sections were homogenized in bead beater tubes, weighed, and supernatants were titrated per standard protocol. ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26354-0 Respiratory Viruses (Institut Pasteur, Paris, France) as previously described51 and produced by two passages on Vero E6 cells in DMEM (Dulbecco’s Modified Eagles Medium) without FBS, supplemented with 1% P/S (penicillin at 10,000 U ml−1 and streptomycin at 10,000 μg ml−1) and 1 μg ml−1 TPCK-trypsin at 37 °C in a Respiratory Viruses (Institut Pasteur, Paris, France) as previously described51 and produced by two passages on Vero E6 cells in DMEM (Dulbecco’s Modified Eagles Medium) without FBS, supplemented with 1% P/S (penicillin at 10,000 U ml−1 and streptomycin at 10,000 μg ml−1) and 1 μg ml−1 TPCK-trypsin at 37 °C in a humidified CO2 incubator and titrated on Vero E6 cells. Whole genome sequen- cing was performed as described51 with no modifications observed compared with the initial specimen and sequences were deposited after assembly on the GISAID EpiCoV platform under accession number ID EPI_ISL_406596. Sequencing ana- lysis revealed two clonal mutations, one in the S gene (22661 G > T: V367F, non- synonymous) and one in the ORF3a gene (26144 G > T: G251V, non-synonymous), which were already present in the challenge inoculum. epiglottis. All specimens were stored between 2 °C and 8 °C until analysis by RT- qPCR with a plasmid standard concentration range containing an RdRp gene fragment including the RdRp-IP4 RT-PCR target sequence. SARS-CoV-2 E gene subgenomic mRNA (sgRNA) levels were assessed by RT-qPCR using primers and probes previously described52,53 (Supplementary Table 4). The protocol describing the procedure for the detection of SARS-CoV-2 is available on the WHO website54. p y μg μg yp humidified CO2 incubator and titrated on Vero E6 cells. Whole genome sequen- cing was performed as described51 with no modifications observed compared with the initial specimen and sequences were deposited after assembly on the GISAID EpiCoV platform under accession number ID EPI_ISL_406596. Sequencing ana- lysis revealed two clonal mutations, one in the S gene (22661 G > T: V367F, non- synonymous) and one in the ORF3a gene (26144 G > T: G251V, non-synonymous), which were already present in the challenge inoculum. Chest CT and image analysis. Lung images were acquired using a computed tomography (CT) system (Vereos-Ingenuity, Philips) as previously described25,26, andanalysed using INTELLISPACE PORTAL 8 software (Philips Healthcare). All images had the same window level of enuity, window width of 1,600. Lesions were defined as ground glass opacity, crazy-paving pattern, consolidation or pleural thick- ening as previously described35,55. ARTICLE All animals were then exposed to a total dose of 106 PFU of SARS- CoV-2 (BetaCoV/France/IDF/0372/2020; passaged twice in VeroE6 cells) via the combination of intranasal and intratracheal routes (day 0), using atropine (0 04 k 1) f di i d k i ( k 1) i h d idi Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article. Data availability The viral sequencing data used in Supplementary Fig. 3 have been deposited in the SRA repository under the accession code PRJNA758764 (PRJNA758764 - SRA - NCBI (nih.gov)). All the other raw data generated in this study are provided in the Source Data file. Source data are provided with this paper. Code availability The code used for the modeling part has been deposited in the Zenodo repository57 under the https://doi.org/10.5281/zenodo.5140032. Received: 15 February 2021; Accepted: 24 September 2021; Received: 15 February 2021; Accepted: 24 September 2021; One male and one female cynomolgus macaques aged 3–6 years received the treatment as described above for the pharmacokinetic and pharmacodynamics (PK/PD) study. Blood was sampled before and 2, 4, 6, and 24 h post-treatment. Saliva, nasopharyngeal and tracheal fluids were sampled before and 24 h post- treatment. Twenty-four hours post-treatment, animals were euthanized and their lungs, heart, kidney, liver, spleen, trachea, and brain were sampled, rinsed with PBS and around 100 mg of tissue was homogenized in 500 µl of PBS with a Precellys and stored at −80 °C. Methods I G F b The study was authorized by the “Research, Innovation and Education Ministry” under registration number APAFIS#24434- 2020030216532863. All information on the ethics committee is available at https:// cache.media.enseignementsup-recherche.gouv.fr/file/utilisation_des_animaux_ fins_scientifiques/22/1/comiteethiqueea17_juin2013_257221.pdf. Detection of human IgG in NHP fluid. Detection of COVA1-18 in NHP samples determined by ELISA using a protocol adapted from others33. Briefly, half area high binding 96-well plates (Greiner Bio-One) were coated overnight with goat anti-Human IgG H+L (monkey pre-adsorbed) at 1 µg ml−1 in PBS. The plates were then blocked in casein buffer (Thermo Scientific) for 2 h at RT. Serum and mucosal samples were serially diluted and loaded onto the plates as well as serially diluted COVA1-18 as the standard. Following a 1 h RT incubation, goat anti- Human IgG (monkey adsorbed)-HRP secondary antibody (Southern Biotech) was added for serum samples (1:4000). For mucosal samples, goat anti-Human IgG (monkey adsorbed)-BIOT (Southern Biotech) was added at 1:10000 dilution. After 1 h RT incubation, serum sample plates were ready for development. For mucosal samples, an additional 1 h incubation with poly-HRP40 (Fitzgerald) (1:10000) was necessary. The plates were then developed for 5 min, and the optical densities measured at 450 nm on a spectrophotometer. The raw data were exported and analyzed using Microsoft Excel and GraphPad Prism (v8.3.0) software. The COVA1-18 concentration in a specific sample was determined by interpolating OD values from dilutions that fell into the linear range of the standard curve of the matching ELISA plate. Viruses and cells. For the macaques studies, SARS-CoV-2 virus (hCoV-19/ France/ lDF0372/2020 strain) was isolated by the National Reference Center for 7 7 TURE COMMUNICATIONS | (2021) 12:6097 | https://doi.org/10.1038/s41467-021-26354-0 | www.nature.com/naturecommunications ARTICLE Briefly, of 10-fold dilutions of supernatants at 100 µl per well were placed atop of Vero-E6 monolayers in 96-well plates, the plates were incubated for 1 h, supernatants were replaced by methyl cellulose overlay, incubated for 3 days at 5% CO2 and 37 °C. The plates were fixed with formalin, removed from BSL-4 according the approved protocol, and plaques counted to determine the viral titers. Statistical analysis. Statistical analysis of Syrian hamsters and hACE2 mice lung viral titers as well as for NHP gRNA and sgRNA were carried out using Mann- Whitney unpaired t-test in GraphPad Prism software (v8.3.0). g pp p p q Ten female cynomolgus macaques aged 3–6 years were randomly assigned between the control and treated groups to evaluate the efficacy of COVA1-18 prophylaxis. The treated group (n = 5) received one bolus dose of COVA-18 human IgG1 monoclonal antibody (10 mg kg−1) by the intravenous route in the saphenous vein one day prior to challenge, while control animals (n = 5) received no treatment. All animals were then exposed to a total dose of 106 PFU of SARS- CoV-2 (BetaCoV/France/IDF/0372/2020; passaged twice in VeroE6 cells) via the combination of intranasal and intratracheal routes (day 0), using atropine (0.04 mg kg−1) for pre-medication and ketamine (5 mg kg−1) with medetomidine (0.05 mg kg−1) for anesthesia. Animals were observed daily and clinical exams were performed at baseline, daily for one week, and then twice weekly, on anaesthetized animals using ketamine (5 mg kg−1) and metedomidine (0.05 mg kg−1). Body weight and rectal temperature were recorded and blood, as well as nasopharyngeal, tracheal and rectal swabs, were collected. Broncho-alveolar lavages (BAL) were performed using 50 ml sterile saline on 3 d.p.i. Chest CT was performed at 3 d.p.i. in anesthetized animals using tiletamine (4 mg kg−1) and zolazepam (4 mg kg−1). Blood cell counts, hemoglobin, and hematocrit, were determined from EDTA blood using a DHX800 analyzer (Beckman Coulter). Ten female cynomolgus macaques aged 3–6 years were randomly assigned between the control and treated groups to evaluate the efficacy of COVA1-18 prophylaxis. The treated group (n = 5) received one bolus dose of COVA-18 human IgG1 monoclonal antibody (10 mg kg−1) by the intravenous route in the saphenous vein one day prior to challenge, while control animals (n = 5) received no treatment. URE COMMUNICATIONS | (2021) 12:6097 | https://doi.org/10.1038/s41467-021-26354-0 | www.nature.com/naturecommunications Acknowledgements p 26. Brouwer, P. J. M. et al. Two-component spike nanoparticle vaccine protects macaques from SARS-CoV-2 infection. Cell 184, 1188–1200 e1119 (2021). g We thank Benoit Delache, Sébastien Langlois, Joanna Demilly, Emma Burban, Quentin Sconosciuti, Maxime Potier, Nina Dhooge, Pauline Le Calvez, Jean-Marie Robert, Tierry Prot and Christina Dodan for their help with the macaque experiments; Laetitia Bos- sevot, Marco Leonec, Laurine Moenne-Loccoz and Julie Morin for the RT-qPCR, and for the preparation of reagents; Blanche Fert and Céline Mayet for her help with the CT scans; Céline Aubenque, Karine Storck, Mylinda Barendji, Julien Dinh and Elodie Guyon for the macaque sample processing; Sylvie Keyser for the transports organization; Nas- tasia Dimant and Brice Targat for their help with the experimental studies in the context of COVID-19-induced constraints; Frédéric Ducancel and Yann Gorin for their help with the logistics and safety management; Isabelle Mangeot for her help with resources management. We thank Sylvie Behillil and Vincent Enouf for contribution to viral stock challenge production, Antoine Nougairede for sharing the plasmid used for the sgRNA assays standardization and Paul Bieniasz for donating cells and reagents for pseudovirus neutralization assays. We thank Matt Hyde and Julie Williams (Animal Resource Center, University of Texas Medical Branch at Galveston) who performed technical procedures with animals. We acknowledge support from CoVIC supported by the Bill and Melinda Gates Foundation. We thank staff at the ISMMS CCMS vivarium for their assistance. We also thank Randy Albrecht for support with the BSL-3 facility and procedures at the ISMMS and Richard Cadagan for excellent technical assistance. The macaque image used in Fig. 2 has been obtained from BioRender. This study was supported by the Nether- lands Organization for Scientific Research (NWO) Vici grant (to R.W.S.), the Bill & Melinda Gates Foundation through the Collaboration for AIDS Vaccine Discovery (CAVD) grant INV-002022 (to R.W.S.), the Fondation Dormeur, Vaduz (to R.W.S. and to M.J.v.G.) and Health Holland PPS-allowance LSHM20040 (to M.J.v.G.). M.J.v.G. is a recipient of an AMC Fellowship, Amsterdam UMC and a COVID-19 grant of the Amsterdam Institute of Infection and Immunity, the Netherlands. R.W.S and M.J.v.G. We thank Benoit Delache, Sébastien Langlois, Joanna Demilly, Emma Burban, Quentin Sconosciuti, Maxime Potier, Nina Dhooge, Pauline Le Calvez, Jean-Marie Robert, Tierry Prot and Christina Dodan for their help with the macaque experiments; Laetitia Bos- sevot, Marco Leonec, Laurine Moenne-Loccoz and Julie Morin for the RT-qPCR, and for 27. Zarifian, A. ARTICLE Lancet Infect. Dis. 20, 697–706 (202 52. Corman, V. M. et al. 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Cell 184, 64–75 e11 (2021). in Europe: a case series. 1Université Paris-Saclay, Inserm, CEA, Center for Immunology of Viral, Auto-immune, Hematological and Bacterial diseases (IMVA-HB/IDMIT), Fontenay-aux-Roses & Le Kremlin-Bicêtre, Paris, France. 2Departments of Medical Microbiology of the Amsterdam UMC, University of Amsterdam, Amsterdam Institute for Infection and Immunity, Amsterdam, The Netherlands. 3Université de Paris, INSERM, IAME, Paris, France. 4Department of Pathology, University of Texas Medical Branch at Galveston, Galveston, TX, USA. 5Galveston National Laboratory, Galveston, TX, USA. 6Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 7Life and Soft, Le Plessis-Robinson, France. 8Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 9Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA. 10Department of Microbiology, University of Texas Medical Branch at Galveston, Galveston, TX, USA. 11Duke Human Vaccine Institute & Department of Surgery, Durham, NC, USA. 12Nexelis, Laval, Québec, Canada. 13Internal Medicine of the Amsterdam UMC, University of Amsterdam, Amsterdam Institute for Infection and Immunity, Amsterdam, The Netherlands. 14Department of Medicine, Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 15The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 16Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 17University of Maryland School of Medicine, Department of Microbiology and Immunology and Center for Vaccine Development and Global Health (CVD), Baltimore, MD, USA. 18Molecular Genetics of RNA Viruses, Department of Virology, Institut Pasteur, CNRS UMR 3569, Université de Paris, Paris, France. 19National Reference Center for Respiratory Viruses, Institut Pasteur, Paris, France. 20Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, NY, USA. 21These authors contributed equally: Pauline Maisonnasse, Yoann Aldon. ✉email: r.w.sanders@amsterdamumc.nl; m.j.vangils@amsterdamumc.nl; roger.le-grand@cea.fr Author contributions Reprints and permission information is available at http://www.nature.com/reprints Additional information Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-021-26354-0. Correspondence and requests for materials should be addressed to Rogier W. Sanders, Marit J.van Gils or Roger Le Grand. Peer review information Nature Communications thanks the anonymous reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Competing interests Amsterdam UMC filed a patent application on SARS-CoV-2 monoclonal antibody COVA1-18. The García-Sastre Laboratory has received research support from Pfizer, Senhwa Biosciences, 7Hills Pharma, Pharmamar, Blade Therapeutics, Avimex, Johnson & Johnson, Dynavax, Kenall Manufacturing, ImmunityBio, Merck, and Nanocomposix. Adolfo García-Sastre has consulting agreements for the following companies involving cash and/or stock: Vivaldi Biosciences, Contrafect, 7Hills Pharma, Avimex, Vaxalto, Pagoda, Accurius, Farmak, Pfizer, and Esperovax. The remaining authors declare no competing interests. ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-26354-0 are recipients of support from the University of Amsterdam Proof of Concept fund (contract no 200421) as managed by Innovation Exchange Amsterdam (IXA). The Infectious Disease Models and Innovative Therapies (IDMIT) research infrastructure is supported by the “Programme Investissements d’Avenir”, managed by the ANR under reference ANR-11-INBS-0008. The Fondation Bettencourt Schueller and the Region Ile- de-France contributed to the implementation of IDMIT’s facilities and imaging tech- nologies. The NHP study received financial support from REACTing, the Fondation pour la Recherche Médicale (FRM, France; AM-CoV-Path) and the European Infrastructure TRANSVAC2 (730964). The virus stock used in NHPs was obtained through the EVAg platform (https://www.european-virus-archive.com/), funded by H2020 (653316). Work performed at Duke University was supported by the CoVPN grant (NIH AI46705) (to D.C.M.). The Ad5-hACE2 mouse work was supported in part by NIAID R21AI157606 (L.C.), and was partially supported by CRIP (Center for Research for Influenza Patho- genesis), an NIAID supported Center of Excellence for Influenza Research and Sur- veillance (CEIRS, contract # HHSN272201400008C) (A.G.S.), by supplements to NIAID grant U19AI135972 and DoD grant W81XWH-20-1-0270, by the Defense Advanced Research Projects Agency (HR0011-19-2-0020), and by the generous support of the JPB Foundation, the Open Philanthropy Project (research grant 2020-215611 (5384) and anonymous donors to A.G.S. Part of this study was supported by the Bill and Melinda Gates Foundation through grants OPP1170236 and INV-004923 (I.A.W.) and through the Global Health Vaccine Accelerator Platforms (GH-VAP) and the Coronavirus Immunotherapy Consortium (CoVIC) (Nexelis). The funders had no role in study design, data collection, data analysis, data interpretation, or data reporting. R.L.G, and R.W.S. conceived, designed, supervised the project, acquired funding, pro- vided resources, and wrote the manuscript. All authors contributed to the review and editing of the final manuscript. R.L.G, and R.W.S. conceived, designed, supervised the project, acquired funding, pro- vided resources, and wrote the manuscript. All authors contributed to the review and editing of the final manuscript. Acknowledgements et al. Chest CT findings of coronavirus disease 2019 (COVID-19): a comprehensive meta-analysis of 9907 confirmed patients. Clin. Imaging 70, 101–110 (2021). 28. Andreano, E. et al. SARS-CoV-2 escape from a highly neutralizing COVID-19 convalescent plasma. Proc. Natl Acad. Sci. USA. 118 (2021). 29. Goncalves, A. et al. SARS-CoV-2 viral dynamics in non-human primates. PLoS Comput Biol. 17, e1008785 (2021). p 30. Wang, P. et al. Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7. Nature 593, 130–135 (2021). 31. Starr, T. N. et al. Prospective mapping of viral mutations that escape antibodies used to treat COVID-19. Science 371, 850–854 (2021). 32. Baum, A. et al. REGN-COV2 antibodies prevent and treat SARS-CoV-2 infection in rhesus macaques and hamsters. Science 370, 1110–1115 (2020). 33. Zost, S. J. et al. Potently neutralizing and protective human antibodies against SARS-CoV-2. Nature 584, 443–449 (2020). 34. Wang, S. et al. Characterization of neutralizing antibody with prophylactic and therapeutic efficacy against SARS-CoV-2 in rhesus monkeys. Nat. Commun. 11, 5752 (2020). 35. Shi, R. et al. A human neutralizing antibody targets the receptor-binding site of SARS-CoV-2. Nature 584, 120–124 (2020). 36. Li, D. et al. In vitro and in vivo functions of SARS-CoV-2 infection-enhancing and neutralizing antibodies. Cell 184, 4203–4219 e4232 (2021). 37. Jones, B. E. et al. The neutralizing antibody, LY-CoV555, protects against SARS-CoV-2 infection in nonhuman primates. Sci. Transl. Med. 13, eabf1906–eabf1906 (2021). 9 NATURE COMMUNICATIONS | (2021) 12:6097 | https://doi.org/10.1038/s41467-021-26354-0 | www.nature.com/naturecommunications © The Author(s) 2021 Reprints and permission information is available at http://www.nature.com/reprints P.M. and Y.A. conceived, designed, performed experiments, analyzed data, managed the project, and wrote the manuscript (original draft). A.M. conceived and developed the predictive model, and wrote the manuscript. R.M., N.D.B. performed, supervised, and analyzed macaque experiments. N.A.K. designed, performed, and analyzed the hamster experiment. M.S. designed, performed, and analyzed the mouse experiment. A.W.F. performed the mouse experiment. J.L.S. produced antibodies and performed ELISAs. A.G. contributed to the predictive model development. J.A.B., M.P., I.B., C.M., M.O.T., N.S.A., and L.G. performed neutralization assays. V.Ch., S.D., and A.I. performed sequencing, analyzed, and interpreted the data. A.J.R. analyzed the hamster histology data. S.J. and R.R. performed the mouse experiment. T.G.C., P.J.M.B., T.P.L.B., J.v.S., M.B., M.J.v.B., H.L., and M.Y. produced proteins. C.E.M. contributed to the hamster experiment. V.Co. contributed to performing and supervising macaque studies. T.N. and J.L. contributed to the macaque experiments and analysis. N.K. and F.R. contributed to the macaque experiment. C.C. and R.H.T.F. provided resources and supervision for the macaque studies contributed to the macaque experiments and analysis. D.C.M., I.A.W., G.J.d.B., and A.G.S. provided resources and funding. E.G. provided resources and supervision for the sequencing. L.C. conceived, designed, and performed the mouse study; acquired funding. A.B. designed and supervised the hamster study, provided funding. S.v.d.W. provided the virus for the macaque study; J.G. conceived and developed the predictive model, supervised, provided funding, and wrote the manuscript. M.J.v.G, Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. © The Author(s) 2021 10 NATURE COMMUNICATIONS | (2021) 12:6097 | https://doi.org/10.1038/s41467-021-26354-0 | www.nature.com/naturecommunicatio
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Impact of Moderna mRNA-1273 Booster Vaccine on Fully Vaccinated High-Risk Chronic Dialysis Patients after Loss of Humoral Response
Vaccines
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    Citation: Patyna, S.; Eckes, T.; Koch, B.F.; Sudowe, S.; Oftring, A.; Kohmer, N.; Rabenau, H.F.; Ciesek, S.; Avaniadi, D.; Steiner, R.; et al. Impact of Moderna mRNA-1273 Booster Vaccine on Fully Vaccinated High-Risk Chronic Dialysis Patients after Loss of Humoral Response. Vaccines 2022, 10, 585. https:// doi.org/10.3390/vaccines10040585 Academic Editors: Lo Presti Elena and Trizzino Antonino Received: 17 February 2022 Accepted: 6 April 2022 Published: 11 April 2022 Publisher’s Note: MDPI stays neutral * Correspondence: patyna@em.uni-frankfurt.de Citation: Patyna, S.; Eckes, T.; Koch, B.F.; Sudowe, S.; Oftring, A.; Kohmer, N.; Rabenau, H.F.; Ciesek, S.; Avaniadi, D.; Steiner, R.; et al. Impact of Moderna mRNA-1273 Booster Vaccine on Fully Vaccinated High-Risk Chronic Dialysis Patients after Loss of Humoral Response. Vaccines 2022, 10, 585. https:// doi.org/10.3390/vaccines10040585 † These authors contributed equally to this work. Abstract: The long-term effect of protection by two doses of SARS-CoV-2 vaccination in patients receiving chronic intermittent hemodialysis (CIHD) is an urging question. We investigated the humoral and cellular immune response of 42 CIHD patients who had received two doses of SARS- CoV-2 vaccine, and again after a booster vaccine with mRNA-1273 six months later. We measured antibody levels and SARS-CoV-2-specific surrogate neutralizing antibodies (SNA). Functional T cell immune response to vaccination was assessed by quantifying interferon-γ (IFN-γ) and IL-2 secreting T cells specific for SARS-CoV-2 using an ELISpot assay. Our data reveal a moderate immune response after the second dose of vaccination, with significantly decreasing SARS-CoV-2-specific antibody levels and less than half of the study group showed neutralizing antibodies six months afterwards. Booster vaccines increased the humoral response dramatically and led to a response rate of 89.2% for antibody levels and a response rate of 94.6% for SNA. Measurement in a no response/low response (NR/LR) subgroup of our cohort, which differed from the whole group in age and rate of immunosuppressive drugs, indicated failure of a corresponding T cell response after the booster vaccine. We strongly argue in favor of a regular testing of surrogate neutralizing antibodies and consecutive booster vaccinations for CIHD patients to provide a stronger and persistent immunity. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Keywords: COVID-19; SARS-CoV-2; vaccination; hemodialysis; booster; mRNA-1273; seroconversion; T cell response 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/). Impact of Moderna mRNA-1273 Booster Vaccine on Fully Vaccinated High-Risk Chronic Dialysis Patients after Loss of Humoral Response Sammy Patyna 1,2,*,†, Timon Eckes 2,† , Benjamin F. Koch 1 , Stephan Sudowe 3, Anke Oftring 2, Niko Kohmer 4, Holger F. Rabenau 4, Sandra Ciesek 4,5,6 , Despina Avaniadi 1, Rahel Steiner 1, Ingeborg A. Hauser 1, Josef M. Pfeilschifter 2 and Christoph Betz 1 1 Division of Nephrology, Department of Internal Medicine III, University Hospital Frankfurt, Goethe University Frankfurt, 60590 Frankfurt, Germany; benjamin.koch@kgu.de (B.F.K.); despina.avaniadi@kgu.de (D.A.); rahel.steiner@kgu.de (R.S.); ingeborg.hauser@kgu.de (I.A.H.); christoph.betz@kgu.de (C.B.) 2 Institute of General Pharmacology and Toxicology, University Hospital Frankfurt, Goethe University Frankfurt, 60590 Frankfurt, Germany; eckes@em.uni-frankfurt.de (T.E.); oftring@med.uni-frankfurt.de (A.O.); pfeilschifter@em.uni-frankfurt.de (J.M.P.) g p 3 GANZIMMUN Diagnostics AG Mainz, 55128 Mainz, Germany; dr.sudowe@ganzimmun.de 4 Institute for Medical Virology, University Hospital Frankfurt, Goethe University Frankfurt, 60590 Frankfurt, Germany; niko.kohmer@kgu.de (N.K.); rabenau@em.uni-frankfurt.de (H.F.R.); sandra.ciesek@kgu.de (S.C.) 5 German Centre for Infection Research, External Partner Site, 60323 Frankfurt, Germany 6 Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Branch Translational Medicine and Pharmacology, 60596 Frankfurt, Germany 2.1. Study Design and Cohorts In a prospective, non-interventional study approach, 42 long-term dialysis patients were observed from January 2021 to December 2021 in a tertiary care dialysis unit at the Department of Nephrology and Dialysis at Frankfurt University Hospital (Frankfurt, Germany). A healthy control group (n = 11; median (IQR) age 34 years (26–49), 27.3% male) was included to compare cellular immune responses after immunization with two doses of BNT162b2 (BioNTech/Pfizer) mRNA vaccine. Antibody levels and SNA were measured four weeks after the second dose (=t0, either with BNT162b2 or ChAdOx1), six months after second dose (=t1) and 14 days after a third dose (=t2, booster vaccine) of mRNA-1273 (Moderna) (Figure 1), exploring the time course of SARS-CoV-2-specific antibody levels and the percentage of neutralizing antibodies after vaccination against SARS-CoV-2 during the COVID-19 pandemic. The study was conducted in accordance with the principles of the Declaration of Helsinki and approved by the local ethics committee (reference number: 20/658). All study participants provided written informed consent before study entry. 1. Introduction The current coronavirus disease 2019 (COVID-19) pandemic confronts our society with a global threat that particularly concerns patients with compromised immune systems, either due to immunosuppressive therapy or diseases associated with impaired immune response. Patients with end-stage renal disease (ESRD) undergoing chronic intermittent https://www.mdpi.com/journal/vaccines Vaccines 2022, 10, 585. https://doi.org/10.3390/vaccines10040585 Vaccines 2022, 10, 585 2 of 9 hemodialysis (CIHD) are affected by lower humoral and cellular immunity [1]. For this rea- son, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and chronic dialysis patients make a particular dangerous relationship. Patients undergoing CIHD represent a vulnerable population which are at an increased risk of SARS-CoV-2 infection and threatened by an almost four-fold higher mortality rate compared to the general pop- ulation [2,3]. Thus, CIHD patients were prioritized for early SARS-CoV-2-vaccination in several countries [4]. Sahin et al. showed that vaccination with BNT162b2 (Tozinameran, BioNTech/Pfizer, Mainz, Germany/New York, NY, USA) induced both a strong antibody and T cell response in healthy adults [5,6]. However, recent data reveal a significantly lower SARS-CoV-2 spike protein antibody titer and reduced vaccination success rates of patients receiving CIHD compared to the general population after vaccination with BNT162b2 [7]. An effective short-term seroconversion rate after SARS-CoV-2 vaccination in the CIHD population was shown but recently published results indicate rapidly decreasing SARS- CoV-2-specific antibody levels in the long term [8]. There is evidence for a beneficial effect of a third dose of a COVID-19 mRNA vaccine on antibody titers in this specific high-risk population [9] and, in addition to the humoral immune response, T cells may also con- tribute to a protective immunity against SARS-CoV-2 and improve vaccine efficacy [10,11]. However, data regarding cellular immune response in this cohort are scarce. g g p Here, we investigated the initial SARS-CoV-2-specific serological and functional T cell immune response after two doses of SARS-CoV-2 vaccination (either BNT162b2 or ChAdOx1 (AstraZeneca, Cambridge, UK)) and its longevity during a time course of up to six months after the second dose in a cohort of high-risk CIHD patients in a tertiary care dialysis unit. In addition, we analyzed antibody titers and surrogate neutralizing antibodies (SNA) after a third dose, often referred to as booster vaccination, with Spikevax (mRNA-1273, Moderna, Cambridge, MA, USA) and measured its effect on the SARS-CoV- 2-reactive T cell response in a subgroup of CIHD patients with no response/low response (NR/LR) (n = 10) after their second vaccination. o positivity is set to . AU/ . We e evated t e cut o to 8.52 AU/ “threshold” range in between. 2.4. Assessment of Cytokine-Producing SARS-CoV-2-Reactive T Cell Responses 2.3. Assessment of SARS-CoV-2-Specific Surrogate Neutralizing Antibodies (SNA) The binding of S-RBD to angiotensin-converting enzyme 2 allowing detection of SARS-CoV-2-neutralizing antibodies was performed using the ELISA-based GenScript SARS-CoV-2 Surrogate Virus Neutralization Test Kit (GenScript Biotech, Piscataway Township, NJ, USA) [12]. Results are expressed as percentage inhibition (%-INH). The adaptive immune response was analyzed by quantifying interferon-γ (IFN-γ) and IL-2 secreting T cells specific for SARS-CoV-2 using a multicolor fluorescence enzyme- linked immunospot (ELISpot) assay (CoV-iSpot, AID GmbH, Strassberg, Germany) [13]. Highly specific sequences of immunodominant epitopes of SARS-CoV-2 structural proteins (=SARS-CoV-2-peptide-mix) were used for stimulation of T cells. Results are expressed as spot forming cells (SFC)/106 lymphocytes. 2.2. Assessment of SARS-CoV-2-Specific Antibodies 2.3. Assessment of SARS-CoV-2-Specific Surrogate Neutralizing Antibodies (SNA) 2.2. Assessment of SARS-CoV-2-Specific Antibodies 2.3. Assessment of SARS-CoV-2-Specific Surrogate Neutralizing Antibodies (SNA) Levels of antibodies targeted against SARS-CoV-2 spike receptor binding domain (S- RBD) were measured with a chemiluminescent microparticle based immunoassay (SARS- CoV-2 IgG II Quant, Abbott GmbH, Wiesbaden, Germany). Results are expressed as standardized binding antibody units (BAU)/mL. According to the manufacturer, a cut-off f i i i i 7 1 BAU/ L W l d h ff 8 52 BAU/ L b ddi The binding of S-RBD to angiotensin-converting enzyme 2 allowing detection of SARS- CoV-2-neutralizing antibodies was performed using the ELISA-based GenScript SARS- CoV-2 Surrogate Virus Neutralization Test Kit (GenScript Biotech, Piscataway Township, NJ, USA) [12]. Results are expressed as percentage inhibition (%-INH). 2.2. Assessment of SARS-CoV-2-Specific Antibodies 2.2. Assessment of SARS-CoV-2-Specific Antibodies Levels of antibodies targeted against SARS-CoV-2 spike receptor binding domain (S- RBD) were measured with a chemiluminescent microparticle based immunoassay (SARS- CoV-2 IgG II Quant, Abbott GmbH, Wiesbaden, Germany). Results are expressed as standardized binding antibody units (BAU)/mL. According to the manufacturer, a cut-off for positivity is set to 7.1 BAU/mL. We elevated the cut-off to 8.52 BAU/mL by adding a “threshold” range in between. 3 of 9 3 of 9 Vaccines 2022, 10, 585 Vaccines 2022, 10, x FO Figure 1. Illustration of the study setup. Samples were collected at three different time points to evaluate the dynamic of the patients’ immunization status by different immunoassays. Abbrevia- tions: CIHD (chronic intermittent hemodialysis); S-RBD (spike receptor binding domain); ELISpot (enzyme-linked immunospot). Figure 1. Illustration of the study setup. Samples were collected at three different time points to evaluate the dynamic of the patients’ immunization status by different immunoassays. Abbreviations: CIHD (chronic intermittent hemodialysis); S-RBD (spike receptor binding domain); ELISpot (enzyme- linked immunospot). Figure 1. Illustration of the study setup. Samples were collected at three different time points to evaluate the dynamic of the patients’ immunization status by different immunoassays. Abbrevia- tions: CIHD (chronic intermittent hemodialysis); S-RBD (spike receptor binding domain); ELISpot (enzyme-linked immunospot). Figure 1. Illustration of the study setup. Samples were collected at three different time points to evaluate the dynamic of the patients’ immunization status by different immunoassays. Abbreviations: CIHD (chronic intermittent hemodialysis); S-RBD (spike receptor binding domain); ELISpot (enzyme- linked immunospot). 2.2. Assessment of SARS-CoV-2-Specific Antibodies 2.3. Assessment of SARS-CoV-2-Specific Surrogate Neutralizing Antibodies (SNA) 2.2. Assessment of SARS-CoV-2-Specific Antibodies 2.3. Assessment of SARS-CoV-2-Specific Surrogate Neutralizing Antibodies (SNA) 3.1. Basic Study Cohort Characteristics We prospectively enrolled 45 CIHD patients, of which 42 met inclusion criteria, with a median (IQR) age of 62 (52–72.5) years. Three patients were excluded due to SARS-CoV-2 positivity (real-time reverse transcriptase PCR). The main clinical and demographic charac- teristics are depicted in Table 1. Due to the incidence of comorbidities, this study cohort is representing a highly vulnerable CIHD group. Five patients were not available for follow- up because of death unrelated to COVID-19 (n = 2, sepsis and cardiac arrest) and change of the dialysis center (n = 3). We observed a good vaccine tolerability, without serious adverse events in this cohort. No patient developed critical side effects or was hospitalized. Table 1. Baseline patient characteristics. NR/LR (no response/low response) group with insufficient humoral response after two doses of SARS-CoV-2 vaccine (t0). Abbreviations: m (male); GN (glomeru- lonephritis); ADPKD (autosomal dominant polycystic kidney disease); Tx (transplant). Data expressed as number (n) and percentage (%) or median (med) and interquartile range (IQR), respectively. Table 1. Baseline patient characteristics. NR/LR (no response/low response) group with insufficient humoral response after two doses of SARS-CoV-2 vaccine (t0). Abbreviations: m (male); GN (glomeru- lonephritis); ADPKD (autosomal dominant polycystic kidney disease); Tx (transplant). Data expressed as number (n) and percentage (%) or median (med) and interquartile range (IQR), respectively. 2.4. Assessmen 2.5. Statistics f y g p The adaptive immune response was analyzed by quantifying interferon-γ (IFN-γ) and IL-2 secreting T cells specific for SARS-CoV-2 using a multicolor fluorescence enzyme-linked immunospot (ELISpot) assay (CoV-iSpot, AID GmbH, Strassberg, Germany) [13]. Highly specific sequences of immunodominant epitopes of SARS-CoV-2 structural proteins (=SARS-CoV-2-peptide-mix) were used for stimulation of T cells. Results are expressed as spot forming cells (SFC)/106 lymphocytes. 2.5. Statistics Continuous variables are shown as median and interquartile range (IQR), categorical variables are reported as frequencies and percentages. Differences between patient cohorts were determined using the Fisher’s exact test for categorical variables; for quantitative variables, a t-test or a Mann–Whitney-U test was used for parametric and nonparametric data, respectively. When comparing more than two groups, one-way ANOVA f ll d b B f i’ h i K k l W lli f ll d b Continuous variables are shown as median and interquartile range (IQR), categor- ical variables are reported as frequencies and percentages. Differences between patient cohorts were determined using the Fisher’s exact test for categorical variables; for quan- titative variables, a t-test or a Mann–Whitney-U test was used for parametric and non- parametric data, respectively. When comparing more than two groups, one-way ANOVA followed by Bonferroni’s post hoc correction or Kruskal–Wallis test followed by Dunn’s post hoc test was performed for parametric and nonparametric data, respectively. For the comparison of related samples comprising more than two groups, a Friedman test was performed. Spearman’s rank correlation analysis was used to determine bivariate relationships by calculating Spearman’s Rho (correlation coefficient). Normality was as- sessed by a Kolmogorov–Smirnov test. All p-values reported are two-sided, the level of significance was set p < 0.05. Statistical analyses were performed using GraphPad Prism version 9.3.1 (GraphPad Software, San Diego, CA, USA) and BiAS (v11.01; epsilon-Verlag, Nordhasted, Germany). Vaccines 2022, 10, 585 4 of 9 3. Results 3.1. Basic Study Cohort Characteristics 3.1. Basic Study Cohort Characteristics 3.1. Basic Study Cohort Characteristics All Patients NR/LR Group p-Value n = 42 n = 10 Age (years) med (IQR) 62 (52–72.5) 74.5 (67.3–79.3) 0.0041 Gender (male) n (%) 29 (69) 6 (60) 0.7109 Vintage on dialysis (years) med (IQR) 2.4 (1–7.2) 6.2 (0.9–12) 0.3893 Days after 2nd vaccination (days) med (IQR) 34.5 (30.5–47.8) 34 (29–47) 0.8189 Ethnicity Caucasian n (%) 31 (73.8) 8 (80) 1.0000 Black n (%) 6 (14.3) 2 (20) 0.6415 Other n (%) 5 (11.9) / Reason for HD Diabetic nephropathy n (%) 9 (21.4) 3 (30) 0.6792 Hypertensive nephropathy n (%) 4 (9.5) 1 (10) 1.0000 ADPKD n (%) 6 (14.3) 2 (20) 0.6415 GN n (%) 3 (7.1) 1 (10) 1.0000 Amyloidosis n (%) 5 (11.9) / Chronic interstitial nephritis n (%) 6 (14.3) / Nephrosclerosis n (%) 6 (14.3) / Vasculitis n (%) 1 (2.4) / Others/unknown n (%) 2 (4.8) 3 (30) 0.0432 Comorbidities Diabetes mellitus n (%) 13 (31.0) 6 (60) 0.1424 Lung disease n (%) 5 (11.9) 2 (20) 0.6079 Smoker n (%) 14 (33.3) 3 (30) 1.0000 Cancer n (%) 5 (11.9) / Severe cardiovascular disease n (%) 23 (54.8) 6 (60) 1.0000 Hypertension n (%) 38 (90.5) 9 (90) 1.0000 Kidney or liver-Tx n (%) 6 (14.3) 4 (40) 0.0847 Immunosupression n (%) 9 (21.4) 6 (60) 0.0243 Data: (%) percentage; (med) median; (IQR) interquartile range; m (male); GN (glomerulonephritis), ADPKD (autosomal dominant polycystic kidney disease). Data: (%) percentage; (med) median; (IQR) interquartile range; m (male); GN (glomerulonephritis), ADPKD (autosomal dominant polycystic kidney disease). 3.2. Dynamics of Antibody Levels in Vaccinated Dialysis Patients 3.2. Dynamics of Antibody Levels in Vaccinated Dialysis Patients Four weeks after the second dose of vaccination (=t0), 37 patients (88.1%) developed antibodies against the SARS-CoV-2 spike protein with a median (IQR) of 128 BAU/mL (43–1027.5), and 32 patients (76.2%) showed functional neutralizing capacity of SNA with a median (IQR) of 69%-INH (15–89), above the predefined threshold for positivity (>8.52 BAU/mL and >30%-INH, respectively; Figure 2). Vaccines 2022, 10, 585 5 of 9 Figure 2. Antibody response in plasma samples of patients taken at three different time points (t0 = four weeks after second dose, t1 = six months after second dose and t2 = 14 days after third dose (=booster vaccine) of mRNA-1273 (Moderna)). Abbreviations: S-RBD-ab (spike receptor binding domain-antibodies); SNA (surrogate neutralizing antibodies). Data are expressed as median (IQR) * p ≤ 0.05, *** p ≤ 0.001, **** p ≤ 0.0001 (Friedman test). Figure 2. Antibody response in plasma samples of patients taken at three different time points (t0 = four weeks after second dose, t1 = six months after second dose and t2 = 14 days after third dose (=booster vaccine) of mRNA-1273 (Moderna)). Abbreviations: S-RBD-ab (spike receptor binding domain-antibodies); SNA (surrogate neutralizing antibodies). Data are expressed as median (IQR). * p ≤0.05, *** p ≤0.001, **** p ≤0.0001 (Friedman test). Figure 2. Antibody response in plasma samples of patients taken at three different time points (t0 = four weeks after second dose, t1 = six months after second dose and t2 = 14 days after third dos (=booster vaccine) of mRNA-1273 (Moderna)). Abbreviations: S-RBD-ab (spike receptor binding domain-antibodies); SNA (surrogate neutralizing antibodies). Data are expressed as median (IQR) * p ≤0.05, *** p ≤0.001, **** p ≤0.0001 (Friedman test). Figure 2. Antibody response in plasma samples of patients taken at three different time points (t0 = four weeks after second dose, t1 = six months after second dose and t2 = 14 days after third dose (=booster vaccine) of mRNA-1273 (Moderna)). Abbreviations: S-RBD-ab (spike receptor binding domain-antibodies); SNA (surrogate neutralizing antibodies). Data are expressed as median (IQR). * p ≤0.05, *** p ≤0.001, **** p ≤0.0001 (Friedman test). During the six-month period, the response rate of positive serology did not change (87.8%) but antibody levels decreased significantly compared to t0 (median (IQR) of 32.3 BAU/mL (9.9–224.1), p < 0.014). 3.2. Dynamics of Antibody Levels in Vaccinated Dialysis Patients Less than half of this study group showed neutralizing antibodies (48.8%) with a median (IQR) of 0%-INH (0–87.8) six months after the second dose of the vaccine. Measuring antibody levels and SNA after the booster vaccine (=t2), we observed a significant elevation compared to timepoint t0 (p < 0.001) and t1 (p < 0.001) with a response rate of 89.2% for antibody levels (median (IQR): 4560 BAU/mL (646.7–7272.5) and a response rate of 94.6% for SNA (median (IQR) of 97%-INH (0–97)). This correspond to a 36 fold i ea e i edia a tibody tite afte the thi d do e o a ed to t During the six-month period, the response rate of positive serology did not change (87.8%) but antibody levels decreased significantly compared to t0 (median (IQR) of 32.3 BAU/mL (9.9–224.1), p < 0.014). Less than half of this study group showed neutralizing antibodies (48.8%) with a median (IQR) of 0%-INH (0–87.8) six months after the second dose of the vaccine. Measuring antibody levels and SNA after the booster vaccine (=t2), we observed a significant elevation compared to timepoint t0 (p < 0.001) and t1 (p < 0.001) with a response rate of 89.2% for antibody levels (median (IQR): 4560 BAU/mL (646.7–7272.5)) and a response rate of 94.6% for SNA (median (IQR) of 97%-INH (0–97)). This corresponds to a 36-fold increase in median antibody titers after the third dose compared to t0. to a 36-fold increase in median antibody titers after the third dose compared to t0. To get a more detailed insight into the distribution of the humoral immune response we predefined different threshold values depending on the level of antibodies o neutralization capacity. Applying these antibodies and SNA cut-off levels (<100 BAU/mL or <50%-INH, no response/low response; 100–500 BAU/mL or 50–70%-IHN, moderat response; >500 BAU/mL or >70%-IHN, strong response) the overall response rates at t0, t and t2 are described in Figure 3, demonstrating a significant reduction in the humora immunity six months after the second dose of the vaccine (=t1), followed by a distinc y p To get a more detailed insight into the distribution of the humoral immune response we predefined different threshold values depending on the level of antibodies or neutralization capacity. 3.2. Dynamics of Antibody Levels in Vaccinated Dialysis Patients Proportion of patients with strong (dark blue: >500 BAU/mL or >70%-INH), moderate (light blue: 100–500 BAU/mL or 50–70%-INH) and no/low (red: <100 BAU/mL or <50%-INH) humoral response at the three different time points (t0, t1 and t2). Data are expressed as percentage. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 compared to t0; ### p ≤ 0.001 compared to t1. Figure 3. Proportion of patients with strong (dark blue: >500 BAU/mL or >70%-INH), moderate (light blue: 100–500 BAU/mL or 50–70%-INH) and no/low (red: <100 BAU/mL or <50%-INH) humoral response at the three different time points (t0, t1 and t2). Data are expressed as percentage. * p ≤0.05, ** p ≤0.01, *** p ≤0.001 compared to t0; ### p ≤0.001 compared to t1. neutralizing capacity for different antibody levels, as illustrated in Figure 4. Data from our study group reveal that levels of SARS-CoV-2-spike-specific antibodies are significantly correlated with the percentage of SNA (t0 rho: 0.83; p < 0.001; t1 rho: 0.9; p < 0.001 and t2 rho: 0.58; p < 0.001). Figure 3. Proportion of patients with strong (dark blue: >500 BAU/mL or >70%-INH), moderate (light blue: 100–500 BAU/mL or 50–70%-INH) and no/low (red: <100 BAU/mL or <50%-INH) humoral response at the three different time points (t0, t1 and t2). Data are expressed as percentage. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 compared to t0; ### p ≤ 0.001 compared to t1. Figure 3. Proportion of patients with strong (dark blue: >500 BAU/mL or >70%-INH), moderate (light blue: 100–500 BAU/mL or 50–70%-INH) and no/low (red: <100 BAU/mL or <50%-INH) humoral response at the three different time points (t0, t1 and t2). Data are expressed as percentage. * p ≤0.05, ** p ≤0.01, *** p ≤0.001 compared to t0; ### p ≤0.001 compared to t1. neutralizing capacity for different antibody levels, as illustrated in Figure 4. Data from our study group reveal that levels of SARS-CoV-2-spike-specific antibodies are significantly correlated with the percentage of SNA (t0 rho: 0.83; p < 0.001; t1 rho: 0.9; p < 0.001 and t2 rho: 0.58; p < 0.001). The neutralizing capacity of SARS-CoV-2-specific antibodies is even more important than only the magnitude of antibody levels. For this reason, we analyzed the fraction of neutralizing capacity for different antibody levels, as illustrated in Figure 4. 3.2. Dynamics of Antibody Levels in Vaccinated Dialysis Patients * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 compared to t0; ### p ≤ 0.001 compared to t1. The neutralizing capacity of SARS-CoV-2-specific antibodies is even more important than only the magnitude of antibody levels. For this reason, we analyzed the fraction of neutralizing capacity for different antibody levels, as illustrated in Figure 4. Data from our study group reveal that levels of SARS-CoV-2-spike-specific antibodies are significantly correlated with the percentage of SNA (t0 rho: 0.83; p < 0.001; t1 rho: 0.9; p < 0.001 and t2 rho: 0.58; p < 0.001). Figure 3. Proportion of patients with strong (dark blue: >500 BAU/mL or >70%-INH), moderate (light blue: 100–500 BAU/mL or 50–70%-INH) and no/low (red: <100 BAU/mL or <50%-INH) humoral response at the three different time points (t0, t1 and t2). Data are expressed as percentage. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 compared to t0; ### p ≤ 0.001 compared to t1. Figure 3. Proportion of patients with strong (dark blue: >500 BAU/mL or >70%-INH), moderate (light blue: 100–500 BAU/mL or 50–70%-INH) and no/low (red: <100 BAU/mL or <50%-INH) humoral response at the three different time points (t0, t1 and t2). Data are expressed as percentage. * p ≤0.05, ** p ≤0.01, *** p ≤0.001 compared to t0; ### p ≤0.001 compared to t1. Figure 3. Proportion of patients with strong (dark blue: >500 BAU/mL or >70%-INH), moderate (light blue: 100–500 BAU/mL or 50–70%-INH) and no/low (red: <100 BAU/mL or <50%-INH) humoral response at the three different time points (t0, t1 and t2). Data are expressed as percentage. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 compared to t0; ### p ≤ 0.001 compared to t1. The neutralizing capacity of SARS-CoV-2-specific antibodies is even more important than only the magnitude of antibody levels. For this reason, we analyzed the fraction of neutralizing capacity for different antibody levels, as illustrated in Figure 4. Data from our study group reveal that levels of SARS-CoV-2-spike-specific antibodies are significantly correlated with the percentage of SNA (t0 rho: 0.83; p < 0.001; t1 rho: 0.9; p < 0.001 and t2 rho: 0.58; p < 0.001). p y de of an vels. F d the Figure 3. 3.2. Dynamics of Antibody Levels in Vaccinated Dialysis Patients Proportion of patients with strong (dark blue: >500 BAU/mL or >70%-INH), moderate (light blue: 100–500 BAU/mL or 50–70%-INH) and no/low (red: <100 BAU/mL or <50%-INH) humoral response at the three different time points (t0, t1 and t2). Data are expressed as percentage. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 compared to t0; ### p ≤ 0.001 compared to t1. The neutralizing capacity of SARS-CoV-2-specific antibodies is even more important than only the magnitude of antibody levels. For this reason, we analyzed the fraction of neutralizing capacity for different antibody levels, as illustrated in Figure 4. Data from our study group reveal that levels of SARS-CoV-2-spike-specific antibodies are significantly correlated with the percentage of SNA (t0 rho: 0.83; p < 0.001; t1 rho: 0.9; p < 0.001 and t2 rho: 0.58; p < 0.001). Figure 4. Fraction of neutralizing capacity for different antibody levels. The inner circle is showing levels of antibody response (BAU/mL) with the corresponding percentage of neutralizing antibodies (%-INH) in the outer circle for t0, t1 and t2, respectively. Data are expressed as percentages. Applying the following antibody and SNA cut-off levels: negative (<7.1 BAU/mL or <30%-INH), border- line (>7.1–8.5 BAU/mL or >30–35%-IHN), low (>8.5–100 BAU/mL or >35–50%-INH), moderate (>100–500 BAU/mL or >50–70%-INH), strong (>500 BAU/mL or >70%-INH). Figure 3. Proportion of patients with strong (dark blue: >500 BAU/mL or >70%-INH), moderate (light blue: 100–500 BAU/mL or 50–70%-INH) and no/low (red: <100 BAU/mL or <50%-INH) humoral response at the three different time points (t0, t1 and t2). Data are expressed as percentage. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 compared to t0; ### p ≤ 0.001 compared to t1. Figure 3. Proportion of patients with strong (dark blue: >500 BAU/mL or >70%-INH), moderate (light blue: 100–500 BAU/mL or 50–70%-INH) and no/low (red: <100 BAU/mL or <50%-INH) humoral response at the three different time points (t0, t1 and t2). Data are expressed as percentage. * p ≤0.05, ** p ≤0.01, *** p ≤0.001 compared to t0; ### p ≤0.001 compared to t1. Figure 3. Proportion of patients with strong (dark blue: >500 BAU/mL or >70%-INH), moderate (light blue: 100–500 BAU/mL or 50–70%-INH) and no/low (red: <100 BAU/mL or <50%-INH) humoral response at the three different time points (t0, t1 and t2). Data are expressed as percentage. 3.2. Dynamics of Antibody Levels in Vaccinated Dialysis Patients Applying these antibodies and SNA cut-off levels (<100 BAU/mL or <50%- INH, no response/low response; 100–500 BAU/mL or 50–70%-IHN, moderate response; >500 BAU/mL or >70%-IHN, strong response) the overall response rates at t0, t1 and t2 are described in Figure 3, demonstrating a significant reduction in the humoral immunity six months after the second dose of the vaccine (=t1), followed by a distinct increase two weeks after the booster vaccine (=t2) with a significant difference, respectively. increase two weeks after the booster vaccine (=t2) with a significant difference respectively. g p y The neutralizing capacity of SARS-CoV-2-specific antibodies is even more important than only the magnitude of antibody levels. For this reason, we analyzed the fraction of neutralizing capacity for different antibody levels, as illustrated in Figure 4. Data from our study group reveal that levels of SARS-CoV-2-spike-specific antibodies are significantly correlated with the percentage of SNA (t0 rho: 0.83; p < 0.001; t1 rho: 0.9; p < 0.001 and t2 rho: 0.58; p < 0.001). Vaccines 2022, 10, 585 Vaccines 2022, 10, x FOR 6 of 9 6 of 9 Figure 3. Proportion of patients with strong (dark blue: >500 BAU/mL or >70%-INH), moderate (light blue: 100–500 BAU/mL or 50–70%-INH) and no/low (red: <100 BAU/mL or <50%-INH) humoral response at the three different time points (t0, t1 and t2). Data are expressed as percentage. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 compared to t0; ### p ≤ 0.001 compared to t1. The neutralizing capacity of SARS-CoV-2-specific antibodies is even more important than only the magnitude of antibody levels. For this reason, we analyzed the fraction of neutralizing capacity for different antibody levels, as illustrated in Figure 4. Data from our study group reveal that levels of SARS-CoV-2-spike-specific antibodies are significantly correlated with the percentage of SNA (t0 rho: 0.83; p < 0.001; t1 rho: 0.9; p < 0.001 and t2 rho: 0.58; p < 0.001). Figure 3. Proportion of patients with strong (dark blue: >500 BAU/mL or >70%-INH), moderate (light blue: 100–500 BAU/mL or 50–70%-INH) and no/low (red: <100 BAU/mL or <50%-INH) humoral response at the three different time points (t0, t1 and t2). Data are expressed as percentage. * p ≤0.05, ** p ≤0.01, *** p ≤0.001 compared to t0; ### p ≤0.001 compared to t1. Figure 3. 3.3. T Cell Response after Vaccination 3.3. T Cell Response after Vaccination F k ft th d d Four weeks after the second dose, SARS-CoV-2-specific T cell reactivity of CIHD patients showed significantly lower levels of IFN-γ release compared to the healthy control group (HC, n = 11) (median (IQR) 7.5 SFC/106 cells (2.5–42.5) vs. 72.5 SFC/106 cells (21.25–101.25) (p < 0.001)), but no difference in IL-2 release between both groups (median (IQR) 40 SFC/106 cells (8.75–111.25) vs. 47.5 SFC/106 cells (28.75–68.75) (p < 0.001)); Figure 5. A positive IFN-γ release was only observed for nine patients (21.4%). A positive release of IL-2 after the booster vaccine occurred in 17 patients (40.5%). Four weeks after the second dose, SARS-CoV-2-specific T cell reactivity of CIHD patients showed significantly lower levels of IFN-γ release compared to the healthy control group (HC, n = 11) (median (IQR) 7.5 SFC/106 cells (2.5–42.5) vs. 72.5 SFC/106 cells (21.25–101.25) (p < 0.001)), but no difference in IL-2 release between both groups (median (IQR) 40 SFC/106 cells (8.75–111.25) vs. 47.5 SFC/106 cells (28.75–68.75) (p < 0.001)); Figure 5. A positive IFN-γ release was only observed for nine patients (21.4%). A positive release of IL-2 after the booster vaccine occurred in 17 patients (40.5%). Figure 5. Cytokine profile of T cell response specific to SARS-CoV-2 at different time points and in comparison to a healthy control group. Abbreviations: ELISpot (enzyme-linked immunospot); ctrl (control); IFN-γ (Interferon-γ); IL-2 (Interleukin-2). Data are expressed as median (IQR). Intragroup paired analysis: * p ≤ 0.05, ** p ≤ 0.01 as indicated (Kruskal–Wallis test). Figure 5. Cytokine profile of T cell response specific to SARS-CoV-2 at different time points and in comparison to a healthy control group. Abbreviations: ELISpot (enzyme-linked immunospot); ctrl (control); IFN-γ (Interferon-γ); IL-2 (Interleukin-2). Data are expressed as median (IQR). Intragroup paired analysis: * p ≤0.05, ** p ≤0.01 as indicated (Kruskal–Wallis test). Figure 5. Cytokine profile of T cell response specific to SARS-CoV-2 at different time points and in comparison to a healthy control group. Abbreviations: ELISpot (enzyme-linked immunospot); ctrl (control); IFN-γ (Interferon-γ); IL-2 (Interleukin-2). Data are expressed as median (IQR). Intragroup paired analysis: * p ≤ 0.05, ** p ≤ 0.01 as indicated (Kruskal–Wallis test). Figure 5. Cytokine profile of T cell response specific to SARS-CoV-2 at different time points and in comparison to a healthy control group. Abbreviations: ELISpot (enzyme-linked immunospot); ctrl (control); IFN-γ (Interferon-γ); IL-2 (Interleukin-2). Data are expressed as median (IQR). Intragroup paired analysis: * p ≤0.05, ** p ≤0.01 as indicated (Kruskal–Wallis test). 3.3. T Cell Response after Vaccination 3.3. T Cell Response after Vaccination F k ft th d d We measured the T cell response again after the booster vaccination (t2) in a no response/low response NR/LR subgroup (n = 10) of t0. One patient showed a positive IFN- γ release after the second dose, but none of the NR/LR group showed a positive release of IL-2. Although data of NR/LR patients present a robust serological response to the booster vaccination (median (IQR): 990 BAU/mL (235.3–5033.3); 94%-INH (64.3–97)), only two patients showed a positive release of IFN-γ and only one patient for IL-2. Compared to the remaining study group (median (IQR): 5261 BAU/mL (970.8–10,238)), we measured significant lower antibody levels in the NR/LR subgroup (p < 0.049). In summary, only one patient in the NR/LP subgroup developed a T cell response after booster vaccination (IFN-γ: 132.5 SFC/106 lymphocytes; IL-2: 165 SFC/106 lymphocytes). We measured the T cell response again after the booster vaccination (t2) in a no response/low response NR/LR subgroup (n = 10) of t0. One patient showed a positive IFN-γ release after the second dose, but none of the NR/LR group showed a positive release of IL-2. Although data of NR/LR patients present a robust serological response to the booster vaccination (median (IQR): 990 BAU/mL (235.3–5033.3); 94%-INH (64.3–97)), only two patients showed a positive release of IFN-γ and only one patient for IL-2. Compared to the remaining study group (median (IQR): 5261 BAU/mL (970.8–10,238)), we measured significant lower antibody levels in the NR/LR subgroup (p < 0.049). In summary, only one patient in the NR/LP subgroup developed a T cell response after booster vaccination (IFN-γ: 132.5 SFC/106 lymphocytes; IL-2: 165 SFC/106 lymphocytes). 3.2. Dynamics of Antibody Levels in Vaccinated Dialysis Patients Data from our study group reveal that levels of SARS-CoV-2-spike-specific antibodies are significantly correlated with the percentage of SNA (t0 rho: 0.83; p < 0.001; t1 rho: 0.9; p < 0.001 and t2 rho: 0.58; p < 0.001). The neutralizing capacity of SARS-CoV-2-specific antibodies is even more important than only the magnitude of antibody levels. For this reason, we analyzed the fraction of neutralizing capacity for different antibody levels, as illustrated in Figure 4. Data from our study group reveal that levels of SARS-CoV-2-spike-specific antibodies are significantly correlated with the percentage of SNA (t0 rho: 0.83; p < 0.001; t1 rho: 0.9; p < 0.001 and t2 rho: 0.58; p < 0.001). Figure 4. Fraction of neutralizing capacity for different antibody levels. The inner circle is showing levels of antibody response (BAU/mL) with the corresponding percentage of neutralizing antibodies (%-INH) in the outer circle for t0, t1 and t2, respectively. Data are expressed as percentages. Applying the following antibody and SNA cut-off levels: negative (<7.1 BAU/mL or <30%-INH), border- line (>7.1–8.5 BAU/mL or >30–35%-IHN), low (>8.5–100 BAU/mL or >35–50%-INH), moderate (>100–500 BAU/mL or >50–70%-INH), strong (>500 BAU/mL or >70%-INH). izing capacity of SARS-CoV agnitude of antibody levels th ntibodies is even more imp son we analyzed the frac ntibodies is even more imp son we analyzed the frac ntibodies is even more imp son we analyzed the frac Figure 4. Fraction of neutralizing capacity for different antibody levels. The inner circle is showing levels of antibody response (BAU/mL) with the corresponding percentage of neutralizing antibodies (%-INH) in the outer circle for t0, t1 and t2, respectively. Data are expressed as percentages. Applying the following antibody and SNA cut-off levels: negative (<7.1 BAU/mL or <30%-INH), border- line (>7.1–8.5 BAU/mL or >30–35%-IHN), low (>8.5–100 BAU/mL or >35–50%-INH), moderate (>100–500 BAU/mL or >50–70%-INH), strong (>500 BAU/mL or >70%-INH). Figure 4. Fraction of neutralizing capacity for different antibody levels. The inner circle is showing levels of antibody response (BAU/mL) with the corresponding percentage of neutralizing antibodies (%-INH) in the outer circle for t0, t1 and t2, respectively. Data are expressed as percentages. Applying the following antibody and SNA cut-off levels: negative (<7.1 BAU/mL or <30%-INH), border- line (>7.1–8.5 BAU/mL or >30–35%-IHN), low (>8.5–100 BAU/mL or >35–50%-INH), moderate (>100–500 BAU/mL or >50–70%-INH), strong (>500 BAU/mL or >70%-INH). Vaccines 2022, 10, 585 7 of 9 derline 00–500 3.3. T Cell Response after Vaccination 3.3. 3.2. Dynamics of Antibody Levels in Vaccinated Dialysis Patients T Cell Response after Vaccination F k ft th d d 4. Discussion 4. Discussion Patients undergoing CIHD represent a vulnerable population with an increased risk of SARS-CoV-2 infection, higher disease severity and COVID-19-related mortality risk Patients undergoing CIHD represent a vulnerable population with an increased risk of SARS-CoV-2 infection, higher disease severity and COVID-19-related mortality risk [14]. For this group, there is still lacking data for long-term humoral and cellular immune responses, especially relating to the presence of neutralizing antibodies. In this study, we analyzed the magnitude and kinetics of antibody and T cell responses to the initial immunization (t0), the status quo after 6 months (t1) and the state following a booster with Moderna’s mRNA-1273 (t2). In our cohort of hemodialysis patients, 88.1% developed antibodies against the SARS-CoV-2 spike protein 4 weeks after the primary vaccine series, and 76.2% showed functional neutralizing capacity of SNA (surrogate neutralizing antibod- Vaccines 2022, 10, 585 8 of 9 ies). It was reported that neutralizing antibody titers decreased over time in patients after SARS-CoV-2 infection [15]. However, after 6 months we can clearly see a significant decline, not only in antibody levels, but even more decisively, a loss in neutralizing capacity. Impor- tantly, more than half of our patients (51.2%) had no levels of SARS-CoV-2-neutralizing antibodies at that time. Comparable results have been reported by Ducloux et al. and Kohmer et al. for Pfizer-BioNTech’s BNT162b2 [9,16]. In line with our data, both studies confirm that levels of SARS-CoV-2-specific antibodies were significantly increased by ad- ministering a third dose of a COVID-19 mRNA vaccine. However, cell-mediated immunity was not analyzed in these studies. y We conclude that a decline in neutralizing antibody levels could be prevented by a timed booster intervention for our cohort. However, measurement in a NR/LR subgroup of our cohort also indicates failure of a corresponding T cell response after the booster vaccine. These findings are in line with data of Espi et al. that describe a missing increase in T cell response after third dose vaccination in dialysis patients [17]. Notably, the NR/LR subgroup showed a higher rate of immunosuppressive medication (21.4% vs. 60%, p = 0.024) and a higher age (median (IQR): 62 (52–72.5) vs. 74.5 (67.3–79.3)) compared to the whole study group, which could be a confounder and an explanation for the diminished T cell response after booster vaccination. 4. Discussion 4. Discussion To analyze the influence of these possible confounders, a higher number of participants in the NR/LR subgroup would be required, which poses a limitation of this study. y Our data underline the importance of SNA when screening for the response of pa- tient´s adaptive immunity after SARS-CoV-2 vaccination, especially in this highly vulner- able group of CIHD patients. Hence, CIHD patients are not only particularly threatened by the COVID-19 pandemic because of a more severe course of disease and a higher risk of infection caused by higher frequency of medical consultations but, most notably, by a lower response after the second dose of vaccination, combined with a relevant loss of both serological and specific T cell immunity. T cell mediated immunity seems to play an important role in SARS-CoV-2 infection. However, to what extent is not clear. The presented results are highlighting the importance of an early booster vaccination of CIHD patients. Further studies are required to generate convincing data for CIHD patients on long- term T cell response after SARS-CoV-2 vaccination. This study only provides data on T cell response after booster vaccination in a rather small subgroup of initial NR/LR patients. In addition, more investigations regarding protection against newly arising variants (e.g., Omicron) are needed. References 1. Losappio, V.; Franzin, R.; Infante, B.; Godeas, G.; Gesualdo, L.; Fersini, A.; Castellano, G.; Stallone, G. Molecular Mechanisms of Premature Aging in Hemodialysis: The Complex Interplay Between Innate and Adaptive Immune Dysfunction. Int. J. Mol. Sci. 2020, 21, 3422. [CrossRef] [PubMed] 2. Taji, L.; Thomas, D.; Oliver, M.J.; Ip, J.; Tang, Y.; Yeung, A.; Cooper, R.; House, A.A.; McFarlane, P.; Blake, P.G. COVID-19 in patients undergoing long-term dialysis in Ontario. Can. Med Assoc. J. 2021, 193, E278–E284. [CrossRef] [PubMed] 3. 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Sahin, U.; Muik, A.; Vogler, I.; Derhovanessian, E.; Kranz, L.M.; Vormehr, M.; Quandt, J.; Bidmon, N.; Ulges, A.; Türeci, Ö.; et al. BNT162b2 vaccine induces neutralizing antibodies and poly-specific T cells in humans. Nature 2021, 595, 572–577. [CrossRef] [PubMed] 7. References Simon, B.; Rubey, H.; Treipl, A.; Gromann, M.; Hemedi, B.; Zehetmayer, S.; Kirsch, B. Haemodialysis patients show a highly diminished antibody response after COVID-19 mRNA vaccination compared to healthy controls. Nephrol. Dial. Transplant. 2021, 36, 1709–1716. [CrossRef] [PubMed] 8. Speer, C.; Schaier, M.; Nusshag, C.; Töllner, M.; Buylaert, M.; Kälble, F.; Reichel, P.; Grenz, J.; Süsal, C.; Benning, L.; et al. Longitudinal Humoral Responses after COVID-19 Vaccination in Peritoneal and Hemodialysis Patients over Twelve Weeks. Vaccines 2021, 9, 1130. [CrossRef] [PubMed] 9. Ducloux, D.; Colladant, M.; Chabannes, M.; Yannaraki, M.; Courivaud, C. Humoral response after 3 doses of the BNT162b2 mRNA COVID-19 vaccine in patients on hemodialysis. Kidney Int. 2021, 100, 702–704. [CrossRef] [PubMed] l dd h d l h l 10. Kalimuddin, S.; Tham, C.Y.; Qui, M.; de Alwis, R.; Sim, J.X.; Lim, J.M.; Tan, H.-C.; Syenina, A.; Zhang, S.L.; Low, J.G.; et al. Early T cell and binding antibody responses are associated with COVID-19 RNA vaccine efficacy onset. Med 2021, 2, 682–688.e4. [CrossRef] [PubMed] [ ] [ ] 11. Moss, P. The T cell immune response against SARS-CoV-2. Nat. Immunol. 2022, 23, 186–193. [CrossRef] [PubMed] ] ell immune response against SARS-CoV-2. Nat. Immunol. 2022, 23, 186–193. [CrossRef] [PubMed] 11. Moss, P. The T cell immune response against SARS-CoV-2. Nat. Immunol. 2022, 23, 186–193. [CrossRef] 12. Kohmer, N.; Rühl, C.; Ciesek, S.; Rabenau, H.F. Utility of Different Surrogate Enzyme-Linked Immunosorbent Assays (sELISAs) for Detection of SARS-CoV-2 Neutralizing Antibodies. J. Clin. Med. 2021, 10, 2128. [CrossRef] [PubMed] 13. Lehmann, P.V.; Zhang, W. Unique strengths of ELISPOT for T cell diagnostics. Methods Mol. Biol. 2012, 792, 3–23. [PubMed] 14. Jdiaa, S.S.; Mansour, R.; El Alayli, A.; Gautam, A.; Thomas, P.; Mustafa, R.A. COVID–19 and chronic kidney disease: An updated overview of reviews. J. Nephrol. 2022, 35, 69–85. [CrossRef] [PubMed] p 15. Wu, L.-P.; Wang, N.-C.; Chang, Y.-H.; Tian, X.-Y.; Na, D.-Y.; Zhang, L.-Y.; Zheng, L.; Lan, T.; Wang, L.-F.; Liang, G.-D. Duration of Antibody Responses after Severe Acute Respiratory Syndrome. Emerg. Infect. Dis. 2007, 13, 1562–1564. [CrossRef] [PubMed] 16 K h N R b H F Ci k S K ä B K Gött U K ll C R D Bl C Th M L t A t l y p p y y g f 16. Kohmer, N.; Rabenau, H.F.; Ciesek, S.; Krämer, B.K.; Göttmann, U.; Keller, C.; Rose, D.; Blume, C.; Thomas, M.; Lammert, A.; et al. 5. Conclusions In summary, a third dose of SARS-CoV-2 mRNA-1273 vaccine in high-risk CIHD patients increased the magnitude and the neutralizing capacity of SARS-CoV-2-specific antibodies, even in the NR/LR subgroup. The relevance of a repeated booster vaccination after a certain period to enhance immunity against SARS-CoV-2 provides an incent for the next inoculation campaign for these immunocompromised CIHD patients. Author Contributions: Conceptualization, C.B. and S.P.; methodology, S.P., B.F.K. and S.S.; validation, T.E., H.F.R. and S.S.; formal analysis, S.P; investigation, S.P., S.S., H.F.R., D.A. and R.S.; resources, C.B., I.A.H., S.S. and J.M.P.; data curation, S.P.; writing—original draft preparation, S.P.; writing—review and editing, T.E., B.F.K., I.A.H., J.M.P., A.O., N.K., H.F.R., S.S. and S.C.; visualization, S.P. and A.O.; supervision, C.B.; project administration, C.B. and I.A.H. All authors have read and agreed to the published version of the manuscript. Funding: The APC was funded by the University Library of Goethe University Frankfurt, Germany. Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of University Hospital Frankfurt, Germany (reference number: 20/658; 14 January 2021). Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of University Hospital Frankfurt, Germany (reference number: 20/658; 14 January 2021). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Vaccines 2022, 10, 585 9 of 9 Acknowledgments: We thank Rosemarie Preyer and Ulrike Loewe from AID Autoimmun Diagnos- tika (Strassberg, Germany) for their contribution to this study. Conflicts of Interest: The authors declare no conflict of interest. References Heterologous immunization with BNT162b2 followed by mRNA-1273 in dialysis patients: Seroconversion and presence of neutralizing antibodies. Nephrol. Dial. Transplant. 2022, gfac144. [CrossRef] [PubMed] 17. Espi, M.; Charmetant, X.; Barba, T.; Mathieu, C.; Pelletier, C.; Koppe, L.; Chalencon, E.; Kalbacher, E.; Mathias, V.; Ovize, A.; et al. A prospective observational study for justification, safety, and efficacy of a third dose of mRNA vaccine in patients receiving maintenance hemodialysis. Kidney Int. 2022, 101, 390–402. [CrossRef] [PubMed]
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https://zenodo.org/records/6990634/files/IJHSS%203(8)%201-6.pdf
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Motivation in Writing Scientific Articles: A Case Study at Junior Higher School (SMP) PGRI 6 Surabaya
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INTRODUCTION Writing scientific articles is an academic activity for teachers, mainly junior high school (SMP) teachers. This is an activity that gives teachers skills in writing their ideas in the form of popular scientific articles. This can be the same skill as writing textbooks as student textbooks if these scientific writing skills are owned by every teacher (Moeliono, 2017). In terms of writing skills, the activity of writing popular scientific articles can also become a habit of reading or literacy. This is because writing activities will be carried out smoothly if their reading activities are also higher. In essence, writing requires sufficient reference by reading so that writers can express their ideas in words that are appropriate to their context or field (Handayani et al., 2014). Therefore, the activity of writing scientific papers is a teacher's skill that supports mainly when writing textbooks for the rest and this must be strongly supported by reading activities. Based on the condition of the location when observed twice, namely on May 21, 2022 and July 18, 2022 at SMP PGRI 6 Surabaya, there are several obstacles as follows. First, there is the principal's interest in improving the quality of teachers. It is expected that the improvement of the quality of teachers is also related to the writing of scientific papers. Second, there are no activities held by the government specifically related to the writing of popular scientific papers so far. And, thirdly, there is high interest in teachers when they were visited by researchers, especially the second visit on July 18, 2022. Therefore, this research is also related to the popular scientific writing training program for teachers of SMP PGRI 6 Surabaya. First, they want to know the extent of their knowledge and skills in writing popular scientific papers. Second, this research will provide strategic suggestions for improving the writing skills of popular scientific papers so that they are accustomed to writing and directly try to read as a source of writing material. So far, not all schools have got training in scientific writing skills in the places where their schools they are teaching. Therefore, not all of them have the opportunity to even write in the media (Interview with the Principal of SMP PGRI 6 Surabaya). In addition, there are not always trainings that teach them to write scientific papers. Research Article Research Article Citation Dijuwari., Saputri, T., Authar, N., Hardiningrum, A., & Arifah, L. Z. (2022). Motivation in Writing Scientific Articles: A Case Study at Junior Higher School (SMP) PGRI 6 Surabaya. Indiana Journal of Humanities and Social Sciences, 3(8), 1-6. Keywords: Scientific writing, internal motivation, external motivation, Policy. and Social Sciences, 3(8), 1-6. Copyright © 2022 The Author(s): This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC BY-NC 4 0) and Social Sciences, 3(8), 1-6. Copyright © 2022 The Author(s): This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC BY-NC 4 0) and Social Sciences, 3(8), 1-6. Copyright © 2022 The Author(s): This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 Interna 4 0) , ( ), Copyright © 2022 The Author(s): This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC BY-NC 4.0). promoting school literacy is very intense (Priyasti & Suyatno, 2021). It is based on their research at the Sidereja 1 State Elementary School. It was stated that this program was also developed through a literacy strategy with professional development related to the teaching of teachers and students in their schools. Environmental conditions also support literacy activities. Therefore, reading and later activities to improve writing skills will be in line with the training activities for teachers of SMP PGRI 6 Surabaya. Article History Received: 04.08.2022 Accepted: 08.08.2022 Published: 12.08.2022 Citation Dijuwari., Saputri, T., Authar, N., Hardiningrum, A., & Arifah, L. Z. (2022). Motivation in Writing Scientific Articles: A Case Study at Junior Higher School (SMP) PGRI 6 Surabaya. Indiana Journal of Humanities and Social Sciences, 3(8), 1-6. *Corresponding Author: Djuwari OPEN ACCESS JOURNALS Indiana Journal of Humanities and Social Sciences Abbriviate Tittle- Ind J Human Soc Sci ISSN (Online)- 2582-8630 Journal Homepage Link- https://indianapublications.com/journal/IJHSS DOI: 10.5281/zenodo.6990634 INDIANA PRODUCTIVE AND QUALITY RESEARCH OPEN ACCESS JOURNALS Indiana Journal of Humanities and Social Sciences Abbriviate Tittle- Ind J Human Soc Sci ISSN (Online)- 2582-8630 Journal Homepage Link- https://indianapublications.com/journal/IJHSS DOI: 10.5281/zenodo.6990634 INDIANA PRODUCTIVE AND QUALITY RESEARCH OPEN ACCESS JOURNALS Indiana Journal of Humanities and Social Sciences Abbriviate Tittle- Ind J Human Soc Sci ISSN (Online)- 2582-8630 Journal Homepage Link- https://indianapublications.com/journal/IJHSS DOI: 10.5281/zenodo.6990634 INDIANA PRODUCTIVE AND QUALITY RESEARCH INDIANA Volume-03|Issue-08|2022 Volume-03|Issue-08|2022 Volume-03|Issue-08|2022 uwari*1, Tiyas Saputri1, Nailul Authar1, Andini Hardiningrum1, Lia Zannuba Arifah1 Djuwari*1, Tiyas Saputri1, Nailul Authar1, Andini Hardiningrum1, Lia Zannuba Arifah1 1Universitas Nahdlatul Ulama Surabaya (UNUSA), Indonesia 1Universitas Nahdlatul Ulama Surabaya (UNUSA), Indonesia Abstract: Writing scientific articles is a good way for improving the teachers’ quality in their profession. Therefore, teachers need this skill in order they can increase their reasoning for the profession. This study tried to explore the teachers’ interest in writing scientific articles at Junior High School (SMP) PGRI 6 Surabaya, Indonesia. It also tried to find out what factors made them interested in writing scientific articles offered to them. This study took 22 teachers as the respondents. They were interviewed in focus-group discussion (FGD). The data were also taken from the interview with the School Principal. These two sources of the data triangulated, and interpreted. The results showed that both the teachers and the school principal have the same interest and willingness to have training on scientific articles. It was also found out that there was no policy taken by the school and the government for that school related to increasing their skill of writing scientific articles. It is recommended that the training program on writing scientific articles be done soon. The school and government policy is needed. This can increase the teachers’ motivation in writing scientific article. For further research, exploring the effectiveness of the training program is required. Article History Received: 04.08.2022 Accepted: 08.08.2022 Published: 12.08.2022 INTRODUCTION This is actually in accordance with the condition in Indonesia, in general, regarding student literacy in primary and secondary schools. Due to this condition, currently the government's program for 1 1 Djuwari et al.; Ind J Human Soc Sci; Vol-3, Iss-8 (Aug, 2022): 1-6 School Policy The policy of the school where the teacher teaches is also an external motivation. The awareness of teachers and schools in terms of the importance of writing is also an attraction that can encourage someone to write. In essence, school work programs can encourage students' interest in writing. This can be implemented in the form of school policies so that students have an interest in writing. Policies are very important related to motivation to write (Akyol & Aktaş, 2018). In relation to this research, there is also research conducted by Wandasari et al. (2019). They stated that one of the external factors that could increase students' interest in writing was also school policy. If the school has policies related to scientific writing, for example literacy programs, the students' interest in writing will increase. So, teachers and schools and even the government are also factors that can increase students' writing creativity. This is implemented in the form of school policies. In the framework, this study can be drawn in Figure 1. Writing Interest Writing strategies will be more effective if the topic of writing is related to one's daily life. In relation to writing interest, Boscolo & Hidi (2007), in his research, it was revealed that there was a relationship between interest in writing and interest in the chosen topic. The chosen topic will help the authors if the topic is interesting. Interesting topics are topics that are liked by the authors. Therefore, so that there is an attraction for teachers' writing motivation, it would be better if they were given interesting topics for them. Meanwhile, according to Akyol & Aktaş (2018), interest in writing is also determined by themes related to stories related to students' lives. In their research, it was found that stories of students' lives can provide them with high motivation in writing. From this empirical evidence, the writing strategy will be more effective if the topic is interesting and related to the daily life of writers, such as teachers or students. Interest in writing is very important for all scientists, ranging from high school students, students, teachers, lecturers, and scientists. This is also according to Afianto (2018); it is also very helpful in the development of emotional intelligence as well as to increase interest in writing. So, writing skills or intelligence, for example, writing arguments also supports emotional intelligence and interest in writing. If this is supported by the results of research such as that conducted by Afianto, then scientific writing training is an activity program that greatly supports not only writing skills but also develops a person's emotions and interest in writing. Meanwhile, there are several factors that influence the increasing interest in writing in human life, thus Jatmika (2019), in his research, recommends that to increase literacy and interest in reading and especially writing, preconditions are needed, such as infrastructure in schools, communities, and parents. From these findings and recommendations, it seems that there are many factors, including the condition of the school in terms of infrastructure, the condition of the community, and even the parents. In relation to these factors, the training of writing scientific papers is one of the supports for the increasing interest in reading and writing for teachers as well (Moeliono et al., 2017). Theoretical Framework Writing strategies will be more effective if the topic of writing is related to one's daily life. In relation to writing interest, Boscolo & Hidi (2007), in his research, it was revealed that there was a relationship between interest in writing and interest in the chosen topic. The chosen topic will help the authors if the topic is interesting. Interesting topics are topics that are liked by the authors (Djuwari, 2021). Therefore, there is an attraction for teachers' writing motivation, it would be better if they were given interesting topics for them. Meanwhile, according to Akyol & Aktaş (2018), interest in writing is also determined by themes related to stories related to students' lives. In their research, it was found that stories of students' lives can provide them with high motivation in writing. From this empirical evidence, the writing strategy will be more effective if the topic is interesting and related to the daily life of writers, such as teachers or students (Djuwari, 2013). Scientific writing can be divided into several types. In relation to the type of scientific writing, Djuwari (2021) explains that there are pure scientific works (academic writing) such as dissertations, theses, theses, and scientific papers in seminars. These papers are often done by researchers, lecturers, students, and other scientists. In addition, there are also informal scientific writing. This type, for example, is a scientific work in the form of work reports. Finally, there are also popular scientific works. Examples of this popular scientific work are the articles published in the mass media, namely newspapers, magazines, or tabloids which are written according to systematic procedures as well (Djuwari, 2010). Therefore, there are many kinds or types of scientific works. The writing of popular scientific works is included in informal scientific works as a systematic expression of ideas as well. *Corresponding Author: Djuwari First Visit Data It was done on the first visit (25 May 2022). Some information was obtained from taking the policy, in this case the school represented by the Principal related to the policies and real conditions of the school regarding the scientific work writing program as follows. Table 1: Summary of the Data from the School Represented by Principal Question Theme Data or information 1 Policy (External Motivation) Considering the importance of writing scientific for increasing the teachers quality but it is important for accreditation status 2 Policy (External Motivation) Awareness of the benefits of writing scientific articles for the teachers quality and school accreditation status 3 School Condition There is no formal program that is stipulated in the school plan and also the problem about the time schedule Table 1: Summary of the Data from the School Represented by Principal Question 1: Sir, what if this school held a scientific writing training program for its teachers? motivation in the form of policy programs (Wandasari et al., 2019). It was answered that the program offered would have an impact on the quality of teachers and the quality of schools. It's scientifically like that. Principal: We consider that scientific writing for teachers in our school is very important. Writing is the same as creativity. That's why, yes, we will improve it. If there is a program, please hold it at our school. We'll schedule it later. In addition, there is an awareness of the principal that writing skills are important because it is the same as increasing the reasoning power of teachers. If they have high reasoning power, then it is the same as increasing their creative power. The point is, from the principal's point of view, there are indications that so far there has been no policy for writing scientific papers in schools. However, he realized that this program was very important for the creativity of teachers in increasing the power of reasoning through writing popular scientific papers. So, this is the initial capital to improve the quality of teachers through external encouragement in the form of policies (Jatnika, 2019). From question 1 and the principal's answer, there is data information related to school policies and conditions related to the interests and programs of writing scientific papers for teachers at the school. First, there is no formal policy from schools and the government. RESEARCH METHODOLOGY There are several findings in the field from the results of interviews when the researchers conducted visits to schools. From the results of this visit, researchers obtained data both in the form of data from the Principal and Teachers. The first visit, the interview was addressed to the Principal. The second visit, the interview was addressed to a group of teachers (Focus Group Discussion/FGD) This research is a qualitative research that aims to explore the interest in writing scientific papers of the teachers of SMP PGRI 6 Surabaya. Research seeks to explore related to writing interest and strategies to increase writing creativity (Williams & Moser, 2019). They are the teachers who teach at the school. Data were taken from field surveys and interviews with principals as representatives of school policy makers and interviews with teachers. Data were collected by means of field visits and discussions or interviews with the principal and teachers at the school. The information from both sides of sources were summarized in t= terms of the themes (Policy, Motivation, and Problem), Then these themes were described. There were 22 teachers in this school and then they were interviewed in the form of a discussion with them. The first visit was conducted on May 25, 2022 and the second visit was conducted on Writing Strategy There are two ways to increase someone's interest in writing. The strategy can be started by finding internal motivation and external motivation. Internal motivation can be generated from within through for example someone's interest or interest in the topic or story of their life. In relation to this motivation, Esra & Sevilen (2021), in their research, state that internal motivation is related to the likes and daily lives of students while external motivation is related to even organization and policies where students learn. 2 Djuwari et al.; Ind J Human Soc Sci; Vol-3, Iss-8 (Aug, 2022): 1-6 June 16, 2022. Apart from the visit, additional data was also obtained by asking through WhatsApp (WA) related to school problems and policies. The, they were triangulated. This is one way in qualitative research (Orel & Mayerhoffer, 2021). Figure 1. Research Framework The data were then analyzed and inferred based on the problems and objectives of this study, namely the condition of their writing interest and how to increase their writing interest. In the end, they could be given training on how to write scientific papers, especially popular scientific works in the media. Figure 1. Research Framework *Corresponding Author: Djuwari First Visit Data For example, the principal approves the program plan offered by the researcher to the school. This will encourage the success of the scientific paper writing program as external motivation or external 3 Djuwari et al.; Ind J Human Soc Sci; Vol-3, Iss-8 (Aug, 2022): 1-6 Question 2: Do you think it has any use or benefits other than the teachers, sir? Question 2: Do you think it has any use or benefits other than the teachers, sir? From the third question, information related to quality improvement problems was obtained through the teacher's scientific writing training program. It was stated that so far there has been no training program for writing scientific papers for teachers in their schools. In addition, there is information concerning their awareness about the importance of writing skills for teachers because they are considered it the same as increasing the reasoning skill of teachers. More importantly, there is an indication that the principal is very interested in holding scientific writing training for his school teachers. He also wants to set a time or schedule for the program. In essence, there are factors that support the program in terms of the policies that will be adopted by the principal at the school. This is the same as the internal motivation of the principal. This motivation is also important for increasing creativity in the form of writing scientific papers (Jatnika, 2019). Principal: What is clear, in addition to improving the quality of teachers in scientific writing, this training program can also increase school accreditation. It is because our schools are also currently accredited by the government. From the second question, the researchers obtained a confirmation of the principal's intention for his commitment to the program to improve scientific writing skills. He has historically clearly stated that his school's scientific writing training program will be useful for improving the quality of teachers in writing scientific papers. In addition, he is also aware that scientific writing training will support the improvement of school accreditation. This means, scientifically, it is found that the motivational factor for writing also comes not only from school policies, but also government policies. The policy is disclosed in the form of school evaluation related to accreditation status. It is also realized by the principal that with the existence of a scientific writing training program, it is not directly the same as increasing the school's accreditation status. First Visit Data So, the government and its policies can also not great support increasing motivation to write scientific papers of teachers in schools. This is what is suggested by the results of the studies by Wandasai et al. (2019). Thus, the survey data for the first visit, addressed to the principal, can be said that so far there has been no real policy related to special training in scientific writing. Second, it is also realized that writing scientific articles is very important for improving the quality of teachers. Indirectly, by improving the quality of teachers, it can also obtain an increase in the quality of schools through the status of school accreditation. This means that there is a relationship between school policy and improving the quality of teachers through increasing scientific writing skills. This is in accordance with the recommendation of the research results of Wandasri et al. (2019) Question 3: Are there any obstacles experienced in improving the quality, especially writing scientific papers for teachers in this school. *Corresponding Author: Djuwari Second Visit Data Second visit, it was an interview for data from teachers (18 June, 2022). This particular visit, at that time, was attended by 9 teachers. Researchers were accepted into their study and sat at a table. At this time, initially, researchers only talked about strategies for writing popular scientific papers. Researchers were warmly received and even took pictures with them at one table. Principal: The problem is, so far, there is no such program. However, we really like the training program to improve the quality of teachers through writing scientific papers. That's important. Please hold. We will arrange the schedule later and we will adjust it to the conditions of the teachers' schedule. Given, now in anemic condition... Table 2: Summary of the Data from the Teachers Question Theme Data or Information 1 Internal Motivation They are enthusiastic when being offered training o scientific writing. However, there is a problem with the sc 2 Condition of the school There is not yet a program that is planned. But, they wan Yet, they want it. It seems that they cannot decide by them 3 Problem with scheduling This is to confirm also that they have problem with the and time. It seems that they just depend on their school pr Question 1: What if we hold training in writing scientific papers, namely popular scientific works? Teachers: Wow, please, sir. We want it. When is it? (With a smile and sitting with the researcher). Now it's still Student Admission program, they are on holiday today until July 18th. From the information on the teachers, the answer was t interested if a popular scientific held in their school (Using Javan Please do it). They said that they time but it was adjusted to their te not to disturb their teaching an Table 2: Summary of the Data from the Teachers Question Theme Data or Information 1 Internal Motivation They are enthusiastic when being offered training on writing scientific writing. However, there is a problem with the schedule. 2 Condition of the school There is not yet a program that is planned. But, they want to get it, Yet, they want it. It seems that they cannot decide by themselves 3 Problem with scheduling This is to confirm also that they have problem with the schedule and time. Second Visit Data It seems that they just depend on their school principal Question 1: What if we hold training in writing scientific papers, namely popular scientific works? Teachers: Wow, please, sir. We want it. When is it? (With a smile and sitting with the researcher). Now it's still Student Admission program, they are on holiday today until July 18th. From the information on the first qu the teachers, the answer was that they w interested if a popular scientific writing trai held in their school (Using Javanese, Wah M Please do it). They said that they were willin time but it was adjusted to their teaching sched not to disturb their teaching and learning Table 2: Summary of the Data from the Teachers Question Theme Data or Information 1 Internal Motivation They are enthusiastic when being offered training on writing scientific writing. However, there is a problem with the schedule. 2 Condition of the school There is not yet a program that is planned. But, they want to get it, Yet, they want it. It seems that they cannot decide by themselves 3 Problem with scheduling This is to confirm also that they have problem with the schedule and time. It seems that they just depend on their school principal Question 1: What if we hold training in writing From the information on the first qu Question 1: What if we hold training in writing scientific papers, namely popular scientific works? Question 1: What if we hold training in writing scientific papers, namely popular scientific works? From the information on the first question to the teachers, the answer was that they were very interested if a popular scientific writing training was held in their school (Using Javanese, Wah Monggo / Please do it). They said that they were willing at any time but it was adjusted to their teaching schedule so as not to disturb their teaching and learning process. Teachers: Wow, please, sir. We want it. When is it? (With a smile and sitting with the researcher). Now it's still Student Admission program, they are on holiday today until July 18th. *Corresponding Author: Djuwari 4 Djuwari et al.; Ind J Human Soc Sci; Vol-3, Iss-8 (Aug, 2022): 1-6 Second, there was information about obstacles at that time, namely they were still busy with new student registration or PMB. REFERENCE 1. Arvianto, F. (2018). Sumbangan Kecerdasan Emosional dan Minat Menulis Terhadap Kemampuan Menulis Argumentasi Pada Mahasiswa Fakultas Hukum, Universitas Sebelas Maret, Surakarta. Metalingua: Jurnal Penelitian Bahasa, 14(2), 151-165. 2. Akyol, H., & Aktaş, N. (2018). The Relationship between Fourth-Grade Primary School Students’ xx Story-writing Skills and Their Motivation to Write. Universal Journal of Educational Research. Question 3: How about scheduling the training program on writing scientific articles? Question 3: How about scheduling the training program on writing scientific articles? f 3. Boscolo, P., & Hidi, S. (2007). The multiple meanings of motivation to write. Writing and motivation, 19(1). 3. Boscolo, P., & Hidi, S. (2007). The multiple meanings of motivation to write. Writing and motivation, 19(1). Teachers: New are now busy with the new student admission and we are also in holiday. Teachers: New are now busy with the new student admission and we are also in holiday. 4. Kunjungan, P. (2022). Wawancara Pribadi Kunjungan awal di Lokasi SMP PGRI 6 Surabaya, pada 21 Mei 2022. In reference to the third question, it seems that they have problem with their time. They said politely and calmly that they are busy with the new students’ admission. Besides that, when visited at that time, it was during the period of holiday. Therefore, they cannot determine yet when the training program can be started. It seems that they depend on the school principal for the decision. 5. Kunnungan, P. (2022) Wawancara Kedua dengan para Guru. 18 Juni 2022. SMP PGRI 6 Surabaya. 6. Djuwari, D. (2021). Synthesis and Novelty for Developing the Framework in Academic Writing. Philippine Social Science Journal, 4(2), 53-60. 7. Djuwari, D. (2013). Error Analysis of English Sentences Written by Administrators at STIE Perbanas Surabaya, Indonesia. IAMURE International Journal of Education, 5(1), 1-1. Second Visit Data When they were interviewed, they were on vacation which would end on July 18, 2022. During this period, they were busy with the admissions program. school itself, both the principal and the teachers have a high interest in holding scientific writing training. There is synchronization of both the Principal and the teachers. As for the conditions and policies that exist in schools, So far, there has been no formal policy related to scientific writing training for the teachers, both by the school and the government at their school. On one hand, this is very important as in the research conducted by Afianto (2018); & Jatnika (2019). On the other hand, they had schedule problems at that time because they were still busy with the new student admissions program and the school holidays. So, there is high interest but constrained by their very busy schedule, not only teaching but also being involved in the process of finding new students, or registering new students. In addition, there is an important factor to consider, namely the high willingness of teachers and school principal to the importance of popular scientific writing skills. They also realize that scientific writing skills are the same as increasing reasoning power. This is at the same time improving the quality of teachers and schools. The quality of schools is indicated by the existence of school accreditation by the government. So, there is a relationship between the internal motivation of teachers in scientific writing with external motivation in the form of school policies and government policies in writing. It needs a policy to improve the scientific writing skills of teachers in schools. Question 2: Is there any program related to writing scientific papers here? Question 2: Is there any program related to writing scientific papers here? Teachers: Not yet, sir. (While sitting relaxed, they answered it.) Teachers: Not yet, sir. (While sitting relaxed, they answered it.) From the second question, real information was obtained about the condition of the school in relation to school policies in the training program to improve the writing skills of teachers in schools. This is in line with the findings on the first visit with the principal. It was stated explicitly, with a short answer (Not yet, sir!). This means that there is no formal policy on scientific writing training to improve teachers' reasoning power and creative thinking. However, this is a good asset in improving the quality of schools through writing as well as reading or literacy activities (Prasti & Suyatno, 2021). Here, there are indications of similarities with the findings on the first visit with the principal. It was stated that interest was present and very high because they both realized the importance of scientific writing skills. That is why; if there is a program related to scientific writing training in their school, then the program can be implemented properly. A suggestion for further research is the need to search for more data, for example after scientific writing training is held, it needs to find out if there is an increase in scientific writing skills. This is also to evaluate the scientific writing training program at the school. In other words, there is a need for further research related to the real skills of the teachers as well as the effectiveness of the school's scientific writing training program. *Corresponding Author: Djuwari *Corresponding Author: Djuwari CONCLUSION From exploratory data analysis with two field visits, there are some basic conclusions. First, within the *Corresponding Author: Djuwari 5 Djuwari et al.; Ind J Human Soc Sci; Vol-3, Iss-8 (Aug, 2022): 1-6 8. Djuwari, D. (2010). Bahasa Indonesia untuk Perguruan Tinggi: teori dan praktik \. Perpustakaan Nasional. Jakarta: STIE Perbanas Pres. 9. Esra, M. E. Ş. E., & Sevilen, Ç. (2021). Factors influencing EFL students’ motivation in online learning: A qualitative case study. Journal of Educational Technology and Online Learning, 4(1), 11-22. 10. Hildayani, R., Sugianto, M., Tarigan, R., & Handayani, E. (2014). Psikologi perkembangan anak. 11. Jatnika, S. A. (2019). Budaya Literasi untuk Menumbuhkan Minat Membaca dan Menulis. Indonesian Journal of Primary Education, 3(2), 1-6. 12. Moeliono, A. M., Lapoliwa, H., Alwi, H., & Sasangka, S. S. T. W. (2017). Tata bahasa baku bahasa Indonesia. 13. Orel, M., & Mayerhoffer, M. (2021). Qualitative research methods for the exploration of Coworking environments. The Qualitative Report, 26(5), 1364- 1382 14. Priasti, S. N., & Suyatno, S. (2021). Penerapan Pendidikan Karakter Gemar Membaca Melalui Program Literasi di Sekolah Dasar. Jurnal Kependidikan: Jurnal Hasil Penelitian dan Kajian Kepustakaan di Bidang Pendidikan, Pengajaran dan Pembelajaran, 7(2), 395-407. 15. Wandasari, Y., Kristiawan, M., & Arafat, Y. (2019). Policy evaluation of school’s literacy movement on improving discipline of state high school students. International Journal of Scientific & Technology Research, 8(4), 190-198. 16. Williams, M., & Moser, T. (2019). The art of coding and thematic exploration in qualitative research. International Management Review, 15(1), 45-55. 6
https://openalex.org/W1590733440
http://revista.fct.unesp.br/index.php/pegada/article/download/938/1059
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AS FACES DO PLANEJAMENTO URBANO
Revista Pegada Eletrônica/Pegada
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MUNDO DO TRABALHO MUNDO DO TRABALHO RESUMO As decisões ligadas ao planejamento urbano na cidade privilegiam determinadas classes sociais em detrimento de outras, intensificando ainda mais o caráter desigual da cidade. Todavia, o planejamento deve ter como objetivo essencial em sua elaboração, amenizar as desigualdades inerentes do próprio processo de produção do espaço urbano a fim de alcançar a justiça social. No entanto, o planejamento possui faces distintas, as quais dão origem a conflitos e disputas, e apontam novos caminhos para que ele - o planejamento – seja posto em prática no intuito de minimizar as desigualdades existentes. PALAVRAS CHAVE Id l i t pi pl n j m nt rb n j p p g PALAVRAS-CHAVE: Ideologia; utopia; planejamento urbano. 1 Mestranda pelo Programa de Pós-Graduação em Geografia da Universidade Estadual Paulista “Júlio de Mesquita Filho” (FCT/UNESP), campus de Presidente Prudente. Integrante do Grupo de Estudos em Desenvolvimento Regional e Infraestrutura (GEDRI) e bolsista da Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). FACES OF URBAN PLANNING Dayana Aparecida Marques de Oliveira Cruz1 d.dayana@hotmail.com Dayana Aparecida Marques de Oliveira Cruz1 d.dayana@hotmail.com INTRODUÇÃO O planejamento é um elemento fundamental para compreender a produção e organização do espaço urbano, pois ele expressa os interesses dos segmentos coexistentes na cidade e pode justificar ou não a ação deles. Tal contraditório como as ações dos agentes sociais concretos (CORRÊA, 2005), o espaço e a legislação também o são. A legislação é a síntese das intenções e interesses latentes no discurso hegemônico, pois é formulada contendo elementos que facilitam certas iniciativas, sobretudo aquelas ligadas ao mercado imobiliário referentes à valorização de localizações selecionadas pelos que o representam. O planejamento apesar de ser uma possibilidade de alcance da justiça social na cidade é apresentado de forma a facilitar as ações que não contribuem com o alcance da mesma, possibilitando práticas divergentes de algumas determinações presentes na redação das leis que o regem. Prova disso, é a determinação presente no Estatuto da Cidade acerca do cumprimento da função social da propriedade urbana, a qual não é posta em prática nas cidades brasileiras. Diante disso, admitimos a caracterização de três faces ao planejamento urbano: o planejamento ideológico, o planejamento utópico e o planejamento que é posto em prática. Objetivamos com essas reflexões, discutir acerca das características gerais do planejamento realizado nas cidades brasileiras, a partir da reflexão teórica, tendo por base autores que pesquisam acerca do planejamento urbano, e os que escreveram acerca dos conceitos de ideologia e utopia. ABSTRACT Decisions relating to urban planning in the city, privilege certain classes others, further intensifying the iniquity of the city. However, planning has different faces, which give rise to conflicts and disputes, and indicate new avenues for him – the planning – is put in place in order to minimize inequalities. KEY WORDS: Ideology, utopia, urban planning 1 Mestranda pelo Programa de Pós-Graduação em Geografia da Universidade Estadual Paulista “Júlio de Mesquita Filho” (FCT/UNESP), campus de Presidente Prudente. Integrante do Grupo de Estudos em Desenvolvimento Regional e Infraestrutura (GEDRI) e bolsista da Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). Revista Pegada – vol. 12 n.2 dezembro/2011 81 MUNDO DO TRABALHO A IMPORTÂNCIA DO PLANEJAMENTO URBANO E A JUSTIÇA SOCIAL O planejamento é um dos elementos fundamentais para compreender a produção do espaço urbano, pois ele expressa as ações e decisões dos agentes produtores do mesmo, os quais se relacionam de forma contraditória e complexa. Na busca pela manutenção de seus interesses, produzem o espaço de maneira desigual e contraditória, tal como a própria sociedade capitalista em que os interesses individuais se sobressaem sobre o coletivo e o público, o que pode ser visto na própria cidade, bem como nas leis que dela fazem parte, Revista Pegada – vol. 12 n.2 dezembro/2011 82 MUNDO DO TRABALHO dentre as quais destacamos neste trabalho, o Plano Diretor e a Lei de Uso e Ocupação do Solo Urbano. dentre as quais destacamos neste trabalho, o Plano Diretor e a Lei de Uso e Ocupação do Solo Urbano. Segundo SOUZA (2010) o planejamento remete ao futuro, significa tentar simular os desdobramentos de um processo, no intuito de precaver-se dos problemas e de aproveitar da melhor forma os benefícios, por isso é importante distinguir planejamento e gestão2. Estas são ferramentas imprescindíveis para a promoção do desenvolvimento sócio- espacial, cuja expressão se dá a partir da “(...) melhoria de vida e um aumento da justiça social” (SOUZA, 2010, p. 75). Mas o que seria justiça social? Segundo HARVEY (1980, p. 82), a justiça social é um princípio para resolver direitos conflitivos. “O princípio da justiça social, por isso, destina-se à divisão dos benefícios e à alocação de danos surgidos no processo do empreendimento conjunto do trabalho”, para isso, o autor assegura que deve haver dois questionamentos: “O que estamos distribuindo? Entre quem ou que estamos distribuindo renda?” (p. 83). Para tanto, a análise da justiça social está dividida em duas partes, que compreendem o que é a distribuição justa e estabelece um parâmetro para que tal distribuição seja realizada. Em função disso, são estabelecidos os seguintes critérios: desigualdade intrínseca; valorização de serviços em termos de oferta e demanda; necessidade; direitos herdados; mérito; contribuição ao bem comum; contribuição produtiva atual; esforços e sacrifícios. Todavia, apesar de todos esses critérios serem necessários, a essência da justiça social segue respectivamente três: necessidade; contribuição ao bem comum e mérito (HARVEY, 1980). Para alcançar a justiça social é necessária uma forma de organização social, política e econômica apropriada. 2 Planejamento e gestão são complementares, porém distintos. “O planejamento é a preparação para a gestão futura (...) e a gestão a efetivação, ao menos em parte (...) das condições que o planejamento feito no passado ajudou a construir” (SOUZA, 2010, p. 46). É importante enfatizar, portanto, que gestão refere-se ao presente, tem haver com administrar uma situação com recursos disponíveis, tendo em vista necessidades imediatas. A IMPORTÂNCIA DO PLANEJAMENTO URBANO E A JUSTIÇA SOCIAL No território uma das possibilidades seria encontrar uma forma socialmente justa de determinar as fronteiras dos territórios e um modo justo de alocar os recursos para ele. A partir do exame dos princípios de justiça social chega-se ao conceito de justiça social territorial: a distribuição de renda deveria localizar dentro dos territórios a necessidade da população e a mobilização de recursos necessários em diferentes esferas para suprir as necessidades dos territórios. Revista Pegada – vol. 12 n.2 dezembro/2011 83 MUNDO DO TRABALHO No espaço intra-urbano a justiça social deve ser analisada tendo em vista as desigualdades de renda expressas na própria paisagem urbana, cuja origem se dá na divisão social do trabalho e que tem na renda um importante elemento de diferenciação, a qual possibilita aos citadinos o consumo de determinadas mercadorias, equipamentos e do próprio espaço. A nosso ver, o conceito de justiça social aplicado à cidade vem no sentido de tentar contribuir com uma melhor distribuição de renda a partir da disposição mais equitativa dos meios de consumo coletivos, de forma a proporcionar maior acessibilidade aos citadinos, por meio de melhores condições de mobilidade através da reestruturação do sistema de transporte coletivo. O planejamento urbano tem muito a contribuir para o entendimento dessas questões, possui importante papel para o desenvolvimento sócio-espacial, como dito anteriormente, em que a justiça social é um dos objetivos. No entanto, é mister ressaltar que o planejamento (assim como a gestão) não é neutro, é a expressão da sociedade, cujas marcas das desigualdades são visíveis perante o modo de produção vigente e, por isso é carregado de ideologias e interesses. Interesses que os agentes produtores do espaço lutam para assegurá-los. AS FACES DO PLANEJAMENTO URBANO SANTOS (2003) afirma que o planejamento possibilitou a entrada rápida e brutal do capital internacionalizado nos países subdesenvolvidos, colaborando com “o agravamento ou à exacerbação de disparidades sociais” (p. 13), por isso o planejamento é visto como um instrumento do capital. SANTOS (2003) aponta três fases pelas quais passou o planejamento no mundo, na primeira fase o planejamento substituiu a colonização; na segunda, dedicou-se ao desenvolvimento dos monopólios na forma transnacional; na terceira, o planejamento mostra-se em uma roupagem que dá a ilusão de propiciar a superação da pobreza, o que na verdade, gera uma nova forma de pobreza, a pobreza planejada. A experiência brasileira com o planejamento está diretamente vinculada à estrutura política, econômica e social do país; os planos urbanísticos aparecem como tentativas de solucionar determinados problemas da sociedade e envolvem a ideologia dominante nas bases da sociedade hegemônica de cada período em questão. Revista Pegada – vol. 12 n.2 dezembro/2011 84 MUNDO DO TRABALHO O que previsto em lei é resultado e depende dos agentes produtores do espaço urbano e de seus interesses para, de fato, ser implantado, além disso, a própria lei é passível de diferentes interpretações e possui algumas brechas, o que facilita burlá-la. Fato que é explícito no estudo de FIX (2001) que, ao analisar a ação do Estado e os interesses do setor imobiliário nas edificações ao longo do Rio Pinheiros, na cidade de São Paulo fala da construção do bairro “Panamby”, construído a partir de um planejamento com um Plano Diretor próprio apenas para atender a classe alta. Para VILLAÇA (1999, p. 191), por estar atrelado aos interesses da classe dominante, o planejamento urbano no Brasil é “(...) apenas discurso, o planejamento é uma fachada ideológica, não legitimando ação concreta do Estado, mas, ao contrário, procurando ocultá-la”. O autor justifica tal afirmação a partir da exposição dos períodos pelo qual o planejamento passou no país, segundo ele, desde a ascensão dos planos de melhoramentos embelezamentos até a formulação de “superplanos”, a classe dominante fez sobressair seu discurso ideológico reformulando-o para garantir seus interesses. Fazendo um paralelo entre as afirmações de Villaça e a tipologia de SOUZA (2010), observamos que, mesmo com a mudança das fortes tendências entre planejamento e regulatório e perspectivas mercadófilas, um elemento permaneceu: a aliança entre representantes da iniciativa privada e Estado, muitas vezes, havendo dificuldade de diferenciação entre essas duas figuras. Para VILLAÇA (1999, p. AS FACES DO PLANEJAMENTO URBANO 180), só pode ser considerado da esfera do planejamento urbano “(...) apenas aquelas ações do Estado sobre o urbano que tenham sido objeto de algum plano (...)”. Mas, nesse sentido, cabe uma observação: se o planejamento no Brasil é ideológico os planos são, na maior parte das vezes, formas de esconder o planejamento que realmente se pratica, que é aquele pensado a fim de fazer com que as decisões e ações atuais garantam também para o futuro o status quo. Por isso, em nossa concepção essa forma extra-oficial de agir sobre o espaço também é planejada e, por isso é considerada planejamento. Para avançar um pouco nessas questões nos atentaremos ao significado de ideologia. Para Marx, ideologia é uma forma de falsa consciência que corresponde aos interesses de classe, sendo: “(...) o conjunto de idéias especulativas e ilusórias (socialmente determinadas) que os homens formam sobre a realidade, através da moral, da religião, da metafísica, dos sistemas filosóficos, das doutrinas políticas e econômicas etc”. (LÖWY, Revista Pegada – vol. 12 n.2 dezembro/2011 85 MUNDO DO TRABALHO 1987, p. 10). A ideologia é um instrumento de dominação utilizado para que a dominação seja mantida, mas que não seja percebida. CHAUÍ (1984, p. 92) resume ideologia como “(...) o processo pelo qual as idéias da classe dominante se tornam idéias de todas as classes sociais, se tornam idéias dominantes”. LÖWY (1987, p. 10 e 11) apresenta que a definição de ideologia é contraposta a de utopia, sendo que a primeira é entendida como “(...) uma forma de pensamento orientada para a reprodução da ordem estabelecida (...)”, e a segunda “(...) define as representações, aspirações e imagens-de-desejo (Wunschbilder) que se orientam na ruptura da ordem estabelecida e que exercem uma função subversiva (um wälzende Funktion)” [grifos do autor]. O pensamento utópico é desacreditado, “(...) visto que no senso comum sempre torna o substantivo „utopia‟ e o adjetivo „utópico‟ como sinônimos de, respectivamente, fantasia inconseqüente e fantasista” (SOUZA, 2006, p. 517). A ideologia possui grande força através da dominação, que se fortalece a partir, principalmente da manipulação por meio da mídia. Neste sentido, SOUZA (2006) busca em Paulo Freire, a concepção de educação bancária para assimilar o educador ao planejador (que chama de educador-social), cujo depósito de idéias e conhecimentos ocorre sem a utilização do diálogo. AS FACES DO PLANEJAMENTO URBANO Souza propõe no âmbito do planejamento urbano o que Paulo Freire propôs em relação à educação libertária, enfatizando a necessidade de um planejador que estimule o diálogo entre ele e a sociedade a fim de construir coletivamente um planejamento, que optamos chamar aqui de utópico. Neste sentido, deve haver maior comprometimento do Estado com as classes sociais menos favorecidas, para que suas ações estejam pautadas no planejamento que objetive promover maior desenvolvimento socioespacial, o que colaborará com a transformação social, desde que não esteja apenas baseado na racionalidade técnica guiada por critérios econômicos (VASCONCELLOS, 2001). O planejamento que não contempla as questões sociais aprofunda ainda mais as desigualdades, gerando o que VILLAÇA (1998) interpretou como a existência de duas cidades no mesmo espaço: a dos segregados involuntariamente e a dos auto-segregados, ou seja, uma desigualdade social e espacial ao mesmo tempo. Embora o autor tenha pontuado dois tipos de segregação, ele afirma que, na verdade, existe apenas uma, já que a voluntária gera a involuntária, isso se deve ao fato de que a segregação se dá a partir de um movimento dialético, seguindo a mesma lógica do escravo e do senhor. A estrutura social no capitalismo demanda essas contradições, pois elas contribuem para o movimento Revista Pegada – vol. 12 n.2 dezembro/2011 86 MUNDO DO TRABALHO circulatório do capital sem o qual o capitalismo não existiria. LOJKINE (1997) apresenta três níveis de segregação: o nível da habitação, o nível dos meios de consumo coletivos e o nível do transporte domicílio-trabalho. circulatório do capital sem o qual o capitalismo não existiria. LOJKINE (1997) apresenta três níveis de segregação: o nível da habitação, o nível dos meios de consumo coletivos e o nível do transporte domicílio-trabalho. Para colocar o planejamento urbano utópico em prática não há apenas dependência dos recursos técnicos e instrumentos legais, mas decisões não apenas na esfera econômica como também e, principalmente política. Deve haver maior comprometimento com as classes menos favorecidas, para que, se possa caminhar rumo à justiça social, a qual atualmente só existe no âmbito da utopia. AS FACES DO PLANEJAMENTO URBANO O planejamento urbano é um importante elemento para discutir questões que se referem à minimização das desigualdades na cidade, pois é a partir dele que as ações e decisões tomadas no âmbito informal têm a possibilidade de serem regulamentadas, afinal é por meio das leis que se viabilizam os projetos que colaboram com mudanças nas condições de vida da população. O PLANEJAMENTO URBANO NO BRASIL Refletir acerca do planejamento urbano no Brasil é necessário, haja visto que as mudanças sofridas por ele, denunciaram a ideologia existente em cada momento histórico distinto. VILLAÇA (1999) ao tratar sobre a história do planejamento urbano, apresenta três períodos, cuja importância se fez notável, a qual permite analisar o caráter do mesmo. Os períodos destacados pelo autor compreendem: 1875 – 1930; 1930 a 1990; e de 1990 em diante. O período compreendido entre 1875 e 1830, foi aquele em que as obras de embelezamento e melhoramento tiveram destaque, sobretudo nas cidades do Rio de Janeiro e de São Paulo. No Brasil, em meados na década de 1930 esse tipo de planejamento representava a ação concreta do Estado, com objetivos que não condiziam com o discurso. Villaça destaca que “Foi sob a égide dos planos de embelezamento que nasceu o planejamento urbano (lato sensu) brasileiro” (p. 193). Ao dar o exemplo do Rio de Janeiro, o autor afirma que, já no início do planejamento no Brasil, constatou-se que “(...) os interesses imobiliários estavam por detrás dos grandes projetos urbanos, os quais patrocinavam, discutiam, defendiam ou atacavam” (p.195). Neste sentido a face ideológica Revista Pegada – vol. 12 n.2 dezembro/2011 87 MUNDO DO TRABALHO do planejamento urbano faz-se presente com a presença de um discurso baseado no privilegiamento dos interesses da classe dominante. O discurso ideológico também se faz presente no segundo período que VILLAÇA (1999) seleciona para caracterizar como um período importante na história do planejamento urbano no Brasil, o qual vai de 1930 a 1990. O autor apresenta a tentativa de esconder a origem dos problemas, atribuindo ao planejamento à função de solucioná-los, os problemas eram, portanto, entendidos como fruto do crescimento caótico, para os quais a solução estaria no planejamento racional com técnicas e métodos bem definidos. No período da ditadura militar o planejamento caracterizou-se como justificativa para construção de obras, principalmente as de infraestruturas ligadas ao transporte. Para SOUZA e RODRIGUES (2004), neste período o planejamento teve caráter conservador, pois as decisões foram tomadas por um pequeno grupo para manutenção de uma ordem vigente. O objetivo deste planejamento, durante a década de 1970 era (...) ordenar a cidade de forma que a ação dos diferentes tipos de capitais (principalmente o imobiliário e o industrial) pudesse ser facilitada, em outras palavras, buscava ordenar a cidade para permitir maiores possibilidades de lucro. O PLANEJAMENTO URBANO NO BRASIL É nesse contexto extremamente autoritário que vai acontecer uma série de intervenções e transformações na cidade, intensificando e consolidando a segregação residencial (SOUZA e RODRIGUES, 2004, p. 42 e 43) Assim, com o passar dos anos, o planejamento vai se tornando cada vez mais próximo dos interesses dos agentes ligados à iniciativa privada, seus traços tomam forma a cada adequação (necessária) no discurso dominante para manter o status quo, atribuindo ao planejamento um caráter ideológico. Nessa perspectiva VILLAÇA (1999, p. 191) destaca que o planejamento urbano na Europa e nos Estados Unidos, corresponde, em parte, a ação concreta do Estado, diferentemente no Brasil que constitui-se “(...) apenas de discurso, o planejamento é uma fachada ideológica, não legitimando ação concreta do Estado, mas, ao contrário, procurando ocultá-la”. O que há de semelhante entre Brasil, Europa e Estados Unidos é que atrás da fachada do planejamento existe a dominação, o poder, “(...) a grande diferença está nos níveis de hegemonia, aceitação e credibilidade desse „poder‟”. Com a construção dos planos, o discurso se tornou cada vez mais hegemônico, todavia, com o processo de urbanização e a conseqüente intensificação da desigualdade, sugiram movimentos urbanos que passaram a contestar esse discurso “(...) especial e Revista Pegada – vol. 12 n.2 dezembro/2011 88 MUNDO DO TRABALHO precisamente naqueles setores nos quais ela vinha tradicionalmente intervindo: nos setores imobiliário e no do sistema viário e de transportes” (VILLAÇA, 1999, p. 204). Diante de tal situação, a classe dominante adaptou seu discurso à nova realidade, essa iniciativa é apontada por Villaça como a gênese planejamento integrado, o qual desencadeou os superplanos. Surge, portanto, uma nova maneira de formular o planejamento em substituição aos antigos planos de “melhoramento e embelezamento” no objetivo de abranger os aspectos gerais da cidade e seus problemas, denominado “plano geral”, cujos primeiros planos surgiram na década de 1930 com os planos Agache no Rio de Janeiro e Prestes Maia em São Paulo, a principal novidade desses planos era o destaque para a infra- estrutura, principalmente a de saneamento e transportes3. Mantem-se, entretanto, o interesse pelas oportunidades imobiliárias que as remodelações urbanas oferecem, e nesse sentido o centro da cidade ainda é o grande foco de atenção dos planos. No discurso, entretanto, pretende-se abordar a cidade inteira. (VILLAÇA, 1999, p. 207). O plano Agache, do Rio de Janeiro é o primeiro a usar a expressão plano diretor. 3 O Plano Prestes Maia foi denominado apenas como “Estudo de um Plano de avenidas” porque várias páginas para o sistema de transportes. O PLANEJAMENTO URBANO NO BRASIL No Brasil, o plano diretor se difunde a partir da década de 1940, é substituído por outros nomes a partir de 1960, período que estendeu-se até o fim da década de 1980, passando pelo período ditatorial em que foram associados à tecnocracia, volta a ter o nome original com a Constituição de 1988. O discurso do plano diretor e do urbanismo multidisciplinar gera o “plano- discurso”, assim chamado por VILLAÇA (1999). Para o autor, os planos representaram três diferentes períodos no Brasil, entre os anos de 1930 a 1990: Urbanismo e plano diretor (1930-1965 – apresentado anteriormente); do planejamento integrado e dos superplanos (1965-1971) e dos planos sem mapa (1971-1992). O planejamento integrado trazia no discurso dos que o defendiam, a necessidade de ver a cidade além de seus aspectos físicos, dessa forma o argumento era que “Os planos não podem limitar-se a obras de modelagem urbana; eles devem ser integrados tanto do ponto de vista interdisciplinar como do ponto de vista espacial, integrando a cidade em sua região” (VILLAÇA, 1999, p. 212). O Planejamento integrado não se insere na esfera política, mas na ideológica porque se limita ao discurso não atingindo a ação real do Estado. Revista Pegada – vol. 12 n.2 dezembro/2011 89 MUNDO DO TRABALHO O apogeu técnico, no entanto, vem com os superplanos, que, devido à abrangência aproximaram-se muito mais de recomendações para criação de outros planos, distanciando o plano e suas propostas, da possibilidade de implantação. O auge foi atingido na atuação do Serviço de habitação e Urbanismo (Serfhau), criado no início do regime militar. Nessa época foi elaborado também o Plano Urbanístico Básico do município de São Paulo (PUB), feito por um corpo técnico que, segundo Villaça após sua finalização acabou indo direto para o arquivo da prefeitura. No início da década de 1970, mas especificamente no ano de 1971 é aprovada a lei nº. O PLANEJAMENTO URBANO NO BRASIL 7.688 que instituía o Plano Diretor de Desenvolvimento Integrado do Município de São Paulo (PDDI), esse plano caracteriza o terceiro período dos planos no Brasil, em que eles passam de técnicos e sofisticados a simplórios, Villaça, apelida-os de planos sem mapa, essa expressão adotada pelo autor busca designar o plano enquanto nova adaptação da ideologia dominante em seu discurso, na tentativa de dar a impressão que o planejamento urbano estava a seus cuidados e que, estava sendo constantemente aperfeiçoado, as características desse novo tipo de plano são facilmente perceptíveis pois apresentam “(...) apenas objetivos, políticas e diretrizes”, essa é uma estratégia interessante porque tenta esconder as disputas e os conflitos, já que “a idéia do plano diretor de princípios e diretrizes está associada à de „posterior detalhamento‟, e isso nunca ocorre”. (VILLAÇA, 1999, p. 221). Com tudo isso, o plano diretor passa a ser uma atividade intelectual, pautada apenas no discurso que, cumpre sua missão ideológica ao maquiar os problemas da maioria e manter os interesses dominantes. Para o autor, o Plano Diretor sobrevive porque está inserido na ideologia dominante em relação aos problemas e suas causas e sobre a isenção e objetividade da técnica. Nos anos oitenta devido à influência neoliberal, o endividamento e estagnação econômica, o planejamento regulatório perde força. Este período, segundo SOUZA, (1988, p. 70), caracterizou-se por “(...) tentativas formalistas e funcionalistas de planejamento, traduzidas na filosofia dos planos diretores, com suas propostas de planejamento, e nos múltiplos planos regionais e estaduais”. No entanto, no fim dos anos de 1980 novas possibilidades surgiram através da Constituição de 1988 a qual representou um avanço na possibilidade do Plano Diretor ser um instrumento que define se a propriedade está ou não cumprindo sua função social. Por esse motivo, o último período que Villaça apresenta, sobre a história do planejamento no Brasil, compreende a década de 1990 em diante e, para ele, representa uma reação às formas anteriores de planejamento, pois trás uma nova visão baseada na retomada dos Revista Pegada – vol. 12 n.2 dezembro/2011 90 MUNDO DO TRABALHO planos diretores e da maior democratização da gestão urbana, sendo assim admite que o processo de politização do planejamento no Brasil só tem início neste século. Para que pudessem ser implantados os princípios presentes na Constituição de 1988, eram necessários instrumentos que a regulamentassem e que incorporasse os princípios constitucionais, previstos no capítulo que dispõe sobre a política urbana. O PLANEJAMENTO URBANO NO BRASIL Com a aprovação do Estatuto da Cidade em 2001 (Projeto de Lei nº. 5.788/90), em combinação com aquele capítulo e com o texto da Medida Provisória nº. 2.220/01 (...) a regulamentação urbanística passa a ser tratada como um processo, com etapas sucessivas: a formulação de instrumentos urbanísticos que serviam para realizar e implementar os princípios, objetivos e diretrizes estabelecidos pelo Plano; sua aprovação na Câmara Municipal; sua fiscalização e revisão periódica a partir do cotejamento entre a estratégia proposta e os resultados alcançados. (AZEVEDO, 2004, p. 156) (...) a regulamentação urbanística passa a ser tratada como um processo, com etapas sucessivas: a formulação de instrumentos urbanísticos que serviam para realizar e implementar os princípios, objetivos e diretrizes estabelecidos pelo Plano; sua aprovação na Câmara Municipal; sua fiscalização e revisão periódica a partir do cotejamento entre a estratégia proposta e os resultados alcançados. (AZEVEDO, 2004, p. 156) (...) a regulamentação urbanística passa a ser tratada como um processo, com etapas sucessivas: a formulação de instrumentos urbanísticos que serviam para realizar e implementar os princípios, objetivos e diretrizes estabelecidos pelo Plano; sua aprovação na Câmara Municipal; sua fiscalização e revisão periódica a partir do cotejamento entre a estratégia proposta e os resultados alcançados. (AZEVEDO, 2004, p. 156) Foi a partir da implantação que os resultados causados pelo planejamento tiveram a possibilidade de serem acompanhados de perto pelo poder público municipal. Com isso, os municípios brasileiros passaram a ser vistos como unidades de planejamento no espaço, sendo que ao poder público local foi dada maior autonomia em relação ao gerenciamento de seu ordenamento territorial, por meio do estabelecimento do Plano Diretor4, o que proporcionou responder de maneira mais efetiva às necessidades da população (RODRIGUES, 2005). Todavia, existem obstáculos para a implantação dos princípios contidos no Estatuto da Cidade, esses obstáculos são colocados, pois os interesses entre os agentes que coexistem na cidade são distintos. SOUZA e RODRIGUES (2004) apontam os obstáculos como sendo de natureza política, econômica e sociopolítica. SILVA (2003) acredita que o Estatuto da Cidade alcançará a plenitude quando incorporar políticas de caráter distintos, dentre muitas que menciona está à política de transporte. Apesar dessa nova direção que toma o planejamento no Brasil, a partir de 1990, SOUZA e RODRIGUES (2004) afirmam que é nessa década, que o planejamento consolida seu caráter mercadófilo, pautado na defesa dos interesses individuais em detrimento do público e do coletivo. 4 O Plano Diretor normatiza os princípios presentes no Estatuto da Cidade, na escala municipal e, consiste em uma lei obrigatória para municípios com mais de vinte e mil habitantes. O PLANEJAMENTO URBANO NO BRASIL Isso se deve a aproximação cada vez maior entre o poder público e a iniciativa privada. Neste sentido, para respaldar algumas ações e decisões Revista Pegada – vol. 12 n.2 dezembro/2011 91 MUNDO DO TRABALHO o discurso ideológico se faz presente, justificando certas iniciativas como necessárias para a população. o discurso ideológico se faz presente, justificando certas iniciativas como necessárias para a população. CONSIDERAÇÕES FINAIS O planejamento que não contempla as questões sociais corrobora com o aumento da injustiça social e da desigualdade. Apesar da elaboração das leis que regulamentam o planejamento urbano estarem suscetíveis aos interesses da iniciativa privada, ainda assim, há necessidade de um planejamento urbano elaborado de forma crítica, que contemple os distintos âmbitos da vida social (CARNEIRO, 1998), tratando de maneira diferente, os diferentes, no intuito de dispor de forma mais eqüitativa os investimentos na cidade. Para colocar o planejamento urbano utópico em prática não há apenas dependência dos recursos técnicos e instrumentos legais, mas decisões não apenas na esfera econômica como também e, principalmente política. A ideologia imposta ao planejamento tem sua raiz no conflito das classes sociais, em que aquelas que possuem maior poder aquisitivo influenciam de forma decisiva na elaboração e aprovação das leis que regulamentam o planejamento urbano, visto que, é maior a quantidade de representantes do poder público que fazem parte dessa classe, confundindo, portanto, os interesses do Estado com os interesses hegemônicos, resultado do estabelecimento de alianças entre iniciativa privada e poder público. O planejamento posto em prática se dá de forma perversa, pois está a serviço do capital, e é maquiado pelas leis e planos que tentam justificar suas ações. Neste sentido, emerge a cada momento histórico a necessidade de um planejamento que contemple as necessidades sociais, o qual tem sido concebido atualmente apenas no âmbito da utopia. Revista Pegada – vol. 12 n.2 dezembro/2011 92 MUNDO DO TRABALHO REFERÊNCIAS BIBLIOGRÁFICAS AZEVEDO, Abílio Moacir de. Territorialidade e plano diretor em São José do Rio Preto. 2004. 275 f. Dissertação (Mestrado em Geografia)- Instituto de Geociências e Ciências Exatas, Universidade Estadual Paulista, Rio Claro, 2004. CARNEIRO, Ruy de Jesus Marçal. Organização da cidade: Planejamento municipal, plano diretor, urbanificação. São Paulo: Max Limonad, 1998. CHAUÍ, M. de S. O que é ideologia. São Paulo: Brasiliense, 1984. CORRÊA, Roberto Lobato. O espaço urbano. 4. ed. São Paulo: Ática, 2005. CORRÊA, Roberto Lobato. O espaço urbano. 4. ed. São Paulo: Ática, 2005. FIX, Mariana. Parceiros da exclusão. São Paulo: Boitempo, 2001, p. 15-107. FIX, Mariana. Parceiros da exclusão. São Paulo: Boitempo, 2001, p. 15-107. HARVEY, David. A justiça social e a cidade. São Paulo: Hucitec, 1980. LOJKINE, Jean. O Estado Capitalista e a Questão Urbana. 2. ed. Tradução de Estrela dos S. Abreu. São Paulo: Martins Fontes, 1997. LÖWY, Michel. As Aventuras de Karl Marx contra o Barão de Münchhausen. São Paulo: Busca Vida, 1987. MARICATO, Ermínia. Brasil, cidades: alternativas para a crise urbana. 2.ed. Petrópolis: Vozes, 2002. RODRIGUES, Arlete Moysés. Direito à e o estatuto da cidade, In: CIDADES: Revista científica/Grupo de estudos urbanos – v. 2, n. 3, Presidente Prudente, 2005, p. 85 – 110. SANTOS, Milton. Economia Espacial: Críticas e Alternativas. 2. ed. São Paulo: Edusp, 2003. SILVA, José Borzacchielo da. Estatuto da cidade versus Estatuto de cidade – eis a questão. In: CARLOS, Ana Fani Alessandri; LEMOS, Amália Inês Geraiges (orgs). Dilemas urbanos: novas abordagens sobre a cidade. São Paulo: Contexto, 2003, p. 29 – 34. SOUZA, Marcelo Lopes de. Mudar a cidade: uma introdução crítica ao planejamento e gestão urbanos. 6. ed. Rio de Janeiro: Bertrand Brasil, 2010. SOUZA, Marcelo Lopes de. A prisão e a agora: reflexões em torno da democratização Revista Pegada – vol. 12 n.2 dezembro/2011 93 MUNDO DO TRABALHO do planejamento e da gestão das cidades. Rio de Janeiro: Bertrand Brasil, 2006. do planejamento e da gestão das cidades. Rio de Janeiro: Bertrand Brasil, 2006. SOUZA, Marcelo Lopes de; RODRIGUES, Glauco Bruce. Planejamento urbano e ativismos sociais. São Paulo: Unesp, 2004. SOUZA, Marcelo Lopes de; RODRIGUES, Glauco Bruce. Planejamento urbano e ativismos sociais. São Paulo: Unesp, 2004. SOUZA, Maria Adélia de. Governo Urbano. São Paulo: Nobel, 1988. VASCONCELLOS, Eduardo Alcântara. Transporte urbano, espaço e equidade: análise das políticas públicas. São Paulo: Annablume, 2001. VILLAÇA, Flávio. Espaço intra-urbano no Brasil. São Paulo: Studio Nobel/FAPESP 1998. VILLAÇA, Flávio. Espaço intra-urbano no Brasil. São Paulo: Studio Nobel/FAPESP, 1998. VILLAÇA, Flávio. Uma contribuição para a história do planejamento urbano no Brasil. In: DEÁK, Csaba; SCHIFFER, Sueli Ramos. (orgs). O processo de urbanização no Brasil. São Paulo: Editora da Universidade de São Paulo, 1999, p. 169 – 243. VILLAÇA, Flávio. Uma contribuição para a história do planejamento urbano no Brasil. In: DEÁK, Csaba; SCHIFFER, Sueli Ramos. (orgs). O processo de urbanização no Brasil. São Paulo: Editora da Universidade de São Paulo, 1999, p. 169 – 243. Revista Pegada – vol. 12 n.2 dezembro/2011 94
https://openalex.org/W4380537295
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Portuguese
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Olinda quer cantar: expressões carnavalescas de uma cidade sem carnaval
GIS - Gesto, Imagem e Som - Revista de Antropologia
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ORCID http://orcid.org/0000-0002-4932-7894 DOI 10.11606/issn.2525-3123. gis.2023.201955 OLINDA QUER CANTAR: EXPRESSÕES CARNAVALESCAS DE UMA CIDADE SEM CARNAVAL DOSSIÊ MUNDOS EM PERFORMANCE: NAPEDRA 20 ANOS FERNANDA DE CARVALHO AZEVEDO MELLO Universidade Federal do Rio Grande do Norte, Rio Grande do Nor- te, Natal, Brasil, 59075-000 – ppgas@cchla.ufrn.br INTRODUÇÃO A linguagem audiovisual é utilizada na disciplina antropológica como um caminho possível de elabora- ção e divulgação das pesquisas etnográficas. A inserção do registro de imagens no fazer da Antropologia foi concretizada com a perspectiva de que a cultura de um FOTOGRAFIA 1 FOTOGRAFIA 1 ORCID http://orcid.org/0000-0002-4932-7894 DOI 10.11606/issn.2525-3123. gis.2023.201955 OLINDA QUER CANTAR: EXPRESSÕES CARNAVALESCAS DE UMA CIDADE SEM CARNAVAL DOSSIÊ MUNDOS EM PERFORMANCE: NAPEDRA 20 ANOS FERNANDA DE CARVALHO AZEVEDO MELLO Universidade Federal do Rio Grande do Norte, Rio Grande do Nor- te, Natal, Brasil, 59075-000 – ppgas@cchla.ufrn.br INTRODUÇÃO A linguagem audiovisual é utilizada na disciplina antropológica como um caminho possível de elabora- ção e divulgação das pesquisas etnográficas. A inserção do registro de imagens no fazer da Antropologia foi concretizada com a perspectiva de que a cultura de um FOTOGRAFIA 1 ORCID http://orcid.org/0000-0002-4932-7894 DOI 10.11606/issn.2525-3123. gis.2023.201955 OLINDA QUER CANTAR: EXPRESSÕES CARNAVALESCAS DE UMA CIDADE SEM CARNAVAL DOSSIÊ MUNDOS EM PERFORMANCE: NAPEDRA 20 ANOS FERNANDA DE CARVALHO AZEVEDO MELLO Universidade Federal do Rio Grande do Norte, Rio Grande do Nor- te, Natal, Brasil, 59075-000 – ppgas@cchla.ufrn.br INTRODUÇÃO A linguagem audiovisual é utilizada na disciplina antropológica como um caminho possível de elabora- ção e divulgação das pesquisas etnográficas. A inserção do registro de imagens no fazer da Antropologia foi concretizada com a perspectiva de que a cultura de um FOTOGRAFIA 1 INTRODUÇÃO A linguagem audiovisual é utilizada na disciplina antropológica como um caminho possível de elabora- ção e divulgação das pesquisas etnográficas. A inserção do registro de imagens no fazer da Antropologia foi concretizada com a perspectiva de que a cultura de um 1 São Paulo, v. 8, e-201955, 2023 1 São Paulo, v. 8, e-201955, 2023 povo não está apenas em seus artefatos, mas em seus hábitos, valores e comportamentos. Em suas primeiras décadas, essa aproximação esteve pautada pelo entendimento racionalista de que a imagem poderia trazer maior objetividade à coleta de dados. “Segundo essa perspectiva, gestos, falas, movimentos e expressões poderiam ser conservados nos filmes assim como se conservam potes de barro e máscaras” (Barbosa e Cunha 2006, 8). Acreditava-se que a utilização da imagem pela Antropologia traduziria melhor que palavras a exatidão, a verdade, a realidade em si. Contudo, neste trabalho, elas representam mais que isso. Para serem apreendidas em sua totalidade, as imagens que trago contam com a emoção dos amantes já há muito saudosos daquele Carnaval, ainda que não se veja (quase) nenhuma folia nas proporções que a cidade costuma ver nessa época. Neste trabalho, trago fotografias tiradas na cidade-alta de Olinda, em Pernambuco, durante o período (que seria) de Carnaval, em 2022 cance- lado pelo segundo ano consecutivo pela prefeitura da cidade por causa da pandemia de Covid-19. O sentimento gerado ao percorrer ruas que estariam em meu próprio roteiro dos quatro dias de festejos de momo era o de nostalgia de coisa futura, saudade de algo que conheço e que anseio re-conhecer todos os anos; presença e ausência ao mesmo tempo. O papel da fotografia, segundo Sylvia Caiuby Novaes (2014), é justamente o de instigar o olhar, implicando-o em sua desnaturalização: as ladei- ras vazias estão cheias de coisas diferentes das tradicionais multidões. A fotografia, dessa forma, não é um retrato fiel da realidade, uma vez que seu sentido é construído no diálogo da forma (como se fotografa e como estão organizadas as fotografias) e do conteúdo (o que se fotografa) com as bagagens subjetivas de quem fotografa (a antropóloga-fotógrafa) e quem interpreta (o destinatário). “É o silêncio eloquente das imagens que podemos levar para nossa disciplina, com tudo que, a seu modo, as fotografias tem (sic) a dizer” (Caiuby Novaes 2014, 64). INTRODUÇÃO Para David MacDougall (1997), os meios visuais diferem de um texto verbal, pois este último é construído por meio de um processo de descrição, e os primeiros permitem que o conhecimento aconteça por meio da familia- rização. Em outras palavras, segundo Fabiene Gama (2016), as imagens contam com quem as observará para conseguir comunicar suas verdades e seus sentidos, apoiando-se na reflexão crescente dentro da Antropologia sobre o espaço que têm a percepção, os afetos e os sentidos na produção do conhecimento etnográfico: Há diferentes níveis de interpretação, que acontecem simul- taneamente de forma denotativa – apontando para as infor- mações presentes na imagem – e conotativa – apontando para diversas outras informações implícitas e simbólicas –, 2 São Paulo, v. 8, e-201955, 2023 e que são de ordem perceptiva (imediatas), cognitiva (relacio- nadas à bagagem cultural ou experiência pessoal) e ideoló- gica ou ética (relacionadas a valores). Isso significa que uma mesma imagem pode ser interpretada de forma diferente por pessoas que possuem conhecimentos diferentes sobre o assunto fotografado (Gama 2016, 119). e que são de ordem perceptiva (imediatas), cognitiva (relacio- nadas à bagagem cultural ou experiência pessoal) e ideoló- gica ou ética (relacionadas a valores). Isso significa que uma mesma imagem pode ser interpretada de forma diferente por pessoas que possuem conhecimentos diferentes sobre o assunto fotografado (Gama 2016, 119). Antecipando algumas diferentes experiências de interpretação – daquelas que conhecem e das que desconhecem o Carnaval nessa cidade –, divido as fotografias em outras três seções, cada uma contando com textos ver- bais que auxiliam as imagens a darem conta de transmitir a experiência vivida. Busco, com isso, chamar a atenção para a montagem deste tra- balho e expô-la como método de construção do relato. Segundo Samain (2012), a imagem é uma “memória de memórias”, uma “forma que pensa”, e neste texto, a montagem, as escolhas cromáticas e as ordenações foram pensadas para enriquecer a percepção de uma experiência subjetiva. Em essência, cada seção cobre a maioria dos significados atribuídos aos grupos de imagens. Ademais, as cores nas imagens estão ausentes ou bastante desbotadas em direção ao preto, branco e cinza com os objetivos de contrastar à experiência colorida, rica e multissensorial da prática em primeira pessoa, e de deixar a melancolia do não Carnaval visível. O critério por trás da diferença entre poucas cores ou nenhuma referencia a intensidade da memória associada a cada conteúdo imagético. 1. A popularização do personagem de Zé Pereira, que dá nome ao sábado de Carnaval, datada do século XIX, ainda é cercada de muitas incertezas e divergências entre os historiadores, contudo, a maioria deles parece concordar tratar-se do português José Nogueira de Azevedo Paredes, um sapateiro que decidiu sair às ruas durante os dias de Carnaval tocando um surdo. Nos anos seguintes, outros instrumentos e seus tocadores foram se juntando ao tipo de folia inaugurada por José Nogueira. Alguns defendem que foi a partir dessa manifestação que surgiram os blocos de rua. Dos bonecos gigantes – figuras tradicionais do Carnaval em Pernambuco –, o de Zé Pereira é o mais antigo do estado, atualmente com mais de 100 anos. É incerto sobre como ‘Zé’ Nogueira se tornou Zé Pereira. Para mais detalhes, acessar o texto disponível em: <https://tribunadosertao.com.br/noticias/2016/02/06/26731-sabado- -de-ze-pereira-misterios-sobre-o-dono-sabado-de-carnaval> 2. Sobre o assunto, consultar Franco e Souza Leão (2018). INTRODUÇÃO A ideia de um período festivo no ano marcado por “inversões” tem origem no velho continente europeu e chegou ao Brasil a partir da colonização. Mas, aos poucos, a festa foi se adaptando à realidade social específica daqui: o entrudo português, uma bagunça generalizada nas ruas onde cada pessoa/grupo experimenta/faz a folia à sua própria moda, foi a forma de festejar com maior participação de populares a tomar as ruas e, após o surgimento de Zé Pereira1, contribuiu com a origem de nossos blocos de rua (Baribiere, Gonçalves e Menezes Neto 2022, 16); e os bailes de máscaras venezianos, por sua vez, foram importados para diferenciar as festas da elite das festas de rua (Lopes Junior 2019), e podemos associá-los à “camarotização”2 – a venda de espaços cercados, elitizados e climatizados no meio da folia – que encontramos facilmente tanto no festejo em Olinda 3 São Paulo, v. 8, e-201955, 2023 quanto no Recife. Nessas cidades, o carnaval é tradicionalmente de rua, ainda que conte com diversos outros modelos mais ou menos populares para além dos camarotes. Cidadãos comuns formam os blocos de fan- farra que tomam as ruas absolutamente lotadas de foliões, animando a multidão ao embalar frevos3 conhecidos de todas/os/es, levando foliões a uma inebriação compartilhada. quanto no Recife. Nessas cidades, o carnaval é tradicionalmente de rua, ainda que conte com diversos outros modelos mais ou menos populares para além dos camarotes. Cidadãos comuns formam os blocos de fan- farra que tomam as ruas absolutamente lotadas de foliões, animando a multidão ao embalar frevos3 conhecidos de todas/os/es, levando foliões a uma inebriação compartilhada. Com a chegada da pandemia de COVID-19 ao Brasil, logo após o Carna- val de 2020, e com a necessidade de isolamento social, passamos a nos organizar de formas diferentes, evitando “aglomerações”. Lives, trans- missões no YouTube e Facebook: nossas festas se tornaram digitais. Com o cancelamento do carnaval de 2021, notamos em várias partes do país que algumas das festas, iniciativas, mobilizações e celebrações de Momo foram para as telas (Sá Gonçalves 2021). Dos blocos olindenses, alguns dos tradicionais fizeram seus desfiles e comemorações de modo virtual. Normalmente contavam com bonecos gigantes tradicionais do Carnaval do estado, orquestras de frevos, passista de frevo, anfitrião e estandartes em um festival de cores. 3. Estilo musical com ritmo alucinante e passos de dança inspirados na capoeira que, desde 2012, é considerado Patrimônio Cultural Imaterial da Humanidade pela Unesco. 4. Foi o caso para os blocos O Homem da Meia Noite, Troça Carnavalesca Mista Cariri Olin- dense, Boi da Macuca, Pitombeira dos Quatro Cantos. Notar Hiperlink para as transmissões disponíveis no YouTube. INTRODUÇÃO Mas sem plateia presente, sem público ao vivo, sem multidão se aglomerando.4 Em 2022, ano das imagens deste ensaio, ainda foi possível observar algu- mas manifestações do chamado “Carnaval digital”, mas a volta às ruas já se iniciava, ainda que timidamente, como veremos nas páginas seguintes. É nesse sentido que as fotografias dialogam com a memória do que deveria ser, acarretando um deslocamento para outro momento além daquele fotografado, especialmente entre aqueles que já viveram algum Carnaval nas Ladeiras de Olinda, evento que causa um forte impacto na vida de todos que o conheceram, gostem ou não da folia aglomerada sob o fervor do sol. Para as pessoas não familiarizadas com esse Carnaval e essas ruas, os textos que antecedem cada conjunto de fotografias ajudarão a reconhecer os elementos em falta e instigar, por meio das imagens, a vontade de (re) visitar aquelas ruas quando finalmente estiverem propriamente cheias de novo. A sequência das fotos que veremos foi pensada com o objetivo de apresentar a presença permanente da população olindense em seu Carnaval através de intervenções artísticas que fazem alusão ao carna- val; remeter aos sentimentos – similares, mas não iguais – de saudade e nostalgia que ocupam aquelas ruas vazias; e, por fim, apontar algumas persistências carnavalescas, sejam elas sanitariamente responsáveis no contexto de pandemia ou aglomerações que trazem risco de contaminação, São Paulo, v. 8, e-201955, 2023 ainda que estejam em tamanho reduzido em relação ao original e restritos a alguns dos locais mais populares das prévias carnavalescas ainda que estejam em tamanho reduzido em relação ao original e restritos a alguns dos locais mais populares das prévias carnavalescas. Antes de seguirmos, trago abaixo um mapa de parte da Cidade Alta com a identificação do local onde cada foto, identificada por sua ordem de apresentação, foi tirada: ainda que estejam em tamanho reduzido em relação ao original e restritos a alguns dos locais mais populares das prévias carnavalescas. Antes de seguirmos, trago abaixo um mapa de parte da Cidade Alta com a identificação do local onde cada foto, identificada por sua ordem de apresentação, foi tirada: Antes de seguirmos, trago abaixo um mapa de parte da Cidade Alta com a identificação do local onde cada foto, identificada por sua ordem de apresentação, foi tirada: IMAGEM 1 Mapa das fotografias na Cidade Alta/ Olinda (PE). Fonte: Google Maps. 7 São Paulo, v. 8, e-201955, 2023 HINO DO ELEFANTE: O CONSTANTE DIÁLOGO ENTRE OLINDA E SEU CARNAVAL Minha primeira parada foi a Praça do Carmo. Estaria cheia de barracas de comidas, ambulantes vendendo bombons, água, refrigerantes e cervejas, fitas e adereços colorindo toda a cidade, frevo em alto e bom som sendo dançado magnificamente em um palco montado na praça. É o mesmo palco da abertura e do encerramento oficiais dos festejos de Momo em Olinda. Naquele último sábado de Zé Pereira, porém, poucas coisas naquela localidade indicavam que estaríamos em festa. A saudade, contudo, pare- cia se fazer ouvir no vento, que, sem a multidão aglomerada, batia mais frequente e fortemente em meu rosto. Era possível ouvir o frevo de outrora em minha imaginação se eu fechasse meus olhos. O título de cada seção a partir desta leva o nome de um famoso frevo e aposta numa experiência de leitura multissensorial, buscando responder à pergunta levantada por Alexsânder Nakaóka Elias (2019, 268) 5 São Paulo, v. 8, e-201955, 2023 sobre a possibilidade de incluir no texto etnográfico “outros elementos culturais importantes relativos ao grupo e/ou comunidade”. Minha pro- posta é, dessa forma, a de uma experiência sinestésica, assim como fez Elias em sua tese. Mesmo vazia, era possível observar uma cidade que ama o carnaval. As manifestações de intimidade de Olinda com o festejo estavam tatuadas, assimiladas, incorporadas no dia a dia da cidade. Os títulos contêm um hiperlink para ouvir os respectivos frevos no Youtube, artifício utilizado já no título desta seção. FOTOGRAFIA 2 6 São Paulo, v. 8, e-201955, 2023 FOTOGRAFIA 3 FOTOGRAFIA 3 FOTOGRAFIA 4 FOTOGRAFIA 3 FOTOGRAFIA 4 FOTOGRAFIA 4 FOTOGRAFIA 4 7 São Paulo, v. 8, e-201955, 2023 9 São Paulo, v. 8, e-201955, 2023 ÚLTIMO REGRESSO: O SENTIDO DO VAZIO Logo minhas pernas me levariam ao lugar em que eu passo a maior parte das minhas horas de folia. A rua 13 de Maio – mais facilmente reconhecida como a “rua dos frangos”, reduto de lésbicas, gays, bissexuais, travestis, transsexuais e pessoas sexualmente curiosas durante o carnaval – está no fim de uma considerável subida desde a perimetral que passa ao lado da Cidade Alta. Vê-la significa que estamos perto da festa e do sucesso. Quando chegamos, meu grupo e eu, normalmente dobramos à direita e nos esprememos até um espaço onde as correntes de pessoas não nos carreguem em direções não intencionadas. Poder transitar livremente, conseguir observar os degraus da escadaria do Museu de Arte Contempo- rânea (MAC), observar a ausência de barracas competindo espaço com as pessoas, a ausência de pessoas se agarrando, não havia ninguém bêbado ou caindo. Faltava algo. Faltava tudo. Naquele dia, a “13” era a rua resi- dencial que normalmente é fora dos dias de festa. Em rápida conversa com a recepcionista da Pousada Auto-Astral, Hoseana, soube que a música era desestimulada – ou proibida, segundo sua con- cepção – nas tradicionais áreas do Carnaval de Olinda. Segundo ela, o sertanejo na praia estava liberado. A medida buscava evitar empolgar transeuntes a se aglomerarem nas ladeiras, haja vista o perigo que a pandemia ainda representava. Carros da Polícia Militar da Companhia Independente de Apoio ao Turista (CIATur) e da fiscalização municipal podiam ser vistos frequentemente, prontos para dispersar qualquer início de aglomeração e intenção de festa. O silêncio ecoava e fazia doer a sau- dade. As imagens que seguem ardem, como propõe Didi-Huberman (2012, 16), por brincarem com sentidos, por transportarem pra outro momento no espaço/tempo, por fazerem sentir a urgência, […] por seu[s] intempestivo[s] movimento[s], incapaz[es] como é[são] de deter[em]-se no caminho (como se costuma dizer “queimar etapas”), capaz[es] como é[são] de bifurcar[em] sempre, de ir[em] bruscamente a outra parte (como se cos- tuma dizer “queimar a cortesia”; despedir-se à francesa). 8 São Paulo, v. 8, e-201955, 2023 FOTOGRAFIA 5 FOTOGRAFIA 6 FOTOGRAFIA 5 FOTOGRAFIA 6 FOTOGRAFIA 6 FOTOGRAFIA 6 9 São Paulo, v. 8, e-201955, 2023 FOTOGRAFIA 7 FOTOGRAFIA 8 FOTOGRAFIA 7 FOTOGRAFIA 7 FOTOGRAFIA 8 10 São Paulo, v. 8, e-201955, 2023 MADEIRA DE LEI: “SE TIVESSE, EU NÃO RESISTIRIA” Por mais que a prefeitura e o governo do estado tivessem montado uma estrutura de desestímulo à festa, entre muitos a vontade de brincar falou mais alto. Em conversas com os moradores do entorno, descobri que tanto na sexta quanto no sábado (26 e 27 de fevereiro de 2022) as ladeiras esti- veram cheias desde o fim da tarde adentrando a noite. Adaptando-me a essa informação, no domingo, cheguei à Rua 13 de Maio no fim da tarde e pude presenciar o início da aglomeração que viria a ser noticiada nos jornais locais. Nem de longe é possível comparar o que se viu apenas durante o fim de semana com o Carnaval a que aquelas ladeiras estão acostumadas. A multidão que presenciei se concentrou nos Quatro Cantos, conforme o Desenho 1. Caminhar mantendo o distanciamento social era difícil, mas longe de ser impossível. Enquanto isso, policiais militares podiam ser vistos fazendo o que apenas posso descrever como segurança pública, pois o efetivo de quatro pessoas era insuficiente para sequer tentar dispersar o grupo grande de pessoas que ocupavam o espaço. Contudo, não se pode dizer que o carnaval passou sem festa nas ladeiras de Olinda ou sem que manifestações carnavalescas pudessem ser vistas em formas de fantasias, adereços e glitter. Enquanto caminhava para fotografar bonecos gigantes que estavam “sentados” numa caminhonete, ouvi um grupo de três pessoas que conversam casualmente na frente de uma casa: Senhora: … porque se tivesse, eu não resistiria. Morador: Eu só não fui pra praia porque não ia ter nada, porque eu também não ia resistir. É gostoso demais! (DIÁRIO DE CAMPO, 27 fev. 22). Ainda que em menores proporções, o frevo, ao entardecer, podia ser ouvido sem fechar os olhos: uma das moradoras colocou uma enorme caixa de som na janela de sua casa na “13” enquanto tomava uma cerveja com duas outras pessoas, numa mesa colocada por ela mesma do outro lado da rua. “A polícia até passa, olha, mas como não tem muita gente, eles não fazem nada”, me explicou Rafael. Ele, sua mãe e a amiga/moradora brindavam ao Carnaval em um local de Carnaval, ainda que não hou- vesse propriamente um Carnaval. Uma criança que passava com a mãe ensaiou alguns passos de frevo, e Margarida, mãe de Rafael, declarou: “É isto, minha filha! Está no sangue de todo olindense”. 11 São Paulo, v. MADEIRA DE LEI: “SE TIVESSE, EU NÃO RESISTIRIA” 8, e-201955, 2023 DESENHO 1 Localizando a aglomeração. Fonte: autoria própria. Material utilizado: iPad e Pencil iPad WB. FOTOGRAFIA 9 DESENHO 1 Localizando a aglomeração. Fonte: autoria própria. Material utilizado: iPad e Pencil iPad WB. DESENHO 1 Localizando a aglomeração. Fonte: autoria própria. Material utilizado: iPad e Pencil iPad WB. FOTOGRAFIA 9 FOTOGRAFIA 9 FOTOGRAFIA 9 12 São Paulo, v. 8, e-201955, 2023 FOTOGRAFIA 10 FOTOGRAFIA 11 FOTOGRAFIA 10 FOTOGRAFIA 10 FOTOGRAFIA 11 FOTOGRAFIA 11 13 São Paulo, v. 8, e-201955, 2023 FOTOGRAFIA 12 FOTOGRAFIA 13 FOTOGRAFIA 12 FOTOGRAFIA 13 14 São Paulo, v. 8, e-201955, 2023 FOTOGRAFIA 14 FOTOGRAFIA 14 KEYWORDS Anthropology and image; Carnival; Pandemics; Olinda-PE. REFERÊNCIAS BIBLIOGRÁFICAS Baribiere, Ricardo José, Renata de Sá Gonçalves e Hugo Menezes Neto. 2022. O carnaval e a pesquisa universitária: antropologia, artes e letras em diálogo. Entrevista com Maria Laura Cavalcanti e Felipe Ferreira e Fred Góes. Sociol. Antropol, vol. 12, no 03: e210070. Barbosa, Andréa e Edgar Teodoro da Cunha. 2006. Antropologia e imagem. Rio de Janeiro: Zahar. Caiuby Novaes, Sylvia. 2014. O silêncio eloquente das imagens fotográficas e sua impor- tância na etnografia. Cadernos de Arte e Antropologia, vol. 3, no. 2: 57-67. Didi-Huberman, Georges. 2012. Quando as imagens tocam o real. Tradução de Patrícia Carmello e Vera Casa Nova. PÓS: Revista do Programa de Pós-graduação em Artes da EBA/UFMG, vol. 2, no. 4: 206-219. Elias, Alexsander Nakaoka. 2019. Por uma etnografia multissensorial. TESSITURAS | Revista de Antropologia e Arqueologia, vol. 7, no. 2: 266-293. Franco, Suelen Matozo e André Luiz Maranhão de Souza Leão. 2018. Lógica de mercado como medida de eficiência da organização do Carnaval de Olinda. Rev. Adm. Con- temp, vol. 22, no 5: 661-682. Gama, Fabiene. 2016. Sobre emoções, imagens e os sentidos: estratégias para experi- mentar, documentar e expressar dados etnográficos. RBSE – Revista Brasileira de Sociologia da Emoção, vol. 15, no. 45: 116-130. 5 São Paulo, v. 8, e-201955, 2023 Lopes Junior, Rubens. 2019. O carnaval como manifestação popular: um paralelo entre a concepção beltraniana de carnaval em Recife e Olinda e o surgimento do carnaval carioca. RIF, vol. 17, no. 39: 181-196. Lopes Junior, Rubens. 2019. O carnaval como manifestação popular: um paralelo entre a concepção beltraniana de carnaval em Recife e Olinda e o surgimento do carnaval carioca. RIF, vol. 17, no. 39: 181-196. Lopes Junior, Rubens. 2019. O carnaval como manifestação popular: um paralelo entre a concepção beltraniana de carnaval em Recife e Olinda e o surgimento do carnaval carioca. RIF, vol. 17, no. 39: 181-196. MacDougall, David. 1997. “The visual in anthropology”. In Rethinking visual anthropology, ed. Marcus Banks e Howard Morphy. New Haven, London: Yale University Press: 276-295. Sá Gonçalves, Renata de. 2021. Descontinuidades e estratégias da nobre dança do carna- val. In A falta que a festa faz: celebrações populares e antropologia na pandemia, org. Maria Laura Cavalcanti e Renata de Sá Gonçalves. Rio de Janeiro: Museu Nacional, Universidade Federal do Rio de Janeiro: 302-316. Samain, Etienne. (org.). 2012. Como pensam as imagens. Campinas: Unicamp. FOTOGRAFIA 15 16 São Paulo, v. 8, e-201955, 2023 RESUMO Em Olinda (PE), durante o mês de momo, as foliãs tomam as ruas com muito glitter, brilho e corpos à mostra. Não há um centímetro sequer de distância entre ninguém, e é muito comum observarmos, de alguma forma, como os apaixonados beijos rápidos que são trocados entre desco- nhecidas conseguem diminuir ainda mais a lonjura. A montagem desse trabalho teve como duplo objetivo mostrar as ruas do trajeto imaginado para o Carnaval de Olinda no ano de 2022; e, a partir da ausência de corpos e da fanfarra, cancelada pelo segundo ano consecutivo pela prefeitura da cidade, refletir sobre os sentidos da festa para a cidade. Em outras pala- vras, o presente trabalho expõe os registros, por meio de fotografias, do não Carnaval, do não beijo, da não multidão, assim como as resistências e insistências em manter acesa a chama da folia, por meio de fotografias do percurso evocado pela memória de carnavais passados. PALAVRAS-CHAVE Antropologia e imagem; Carnaval; Pandemia; Olinda-PE. Recebido: 07/09/2022 Reapresentado: 10/01/2023 Aprovado: 14/02/2023 ABSTRACT In Olinda (PE), during its most festive month, the revelers take to the streets with lots of glitter and bodies on display. There is not even an inch of distance between anyone and it is very common to observe, in some way, how the passionate quick kisses that are exchanged between stran- gers manage to further diminish the distance. The assembly of this paper has as a double objective to show the streets of the imagined route for the Carnival of Olinda in the year 2022; and, from the absence of bodies and fanfare, canceled for the second consecutive year by the city hall, reflect on the meanings of the party for the city. In other words, the present arti- cle exposes the records, through photographs, of the non-carnival, of the non-kiss, of the non-crowd, as well as the resistances and insistences on keeping the flame of the revelry alive, through photographs of the route evoked by the memory of past carnivals. Fernanda de Carvalho Azevedo Mello é doutoranda em Antropologia Social pela Universidade Federal do Rio Grande do Norte (UFRN) e mestra em Antropologia pela Universidade Federal de Pernambuco (UFPE). Cursou graduação em Relações Internacionais na Faculdade Integrada do Recife (FIR) e Ciências Sociais na Universidade Federal Rural de Pernambuco (UFRPE). Atualmente, desenvolve pesquisa no Carnaval de Olinda (PE), etnografando a eroticidade e a paquera entre mulheres. Integra o grupo de estudos Gênero, Corpo e Sexualidade (GCS) da UFRN. E-mail: nandacmello@hotmail.com Licença de uso. Este artigo está licenciado sob a Licença Creative Commons CC-BY. Com essa licença você pode compartilhar, adaptar, criar para qualquer fim, desde que atribua a autoria da obra. Recebido: 07/09/2022 Reapresentado: 10/01/2023 Aprovado: 14/02/2023 17 São Paulo, v. 8, e-201955, 2023
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Training Visual Imagery: Improvements of Metacognition, but not Imagery Strength
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INTRODUCTION Sasaki et al., 2010); can imagery also improve with daily prac- tice? There is some evidence to suggest that perceptual learning can occur from training without physical stimulation. Repeti- tively imagining the crucial part of a visual bisection stimulus (visual spatial judgment) or imagining a low-contrast Gabor pattern (contrast judgment) can improve performance on sub- sequent perceptual tasks (Tartaglia et al., 2009). Similarly, imag- ining motor-acts facilitates performance on corresponding tasks by training relevant parts of motor cortex, and by strengthen- ing associations between processes and actions (Driskell et al., 1994; Weiss et al., 1994; Feltz and Landers, 2007). To date, research has mainly focused on the effects imagery training has on subsequent perceptual tasks. Here, we look directly at the influence of imagery training on the strength of imagery itself. Mental imagery can be described as the retrieval of perceptual information from memory, and the subsequent examination of this information in the “minds eye.” Research has provided a growing body of behavioral and neuroimaging evidence that there is considerable overlap between the “minds eye” and actual perception (Chen et al., 1998; Kreiman et al., 2000; O’Craven and Kanwisher, 2000; Zatorre and Halpern, 2005). For exam- ple, behavioral studies have demonstrated that imagery content can selectively influence perception (Perky, 1910; McDermott and Roediger, 1994; Pearson et al., 2008). Imagery has been shown to affect visual detection thresholds (Ishai and Sagi, 1997), perfor- mance on a visual acuity task (Craver-Lemley and Reeves, 1992), and to induce negative aftereffects (Gilden et al.,1995) in much the same way as a sensory stimulus. Recent neuroimaging studies show that there is considerable spatial overlap between activated areas of the brain during both visual perception and visual imagery, for example information about a pattern held in mind during work- ing memory or imagery can be present in visual sensory cortex (Kosslyn et al., 1995; Slotnick et al., 2005; Harrison and Tong, 2009; Serences et al., 2009; Stokes et al., 2009). Like perception, visual imagery is impaired when visual cortical activity is dis- turbed by means of transcranial magnetic stimulation (Kosslyn et al., 1999). One of the hallmarks of mental imagery is the considerable difference in reported imagery strength and vividness observed across individuals (Galton, 1883; McKellar, 1965; Marks, 1973; Amedi et al., 2005; Cui et al., 2007). Some individuals claim veridical, vivid imagery, while others doubt its entire existence (McKellar, 1965). ORIGINAL RESEARCH ARTICLE published: 10 July 2012 Training visual imagery: improvements of metacognition, but not imagery strength Rosanne L. Rademaker 1,2,3* and Joel Pearson2,3,4 1 Cognitive Neuroscience Department, Maastricht University, Maastricht, Netherlands 2 Psychology Department, Vanderbilt University, Nashville, TN, USA 3 Vanderbilt Vision Research Center, Vanderbilt University, Nashville, TN, USA 4 School of Psychology, The University of New South Wales, Sydney, NSW, Australia Rosanne L. Rademaker 1,2,3* and Joel Pearson2,3,4 1 Cognitive Neuroscience Department, Maastricht University, Maastricht, Netherlands 2 Psychology Department, Vanderbilt University, Nashville, TN, USA 3 Vanderbilt Vision Research Center, Vanderbilt University, Nashville, TN, USA 4 School of Psychology, The University of New South Wales, Sydney, NSW, Australia *Correspondence: Correspondence: Rosanne L. Rademaker, Cognitive Neuroscience Department, Maastricht University, Universiteitssingel 40, 6229 ER Maastricht, Netherlands. e-mail: rosanne.rademaker@ maastrichtuniversity.nl Keywords: visual imagery, training, learning, metacognition, introspection, binocular rivalry, consciousness Keywords: visual imagery, training, learning, metacognition, introspection, binocular rivalry, consciousness Edited by: Visual imagery has been closely linked to brain mechanisms involved in perception. Can visual imagery, like visual perception, improve by means of training? Previous research has demonstrated that people can reliably evaluate the vividness of single episodes of imag- ination – might the metacognition of imagery also improve over the course of training? We had participants imagine colored Gabor patterns for an hour a day, over the course of five consecutive days, and again 2 weeks after training. Participants rated the subjective vividness and effort of their mental imagery on each trial. The influence of imagery on subsequent binocular rivalry dominance was taken as our measure of imagery strength. We found no overall effect of training on imagery strength. Training did, however, improve participant’s metacognition of imagery. Trial-by-trial ratings of vividness gained predictive power on subsequent rivalry dominance as a function of training. These data suggest that, while imagery strength might be immune to training in the current context, people’s metacognitive understanding of mental imagery can improve with practice. Stephen Michael Kosslyn, Stanford University, USA Reviewed by: Stephen Michael Kosslyn, Stanford University, USA Gregoire Borst, Université Paris Descartes, France *Correspondence: Rosanne L. Rademaker, Cognitive Neuroscience Department, Maastricht University, Universiteitssingel 40, 6229 ER Maastricht, Netherlands. e-mail: rosanne.rademaker@ maastrichtuniversity.nl Stephen Michael Kosslyn, Stanford University, USA INTRODUCTION To assess metacognition we use a method derived from signal detection theory (Swets, 1986; Macmillan and Creelman, 1991; Galvin et al., 2003; Kornbrot, 2006) that has been successfully employed in a variety of recent metacognition studies (Fleming et al., 2010; Song et al., 2011). Using this method, we looked at the likelihood that imagery biased subsequent rivalry, given a certain level of imagery vividness. Signal detection allows us to estimate a single quantitative “sensitivity” measure of metacognitive ability, derived from these objective (amount of perceptual bias) and sub- jective (ratings of vividness) variables. This measure of sensitivity is criterion free, which means that it is not prone to changes in cri- terion (rating-magnitude), and it is not affected by irregular use of the rating scale (which generally results in unequal numbers of observations across the various conditions). We have previously demonstrated that imagery can alter future competitive visual interactions in favor of the imagined stimulus on a large percentage of trials (Pearson et al., 2008, 2011), while catch-trial presentations of mock rivalry stimuli do not reveal such bias,rulingoutthepossibilityof demandcharacteristics(Pylyshyn, 2003). Indeed, scores on offline imagery questionnaires predict imagery strength measured using rivalry (Pearson et al., 2011) and rivalry has been utilized to examine the role of imagery during visual working memory (Keogh and Pearson, 2011). Thus, there is compellingevidencethatrivalrybias(or“perceptualbias”)isause- ful way to measure imagery strength in general (e.g.,encompassing perceptual elements and sensations of vividness). In the current study, imagery strength is the underlying construct of interest, and the extent to which imagery biases perception is taken as a reliable measure of imagery strength. The subjective experiences associatedwithimagerystrengthareprobedbyhavingparticipants report the “vividness” of their mental images. By way of preview, here we report that imagery strength – mea- sured as the extent to which imagery biases perception during binocular rivalry – did not increase over the 5-day training period. Interestingly, participant’s metacognition of imagery did signifi- cantly improve over the training period. This dissociation between imagery strength and metacognitive ability suggests a degree of independence between the two processes. Can people evaluate the phenomenal qualities of internally generated experiences, such as whether a mental image is vivid or detailed? Recently, an attempt was made to answer the ques- tion of knowing ones own thoughts (exemplifying the problem of “metacognition;” Flavell, 1979) in relation to mental imagery (Pearson et al., 2011). INTRODUCTION The factors causing such differences in imagery strength remain largely unknown. One hypothesis is that individ- uals who actively practice, or whose everyday activities involve strong use of imagery, might have strengthened their imagery through training and practice (Sacks, 2010). We sought to exam- ine such a proposal in the lab by engaging individuals in an imagery task daily, over a period of 5 days. Can repeated instances of forming visual imagery lead to improved imagery strength? If visual imagery can indeed be defined as the recreation of a perceptual representation in the absence of retinal input (Ishai and Sagi, 1995), one may wonder exactly how similar imagery is to perception. Specifically, prolonged visual practice can improve perceptual skill (Fahle and Poggio, 2002; Fine and Jacobs, 2002; July 2012 | Volume 3 | Article 224 | 1 www.frontiersin.org www.frontiersin.org The training of imagery and metacognition Rademaker and Pearson be stable and task independent, suggesting a common cognitive process (Song et al., 2011). To address this question researchers must be able to reli- ably measure imagery strength from 1 day to the next. Previous work demonstrated that sustained imagery has a pronounced and visually specific impact on subsequent perception (Pearson et al., 2008, 2011). These studies utilized a visual phenomenon called binocular rivalry; when two different patterns are pre- sented one to each eye, only one of the patterns is consciously perceived. Subtle experimental manipulations, such as attention (Meng and Tong, 2004; Mitchell et al., 2004; Chong and Blake, 2006; Kamphuisen et al., 2007), sensory memory (Pearson and Brascamp,2008),or imagery (Pearsonet al.,2008),can bring about a slight imbalance in the neural states, creating a bias that helps one pattern win the race for dominance at the expense of the other. Little is known regarding the stability and independence of metacognition of mental imagery. If metacognition for percep- tual tasks originates from a common cognitive process, might a similar process allow people to have metacognition of mental imagery? Despite the highly subjective and volitional nature of imagery, people are reasonably good at imagery metacognition (Pearson et al., 2011). Is this ability stable, or might metacognition of imagery improve with repeated practice? Here, we also investi- gated the degree of imagery metacognition as a function of daily training. PARTICIPANTS Nine observers (six female) participated in the experiment.All had normal or corrected-to-normal visual acuity and normal stere- ovision, and all provided written informed consent. Observers received payment for their participation ($10 per hour, plus a $5 per hour bonus upon completion) with the exception of a partici- pating author (RR) and participant BW. The study was carried out with the approval of the Institutional Review Board at Vanderbilt University. Frontiers in Psychology | Perception Science INTRODUCTION This study provided compelling new evi- dence that people have accurate metacognitive knowledge at fine-grained scale, regarding specific instances of imagery: On individual trials, higher ratings of imagery vividness predicted a greater likelihood that the imagined pattern would appear domi- nant during subsequent rivalry (Pearson et al.,2011). Interestingly, repeated attempts to form a particular visual image can lead to dif- ferent degrees of success with each try, causing imagery strength to fluctuate from one moment to the next. Despite this variance in imagery strength, people demonstrate good metacognitive under- standing of their imagery, and can readily evaluate how vivid their mental images are on a particular occasion. MATERIALS b Observers viewed the stimuli on a luminance-calibrated CRT monitor with 1152 × 870 resolution and a 75-Hz refresh rate in an otherwise darkened room. Visual stimuli were generated with Matlab 7.5.0 (R2007b) and the Psychophysics toolbox (Brainard, 1997; Pelli, 1997) under Mac OSX. Observers sat at a viewing distance of 56 cm, and used a chinrest to maintain a stable head position. A mirror stereoscope was used to present a different pat- tern to each eye, and binocular convergence of the two images was aided by a white bull’s eye fixation dot (0.95˚) at the center of each monocular half-image. Participants were instructed to maintain steady fixation throughout all experimental trials. At a general level, there has been a growing interest in metacog- nitive judgments of memory and sensory decision-making (Kiani and Shadlen, 2009; Fleming et al., 2010; Rounis et al., 2010; Song et al., 2011). Frontal brain regions are important for introspec- tive or metacognitive ability (Kepecs et al., 2008; Fleming et al., 2010), which suggests that the neural substrates of metacogni- tive ability are distinct from those supporting primary perception. Although the ability to introspect varies substantially across indi- viduals, within a single individual metacognitive ability seems to July 2012 | Volume 3 | Article 224 | 2 The training of imagery and metacognition Rademaker and Pearson Rivalry stimuli consisted of a green and a red grating (spatial frequency = 1.23 c/˚) surrounding a central fixation point, pre- sented against a black background with a mean luminance of 0.09 cd/m2. CIE color values of the stimuli were as follows – green: x = 0.293, y = 0.572; red: x = 0.602, y = 0.353. Gratings were pre- sented at 75% contrast and had a Gaussian-shaped luminance profile (mean luminance = 6.95 cd/m2) that faded to black at the stimulus edge (Gaussian σ = 4.29˚). Five observers were trained with a green grating of orientation 112.5˚ and a red grating of orientation 22.5˚, while on generalization blocks they were presented with 67.5˚ green, and 157.5˚ red gratings. The oppo- site was true for the remaining four observers, meaning that we counterbalanced which grating-pairs were used for training and generalization between participants. On catch trials, a mock rivalry stimulus was presented consisting of a physical blend of the green and red rivalry patterns. This stimulus was pre- sented to both eyes simultaneously in order to avoid interocular competition. MATERIALS b Presentation of the mock-stimulus allowed us to test for decisional bias and demand characteristics (Landsberger, 1958). into the lab at or around the same time on each day of training, and were dark-adapted for a couple of minutes before the start of each experimental session. Duringtheexperiment,participantswerebrieflypresentedwith a randomly chosen (equal number of both) central cue (“G” for green, or “R” for red) at the beginning of each imagery-trial (Figure 1A). Subsequently, participants would engage in visual imagery of the cued pattern for an 8-s period. After completing this imagery period, the word “vividness?” cued participants to first report the quality of their imagery by means of left-handed button presses (1 = almost no imagery, 2 = some weak imagery, 3 = moderate imagery, 4 = strong imagery almost like perception), after which they were cued by the word “effort?” to report the amount of vigor with which they had tried to imagine the pattern (1 = almost no effort, 2 = some effort, 3 = moderate effort, 4 = tried very hard to form a mental image). Observers were instructed to use the full range of the rating scale to the best of their abilities. As soon as a participant had responded to both questions, a rivalry display (90% of trials) or a mock display (10% of trials) was presented for 750 ms. On rivalry trials, the green grating was presented to the left eye, and the red grating to the right eye. On mock trials, the plaid-stimulus was presented to both eyes simul- taneously. Participants reported which image had appeared most dominant, by pressing one of three buttons (1 = green, 2 = mixed, 3 = red). For this response, the right hand was used in order to minimize potential response conflict between the two hands. A “mixed” response could be made on all trials (rivalry and mock trials). On rivalry trials, the observer could give a mixed response in case he or she was unable to distinguish which grating had appeared more dominant due to binocular combination or piece- meal rivalry. This type of mixed percept was reported on 6.49% of rivalry trials (SEM = 2.49%). The dominant eye plays a key role in determining which of two monocular images is likely to be perceived at the onset of binocular rivalry. MATERIALS b Therefore, individual fine-tuning of stimulus contrast was done before the start of the experiment, and before each daily session, to control for differences in ocular dominance between observers. We used the same procedure as in previous research (Pearson et al., 2008, 2011; Keogh and Pearson, 2011), matching the relative strength of the rivalry gratings to the point at which perceptual competition is most balanced, and thus most susceptible to disruption. PROCEDURE To investigate whether visual imagery can be improved by means of training,and to see how this relates to metacognition of imagery over time, we had observers perform a visual imagery task on five consecutive days, for about an hour a day. A sixth follow-up ses- sion was conducted 2–3 weeks after training. Participants came A single training session consisted of two blocks of 70 trials each. Within each block, seven catch trials were randomly inter- leaved between the rivalry trials.We tested potential generalization of learning to non-trained orientations on day 1 and 5 of train- ing, and during follow-up. On these days, observers performed 1s 8s Imagery Cue Subjective ratings 1s R vivid? effort? ~1s ~1s 0.75s Rivalry A B p(self-report | no perceptual bias) p(self-report | perceptual bias) 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 left eye right eye FIGURE 1 | Sample trial sequence and example ROC II curve. (A) Participants were presented with a random cue (“G” or “R”) after which they formed a mental image of the green or red grating over an 8-s period. Participants were then cued to report the vividness of the imagined item, and the effort exerted while imagining the item, on an absolute scale from 1 to 4. After a brief flash of the rivalry display, participants reported which grating had appeared perceptually dominant, or whether their percept was an equal mix of the two. On 10% of the trials – instead of the rivalry display – a mock-stimulus was presented to both eyes simultaneously, consisting of a physical combination of both the green and red grating. (B) To determine how well subjective ratings predict perceptual bias, type II ROC sensitivity was calculated by taking the area under the ROC curve (Aroc). This area is the sum of the area of the half-square triangle (dark-gray shaded region) and the area between the diagonal and the ROC function (light-gray shaded region). 1s 8s Imagery Cue Subjective ratings 1s R vivid? effort? ~1s ~1s 0.75s Rivalry A B p(self-repor p(self-report | perceptual bias) 0 0.2 0 0.2 0.4 0.6 0.8 1 left eye right eye B p(self-report | no perceptual bias) p(self-report | perceptual bias) 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 A of the two. PROCEDURE On 10% of the trials – instead of the rivalry display – a mock-stimulus was presented to both eyes simultaneously, consisting of a physical combination of both the green and red grating. (B) To determine how well subjective ratings predict perceptual bias, type II ROC sensitivity was calculated by taking the area under the ROC curve (Aroc). This area is the sum of the area of the half-square triangle (dark-gray shaded region) and the area between the diagonal and the ROC function (light-gray shaded region). FIGURE 1 | Sample trial sequence and example ROC II curve. (A) Participants were presented with a random cue (“G” or “R”) after which they formed a mental image of the green or red grating over an 8-s period. Participants were then cued to report the vividness of the imagined item, and the effort exerted while imagining the item, on an absolute scale from 1 to 4. After a brief flash of the rivalry display, participants reported which grating had appeared perceptually dominant, or whether their percept was an equal mix of the two. On 10% of the trials – instead of the rivalry display – a mock-stimulus was presented to both eyes simultaneously, consisting of a physical combination of both the green and red grating. (B) To determine how well subjective ratings predict perceptual bias, type II ROC sensitivity was calculated by taking the area under the ROC curve (Aroc). This area is the sum of the area of the half-square triangle (dark-gray shaded region) and the area between the diagonal and the ROC function (light-gray shaded region). FIGURE 1 | Sample trial sequence and example ROC II curve. (A) of the two. On 10% of the trials – instead of the rivalry display – a mock-stimulus was presented to both eyes simultaneously, consisting of a physical combination of both the green and red grating. (B) To determine how well subjective ratings predict perceptual bias, type II ROC sensitivity was calculated by taking the area under the ROC curve (Aroc). This area is the sum of the area of the half-square triangle (dark-gray shaded region) and the area between the diagonal and the ROC function (light-gray shaded region). FIGURE 1 | Sample trial sequence and example ROC II curve. ANALYSES To assess the strength of visual imagery, we looked at the per- ceptual facilitation (or bias) of imagery on rivalry. This was calculated as the percentage of trials in which the imagined grat- ing matched subsequent perception during rivalry (Pearson et al., 2008), excluding trials on which a mixed percept was reported. A perceptual bias greater than 50% (chance) on the rivalry trials but not on the catch trials suggests facilitation due to imagery con- tent. Due to experimenter error, a small number of runs (7 out of 108) were missing from the data. Where necessary, we used tri- linear interpolation to infer the mean percentage of bias. For the day-by-day analysis (Figure 2) only one data point was interpo- lated (percentage perceptual bias for participant CB on day 4); the session-by-session analysis of the same data required interpolation of all seven missing runs. Aroc = 0.25 4 X i=1 hhi+1 −fi 2 − hi −fi+1 2i + 0.5 (1) (1) The bias of the ROC II curve (Broc) was defined as the ratio K B/K A, where K B is the area between the ROC curve and the major diagonal (dashed line in Figure 1B) to the right of the minor diagonal (dotted line in Figure 1B), and K A is the area between the ROC curve and the major diagonal to the left of the minor diagonal. A neutral bias would give Broc equal to zero, while a negative or positive Broc indicates a bias toward lower or higher ratings respectively. Broc = ln   0.25 2P i=1 hhi+1 −fi 2 − hi −fi+1 2i 0.25 4P i=3 hhi+1 −fi 2 − hi −fi+1 2i   (2) Data obtained from subjective ratings of vividness (and effort) were analyzed by constructing type II receiver operating charac- teristic (ROC) curves for each participant on each day of training. This method of assessing metacognitive ability is derived from signal detection methods (Swets, 1986; Macmillan and Creelman, 1991; Galvin et al., 2003; Kornbrot, 2006) and has been success- fully employed in a variety of recent inquiries about metacognition (Fleming et al., 2010; Song et al., 2011). PROCEDURE (A) Participants were presented with a random cue (“G” or “R”) after which they formed a mental image of the green or red grating over an 8-s period. Participants were then cued to report the vividness of the imagined item, and the effort exerted while imagining the item, on an absolute scale from 1 to 4. After a brief flash of the rivalry display, participants reported which grating had appeared perceptually dominant, or whether their percept was an equal mix July 2012 | Volume 3 | Article 224 | 3 www.frontiersin.org The training of imagery and metacognition Rademaker and Pearson twice the amount of trials, with training and generalization blocks presented separately and in a randomized order. twice the amount of trials, with training and generalization blocks presented separately and in a randomized order. is the sum of the area of the half-square triangle (dark-gray shaded region in Figure 1B) and the area between the diagonal and the ROC function (light-gray shaded region in Figure 1B): Frontiers in Psychology | Perception Science ANALYSES Essentially, the only differ- ence between type I and type II tasks lies in the event that is being discriminated: Type I decisions are about the occurrence of events independent of the observer (so,distinguishing between signal and noise), whereas type II decisions are about whether a decision was correct or incorrect (so, making a metacognitive judgment). (2) Previous research has shown that subjective ratings of vivid- ness – but not effort – are predictive of how much perceptual bias someone experiences (Pearson et al., 2011). To confirm this, we also applied the ROC II methods described above to participant’s ratings of exerted effort. This effort-based ROC thus characterizes the probability of a participant being perceptually biased given a certain level of self-reported effort. Finally, to determine whether the ROC II model did a good job accounting for our metacognitive data, we fit a linear regression model: Since it is not possible to be “correct” or “incorrect” about an internally generated image, we adapted the definition of the type II decision to include judgments about the vividness of single episodes of mental imagery and its effect on the per- ceptual outcome during brief instances of subsequent binocular rivalry. Applying the signal detection logic, we categorized trials where participants reported high vividness and where imagery subsequently biased perception as “hits.” Trials where partici- pants reported high vividness but perception was not subsequently biased were categorized as “false alarms.” Here, the ROC II char- acterizes the probability of a participant being perceptually biased during rivalry, given a certain level of self-reported vividness. z (h) = β0 + β1z f  + ε (3) (3) Where z is the inverse of the cumulative normal distribu- tion function. The ROC II model provided a good fit to the self-reported vividness (mean R2 = 0.976 ± 0.004) and effort data (mean R2 = 0.981 ± 0.007). IMAGERY TRAINING Sustained mental imagery can bias the perception of an ambigu- ous display, resulting in a reliable measure of imagery strength on a trial-to-trial basis (Pearson et al., 2008, 2011). When people rate their imagery as more vivid, the likelihood that imagery influences perception is larger (Pearson et al., 2011). Thus, if training men- tal imagery would result in more vivid images, one would expect to see an increase of perceptual bias over time. Figure 2 shows the mean imagery strength (or “perceptual bias”) as a function of days of training and again 2 weeks later. A within-subjects ANOVA revealed that training did not increase the amount of perceptual bias over time [F(5,40) < 1]. To construct the ROC II curves, we calculated p(rating = i | perceptual bias) and p(rating = i | no perceptual bias) for all i, and transformed these into cumulative probabilities before plot- ting them against each other (anchored at [0,0] and [1,1]). Distribution-free methods were employed to characterize type II ROC sensitivity by calculating the area under the ROC curve (Aroc), and type II ROC bias (Broc). These parameters are derived from simple geometry and do not make assumptions about the shape of the distribution (Kornbrot, 2006). The area under the ROC curve (Aroc) quantifies the extent to which metacogni- tive judgments are predictive of perceptual bias during rivalry (Figure 1B); a diagonally flat ROC function indicates little pre- dictive value of the metacognitive judgment on the subsequent perceptual outcome during rivalry. The area under the ROC curve Mental imagery did bias perception in favor of the imagined grating [F(5,40) = 8.861; p = 0.018] which is consistent with pre- vious work demonstrating the effect of mental imagery on rivalry (Pearson et al., 2008, 2011). Unsurprisingly – considering the lack of a training effect – gratings of both trained and untrained July 2012 | Volume 3 | Article 224 | 4 The training of imagery and metacognition Rademaker and Pearson 1 2 3 4 5 2-3 weeks 45 50 55 60 65 70 Day of training Perceptual bias (% of trials) training generalization FIGURE 2 | Imagery biased perception during rivalry in favor of the previously imagined grating, but this perceptual bias did not significantly change over the course of a 5-day training. The blue line represents the mean training data, and the blue shaded regions represent ±1 SEM. METACOGNITIVE JUDGMENTS To assess whether people’s metacognitive insights about imagery strength improve over the course of training, we constructed ROC II curves for each individual observer, on each day of training (Figure 4; Materials and Methods). The extent to which metacog- nitive judgments of vividness predict perceptual bias was quan- tified as the area under each ROC II curve. Data presented in Figure 4 demonstrate that on earlier days of training (darker green lines) the area under the curve is smaller than on later days of training (lighter green lines). The upward bowing profile of the curves observable in over half of our participants demonstrates that vividness judgments indeed predict perceptual bias. There is a clear trend toward more metacognitive abil- ity over time for both the trained [F(5,40) = 1.742; p = 0.147] and untrained (generalization) orientation [F(2,16) = 7.416; p = 0.005]. Trained and untrained orientations do not statisti- cally differ [F(1,8) < 1]. This lack of orientation specificity may not be surprising considering that metacognition for perception is something presumably supported by higher-level frontal areas of the brain (Fleming et al., 2010), where responses are invariant to fine-grained orientation information. Hence, we simplified our analysis by collapsing the data from all orientations before con- structing the ROC II curves as displayed in Figure 4. Estimates of the type II ROC sensitivity Aroc are therefore slightly more reliable on day 1, day 5, and during follow-up, since they are constructed based on more data. FIGURE 2 | Imagery biased perception during rivalry in favor of the previously imagined grating, but this perceptual bias did not significantly change over the course of a 5-day training. The blue line represents the mean training data, and the blue shaded regions represent ±1 SEM. The parts of the plot depicted in red represent data from blocks where we tested training generalization to other orientations. (generalization) orientations yielded similar perceptual biases: a within-subjects ANOVA for training days 1, 5, and follow-up revealed no main effect of orientation [F(1,8) < 1]. Analyzing the data by session did not unveil any hidden differences in perceptual bias over time [F(11,88) = 1.106; p = 0.366], which excludes the possibility that most learning took place between the first couple of sessions. IMAGERY TRAINING The parts of the plot depicted in red represent data from blocks where we tested training generalization to other orientations. negligible influence on participant’s reports of rivalry dominance, as previously documented (Pearson et al., 2011). 1 2 3 4 5 2-3 weeks 45 50 55 60 65 70 Day of training Perceptual bias (% of trials) training generalization METACOGNITIVE JUDGMENTS The information from Figure 4 is summarized in Figure 5, showing the main effect of training: vividness judgments pre- dict perceptual bias increasingly better over time [F(5,40) = 3.075; p = 0.019]. This trend is linear when only looking at training days 1–5 [F(1,8) = 5.846; p = 0.042] but becomes quadratic when follow-up is included [F(1,8) = 8.778; p = 0.018],indicating a drop of the proportion Aroc at follow-up. Nevertheless, planned com- parisons (uncorrected t-tests) show that – with the exception of day 1 – the predictive value of self-reported vividness on the per- ceptual outcome is larger than would be expected by chance (one- tailed one-sample t-test day 1: p = 0.243; all others: p < 0.029), and this ability is still present 2–3 weeks after training (p = 0.021). Additional evidence that mental imagery was not improved by training comes from participant’s introspective judgments of imagery vividness. Mean self-reported vividness of men- tal imagery was statistically the same on all days of training [F(5,40) = 1.224; p = 0.316]. Self-reports of exerted effort did not change over the course of training either [F(5,40) < 1]. In summary, neither the perceptual measure of imagery strength (“perceptual bias”) nor ratings of vividness showed any significant change over the 5-days of training. Thus, it appears that training in this study was unable to increase imagery strength over time. CATCH TRIALS In previous work we demonstrated that people can reliably evaluate the vividness of their mental imagery from one trial to the next (Pearson et al., 2011). Participants in that previous study were tested only once. Thus, one might expect to find evi- dence for metacognition of imagery strength on day 1 of training in the current experiment. However, the difference in analyses used to determine metacognition in our previous and current work (within-subjects analysis of variance, and area under ROC II curve respectively), make it hard to directly compare the findings. A within-subjects analysis of variance performed on the current data shows that on day 1 of training, participants marginally (but not significantly) showed a main effect of vividness on perceptual bias [F(3,15) = 2.83; p = 0.074]. However, a lack of observed power (0.558) indicates that at this sample size there is only a small (44%) chance of finding a significant effect (at α = 0.05) when assuming that people have metacognitive insights into their own imagery Catch trials were presented in a randomly interleaved fashion on 10% of all experimental trials, to determine whether observers showed response bias in favor of the imagined grating. On these trials,a mock rivalry display was presented consisting of a balanced physical combination of the green and red gratings shown to both eyes simultaneously. If the effects observed during rivalry were due to decisional bias or demand characteristics, we expect to find the same degree of response bias on catch trials. We analyzed bias by coding veridical“mixed”responses to the catch trials as 50%,while responses that matched the cued pattern were coded as 100%, and responses opposite to the cued grating were coded as 0%. The percentage of catch trials during which participant’s responses were biased in favor of the cued grating are shown in Figure 3 (for all days of training). On average, this bias was 50.79%. CATCH TRIALS This indicates that demand characteristics and decisional bias have a July 2012 | Volume 3 | Article 224 | 5 www.frontiersin.org The training of imagery and metacognition Rademaker and Pearson 0 10 20 30 40 50 60 70 80 90 100 day 1 day 2 day 3 day 4 day 5 2−3 weeks Perceptual bias (% of trials) MM AA EY DB CB EE RS BW RR FIGURE 3 | Mean percentage of catch trials on which participant’s response to the fake-rivalry display was biased in favor of the imagined grating. A score of 50% indicates a lack of bias. Gray shaded areas show each individual participant, the colored bars represent the 5 days of training plus follow-up 2–3 weeks later. FIGURE 3 | Mean percentage of catch trials on which participant’s response to the fake-rivalry display was biased in favor of the imagined grating. A score of 50% indicates a lack of bias. Gray shaded areas show each individual participant, the colored bars represent the 5 days of training plus follow-up 2–3 weeks later. day 1 day 2 day 3 day 4 day 5 2−3 weeks RR BW RS CB DB EY MM AA p(vividness | no perceptual bias) p(vividness | perceptual bias) 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 EE 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 FIGURE 4 | Participant’s metacognition of imagery over the course of training. ROC II curves based on the vividness ratings reported by our participants (collapsed over Gabor orientations). Each plot represents an individual participant; the colored lines represent the ROC curves for the different days of training. FIGURE 4 | Participant’s metacognition of imagery over the course of training. ROC II curves based on the vividness ratings reported by our participants (collapsed over Gabor orientations). Frontiers in Psychology | Perception Science CATCH TRIALS Each plot represents an individual participant; the colored lines represent the ROC curves for the different days of training. Frontiers in Psychology | Perception Science Frontiers in Psychology | Perception Science July 2012 | Volume 3 | Article 224 | 6 The training of imagery and metacognition Rademaker and Pearson strength at the population level. An a priori power analysis indi- cates that, assuming a medium effect size, 21 subjects would be required to obtain a power of 0.95. that,as expected,effort did not predict perceptual bias [F(1,8) < 1]. The area under the ROC II curve (Aroc), which quantifies the degreetowhichself-reportedeffortpredictsperceptualbiasduring rivalry,did not differ from chance on any of the training days (two- tailed one-sample t-tests all p > 0.081). Neither did we observe a change over time for the trained [F(5,40) < 1], untrained (general- ized) [F(2,16) = 2.711; p = 0.097],or collapsed [F(5,40) < 1] grating orientations. Vividness ratings are predictive of the efficacy that mental imagery has at biasing the perception of rivaling stimuli. By contrast, self-reported effort for imagery was not hypothesized to predict perceptual bias.Attempts to exert greater effort do not nec- essarily result in highly effective imagery, as demonstrated by pre- vious work (Pearson et al., 2011). To ensure that our findings were specific to introspective vividness – and not effort – we constructed ROC II curves (as in Figure 4; Materials and Methods) based on the effort ratings reported by our participants. The pooled (across participants) curves per day are shown in Figure6A;the diagonally flat function indicates a weak link between self-reported effort rat- ings and perceptual bias during rivalry. Figure 6B demonstrates Vividness appears to predict perceptual bias more strongly over the course of training, whereas effort does not predict perceptual bias at all. Can this finding be explained by the way participants used the rating scales? Participant’s average reported vividness (2.57 ± 0.21) and effort (2.86 ± 0.14) did not significantly dif- fer (p = 0.261). In other words, subjective vividness – but not effort – is predictive of how well something was imagined inde- pendent of rating-magnitude. Signal detection theory considers metacognitive ability (sensitivity) and rating-magnitude (bias) as two independent properties (Galvin et al., 2003). In accordance with this notion,we find that individuals with higher self-reported vividness were not better at evaluating their imagery strength and vice versa. CATCH TRIALS FIGURE 5 | Imagery metacognition improves over the course of FIGURE 5 | Imagery metacognition improves over the course of training. The extent to which self-reported vividness predicts perceptual bias (quantified as the area under the ROC II curve after collapsing all FIGURE 5 | Imagery metacognition improves over the course of training. The extent to which self-reported vividness predicts perceptual bias (quantified as the area under the ROC II curve after collapsing all grating orientations, as also shown in Figure 4) plotted against day of training. Shaded areas represent ±1 SEM. training. The extent to which self reported vividness predicts perceptual bias (quantified as the area under the ROC II curve after collapsing all grating orientations, as also shown in Figure 4) plotted against day of training. Shaded areas represent ±1 SEM. p(effort | no perceptual bias) p(effort | perceptual bias) day 1 day 2 day 3 day 4 day 5 2−3 weeks 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 B Proportion A effort 1 2 3 4 5 2-3 weeks Day of training roc 0.4 0.5 0.6 0.7 0.65 0.55 0.45 A FIGURE 6 | Participant’s self-reported effort with which the gratings were imagined does not predict perceptual bias. (A) ROC II curves based on the effort ratings pooled across participants. Green colored lines represent the different days of training. The diagonally flat functions indicate a weak link between self-reported effort ratings and perceptual bias during rivalry. (B) The extent to which self-reported effort predicts perceptual bias (quantified as the area under the ROC II curve) plotted against each day of training. Self-reported effort does not predict perceptual bias, and this does not change over time. Data was collapsed over Gabor orientations, since outcomes did not differ between the two sets of orientations [F (1,8) < 1]. Shaded areas represent ±1 SEM. www.frontiersin.org July 2012 | Volume 3 | Article 224 | 7 B Proportion A effort 1 2 3 4 5 2-3 weeks Day of training roc 0.4 0.5 0.6 0.7 0.65 0.55 0.45 effort predicts perceptual bias (quantified as the area under the ROC II curve) plotted against each day of training. Self-reported effort does not predict perceptual bias, and this does not change over time. Data was collapsed over Gabor orientations, since outcomes did not differ between the two sets of orientations [F (1,8) < 1]. CATCH TRIALS (A) ROC II curves based on the effort ratings pooled across participants. Green colored lines represent the different days of training. The diagonally flat functions indicate a weak link between self-reported effort ratings and perceptual bias during rivalry. (B) The extent to which self-reported effort predicts perceptual bias (quantified as the area under the ROC II curve) plotted against each day of training. Self-reported effort does not predict perceptual bias, and this does not change over time. Data was collapsed over Gabor orientations, since outcomes did not differ between the two sets of orientations [F (1,8) < 1]. Shaded areas represent ±1 SEM. 0.4 0.5 0.6 0.7 1 2 3 4 5 2-3 weeks Day of training Proportion A vividness roc 0.65 0.55 0.45 FIGURE 5 | Imagery metacognition improves over the course of training. The extent to which self-reported vividness predicts perceptual bias (quantified as the area under the ROC II curve after collapsing all grating orientations, as also shown in Figure 4) plotted against day of training. Shaded areas represent ±1 SEM. 0.4 0.5 0.6 0.7 1 2 3 4 5 2-3 weeks Day of training Proportion A vividness roc 0.65 0.55 0.45 FIGURE 5 | Imagery metacognition improves over the course of training. The extent to which self-reported vividness predicts perceptual bias (quantified as the area under the ROC II curve after collapsing all grating orientations, as also shown in Figure 4) plotted against day of training. Shaded areas represent ±1 SEM. 0.4 0.5 0.6 0.7 1 2 3 4 5 2-3 weeks Day of training Proportion A vividness roc 0.65 0.55 0.45 The type II bias of the ROC curve (Broc) provides us with a distribution-free estimate of the criterion used by participants to provide their subjective ratings. A neutral bias would give Broc equal to zero, while a negative or positive Broc indicates a bias toward lower or higher ratings respectively. This estimate corre- sponded very well with the actual rating-magnitude collected dur- ing the experiment: self-reported vividness ratings and estimated vividness bias (Broc vividness) were highly correlated (r = 0.913; p < 0.001), as were self-reported effort and estimated effort bias (r = 0.889; p = 0.001). The close resemblance between these two variables – both measures of participant’s criterion – helps validate the distribution-free approach used to determine ROC estimates in the current paradigm. CATCH TRIALS Specifically, participant’s ability to make accurate metacognitive judgments of their mental imagery (Aroc vivid- ness) and participant’s mean vividness ratings were not correlated (r = 0.063; p = 0.873). ings and perceptual bias during rivalry. Figure 6B demonstrates 0.4 0.5 0.6 0.7 1 2 3 4 5 2-3 weeks Day of training Proportion A vividness roc 0.65 0.55 0.45 FIGURE 5 | Imagery metacognition improves over the course of training. The extent to which self-reported vividness predicts perceptual bias (quantified as the area under the ROC II curve after collapsing all grating orientations, as also shown in Figure 4) plotted against day of training. Shaded areas represent ±1 SEM. effort is predictive of how well something was imagined inde pendent of rating-magnitude. Signal detection theory considers metacognitive ability (sensitivity) and rating-magnitude (bias) as two independent properties (Galvin et al., 2003). In accordance with this notion,we find that individuals with higher self-reported vividness were not better at evaluating their imagery strength and vice versa. Specifically, participant’s ability to make accurate metacognitive judgments of their mental imagery (Aroc vivid- ness) and participant’s mean vividness ratings were not correlated (r = 0.063; p = 0.873). The type II bias of the ROC curve (Broc) provides us with a distribution-free estimate of the criterion used by participants to provide their subjective ratings. A neutral bias would give Broc equal to zero, while a negative or positive Broc indicates a bias toward lower or higher ratings respectively. This estimate corre- sponded very well with the actual rating-magnitude collected dur- ing the experiment: self-reported vividness ratings and estimated vividness bias (Broc vividness) were highly correlated (r = 0.913; p < 0.001), as were self-reported effort and estimated effort bias (r = 0.889; p = 0.001). The close resemblance between these two variables – both measures of participant’s criterion – helps validate the distribution-free approach used to determine ROC estimates in the current paradigm. p(effort | no perceptual bias) p(effort | perceptual bias) day 1 day 2 day 3 day 4 day 5 2−3 weeks 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 B Proportion A effort 1 2 3 4 5 2-3 weeks Day of training roc 0.4 0.5 0.6 0.7 0.65 0.55 0.45 A FIGURE 6 | Participant’s self-reported effort with which the gratings were imagined does not predict perceptual bias. DISCUSSION The research presented here suggests that mental imagery strength does not improve over the course of our 5-day training regime. Over the 5-days, no changes were observed relating to imagery strength as measured by rivalry, nor were there any changes in the average introspective judgments of imagery vividness. We fur- ther demonstrated that self-reported vividness of mental imagery predicts the perceptual consequences of single epochs of imagery. More importantly, this prediction becomes stronger with practice, implying increased metacognition of imagery over the course of training. Self-reported effort of mental imagery on the other hand, did not predict perceptual outcomes. There have been reports of visual imagery increasing perfor- mance on subsequent perceptual tasks (Tartaglia et al., 2009). Yet we were unable to find an increase in facilitation of rivalry domi- nance after 5 days of training. The question is of course, why? The emphasis of the research presented here was on improving imagery strength over time. This is a notably different emphasis from stud- ies that have investigated how imagery training changes perceptual skills (Tartaglia et al., 2009). One obvious explanation for the lack of an imagery training effect in this study is that imagery strength simply cannot improve with practice. This idea is corroborated by the fact that neither imagery bias, nor subjective ratings of imagery strength showed a significant increase as a function of training. Introspective ratings of imagery strength are reflected in the perceptual outcomes during rivalry, and the close relation- ship between the two implies they measure the same underlying construct (Pearson et al., 2011). Thus, several aspects of the data support the hypothesis that it is not possible – or very difficult – to improve imagery strength by means of training. One could hypothesize that imagery strength is liable to improvement, but we simply failed to find any in this study due to the configuration of our task. Research into the process of improv- ing perceptual skill – or perceptual learning – provides useful con- text in support of this hypothesis. One influential view known as the reverse hierarchy theory (Ahissar and Hochstein, 2004), states that learning is gated by top-down, task-related factors: Learning begins at high-level areas of the brain, after which it trickles down the hierarchy, fine-tuning the read out from lower level areas. CATCH TRIALS Shaded areas represent ±1 SEM. B Proportion A effort 1 2 3 4 5 2-3 weeks Day of training roc 0.4 0.5 0.6 0.7 0.65 0.55 0.45 p(effort | no perceptual bias) p(effort | perceptual bias) day 1 day 2 day 3 day 4 day 5 2−3 weeks 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 A p(effort | no perceptual bias) 0 0.2 0.4 0.6 0.8 1 effort predicts perceptual bias (quantified as the area under the ROC II curve) plotted against each day of training. Self-reported effort does not predict perceptual bias, and this does not change over time. Data was collapsed over Gabor orientations, since outcomes did not differ between the two sets of orientations [F (1,8) < 1]. Shaded areas represent ±1 SEM. effort predicts perceptual bias (quantified as the area under the ROC II curve) plotted against each day of training. Self-reported effort does not predict perceptual bias, and this does not change over time. Data was collapsed over Gabor orientations, since outcomes did not differ between the two sets of orientations [F (1,8) < 1]. Shaded areas represent ±1 SEM. FIGURE 6 | Participant’s self-reported effort with which the gratings were imagined does not predict perceptual bias. (A) ROC II curves based on the effort ratings pooled across participants. Green colored lines represent the different days of training. The diagonally flat functions indicate a weak link between self-reported effort ratings and perceptual bias during rivalry. (B) The extent to which self-reported FIGURE 6 | Participant’s self-reported effort with which the i i i d d di l bi (A) ROC GURE 6 | Participant’s self-reported effort with which the July 2012 | Volume 3 | Article 224 | 7 www.frontiersin.org The training of imagery and metacognition Rademaker and Pearson through imagery training, yet, changes at this cortical level may not be sufficient to improve imagery strength itself. Strikingly, Figure 4 shows large differences between individu- als: the degree to which metacognitive vividness judgments predict perceptual bias varies quite a bit from one person to the next (Aroc = 0.48–0.71). This type of variability is not uncommon, and previous studies have reported similarly large individual differ- ences in metacognitive ability for perceptual tasks (Song et al., 2011). Besides large differences related to metacognition, also the overall amount of perceptual bias experienced by our partici- pants varied widely (46–76%). CATCH TRIALS Nonetheless, participant’s overall metacognitive ability and the percent perceptual bias they experi- enced throughout the experiment,were uncorrelated (r = −0.027; p = 0.945). This suggests that participant’s metacognitive ability in this task is independent of imagery strength, as measured by binocular rivalry. Historically,mental imagery has been considered a fainter form of perception (Hume, 1739). Evidence to support this notion comes from functional magnetic resonance imaging (fMRI) stud- ies demonstrating that the magnitude of brain activity is lower during imagery than during bottom-up perception (Goebel et al., 1998; O’Craven and Kanwisher, 2000). Likewise, single neuron recordings in the medial temporal lobe of humans found fewer neurons that were recruited during imagery than during per- ception, and that the firing rate of these cells was lower during imagery compared to perception (Kreiman et al., 2000). In the case of perceptual bias during rivalry, imagery is presumed to influence or boost the memory trace that exists between one rivalry presentation and the next, and the location and orienta- tion specificity of this memory trace implies that it is composed of primarily low-level characteristics (Ishai and Sagi, 1995; Pear- son et al., 2008; Slotnick, 2008). Mechanisms such as a gain in sensitivity for the imagined pattern, or the strengthening of sen- sory traces, would be needed to modify population activity in lower visual areas mediating alternations of conscious percep- tion during rivalry. Imagery may simply lack sufficient impact to induce permanent plastic changes at these lowest sensory levels. Future research directions aiming to improve imagery strength could investigate the necessity of bottom-up information for learning. Specifically, it would be interesting to see if there is a transfer from improving visual perception by means of prolonged training with actual sensory stimuli, to improvements of imagery strength. Frontiers in Psychology | Perception Science DISCUSSION This theory invokes a number of detailed predictions, namely, early (fast) learning should be related to high-level changes, whereas asymptotic (slow) learning should involve plasticity in low-level sensory areas – if required by the task. There is considerable evi- dence supporting this view (Ahissar and Hochstein, 1993, 1997; Dosher and Lu, 1998; Dupuis-Roy and Gosselin, 2007). In light of the reverse hierarchy framework,our training regime issuspecttoacriticalvulnerability.Namely:trainingduration.Five days may have been insufficient time to reach the asymptotic learn- ing phase. The Tartaglia study previously mentioned (Tartaglia et al., 2009) trained participant’s imagery for 10 days, twice as long as in our study, and found an improvement on perceptual tasks. Assuming that specific cellular plastic processes at the hierarchical level of ocular dominance columns can only occur during asymp- totic learning, longer training might be necessary when aiming to influence rivalry perception. The idea that training cannot easily change imagery strength might be explained by the manner in which imagery strength is linked to brain anatomy. The Tartaglia study (Tartaglia et al.,2009) had participants repetitively imagine the crucial part of a bisection stimulus (spatial judgment) or a low-contrast Gabor pattern (con- trast judgment). They found improved perceptual performance on a subsequent perceptual bisection task and a Gabor detection task after imagery training, and this improvement generalized to untrained orientations. This lack of orientation specificity implies that learning through imagery did not involve plastic changes in early visual cortex, but probably involved higher-level extra-striate areas. Higher-level changes may boost perceptual performance Recent research has demonstrated that perceptual learning can also occur without a specific task and outside of awareness, as long as the information of interest is paired with feedback or July 2012 | Volume 3 | Article 224 | 8 The training of imagery and metacognition Rademaker and Pearson a reward signal (Seitz and Watanabe, 2003, 2005; Seitz et al., 2009) or with online-feedback via decoded fMRI signals (Shi- bata et al., 2011). Our experimental design lacked a direct reward signal. Perhaps if successful epochs of imagery were paired with a reward signal, this could facilitate learning. In practice the implementation of a reward may prove difficult to realize. Often, measures of imagery strength are dependent on subjec- tive reports, and offering rewards based on only self-reports could induce strong response and observer biases. DISCUSSION Considering the orthogonal training orientations of our experiment, disruption of consolidation seems an unlikely explanation for the ineffectiveness of imagery training. Introspective or “metacognitive” sensitivity is important to guide actions and to make decisions (Vickers, 1979; Daw et al., 2005; Dayan and Daw, 2008) and being able to adequately esti- mate ones confidence can help drive adaptive behavior (Kepecs et al., 2008). In its simplest form, low confidence that a recent decision was correct may prompt reexamination of the evidence, or seeking a second-opinion. In the event of internally gener- ated experiences such as mental imagery, low confidence that an image was veridical and life-like may lead someone to recon- sider such an experience. A better metacognitive understanding may help the imaginer bridge the gap between first and third- person perspective. For example, people can resolve potential ambiguities about perception by comparing their own percep- tual experience with the subjective experience of another person (Bahrami et al., 2010). Similarly, when the imaginer has a better understanding of the authenticity of his or her mental image, it will be easier to communicate its content to another person. In sum, increasing the efficiency with which people introspect the quality of their mental images can prove a novel and important finding. Can people become better at knowing their own thoughts? We were able to improve subject’s ability to judge the vividness of their imagery. This improvement was still present during a follow-up test, implying a long lasting effect of training on metacognitive evaluation of mental imagery. Furthermore, training of metacog- nition was not orientation specific: metacognition was improved for both trained and untrained sets of orientations. It is likely that the improvement of metacognition reported here originates from higher-level brain areas. This is in concordance with the suspected high-level neural locus of metacognitive ability for per- ception (Kepecs et al., 2008; Kiani and Shadlen, 2009; Fleming et al., 2010) as well as the idea that networks in high-level cortical regions orchestrate strategic choices during early learning, allocat- ing attention and motivation in response to specific task demands (Willingham, 1999; Hochstein and Ahissar, 2002; Doyon et al., 2003). In conclusion, we discussed a variety of reasons why train- ing did not lead to an improvement of imagery strength in the current study. DISCUSSION Neverthe- less, it is possible that our training was insufficient to obtain an effect, and providing feedback, rewards, or some manner of get- ting participants to intentionally try and increase their imagery strength, could have been a more effective way to train mental imagery. (Fleming et al., 2010), and there is a marked dissociation between metacognitive ability and performance on visual perceptual tasks (Lau and Passingham, 2006; Lau, 2008; Rounis et al., 2010; Song et al., 2011). Thus, metacognitive ability can be viewed as a stable and task independent cognitive process that can be improved with practice, independent of performance on other tasks. Changes in high-level neuronal populations are likely candidates for this learning. The ability to introspect on private thoughts is key to human subjective experience. Yet, people’s ability to evaluate internally generated experiences – such as imagery – is not as self-evident as it may appear. Although a large number of studies now demonstrate that something as private as a mental image can be successfully studied from a third-person perspective (Ishai and Sagi, 1995, 1997; Kosslyn et al., 2001; Pearson et al., 2008; Tartaglia et al., 2009), research has only recently begun to tackle issues related to the first-person perspective (Pearson et al., 2011). The core problem from the first-person perspective of the imaginer is that self-generated instances of imagery, unlike perception, cannot be directly compared with a perceptual template. Nevertheless, peo- ple seem quite capable of knowing if a mental image is accurate, vivid, or detailed. And practice further improves this first-person introspective ability. Why might such metacognitive knowledge be important? During memory consolidation, initially fragile memory traces become stabilized due to practice-induced plasticity in task rele- vantbrainareas(Karni,1996;Dudai,2004).Cantheineffectiveness of imagery training be due to somehow disrupted memory consol- idation? Classically, consolidation has been defined as a time lim- ited process directly following learning (Dudai, 2004). However, recent studies indicate that interference is rather time indepen- dent, and can occur at long intervals after training (Goedert and Willingham,2002; Caithness et al.,2004; Zhang et al.,2008). Inter- ference can be considered strongly stimulus dependent, resulting from similarity between the learned and interfering stimulus, and the corresponding neuronal populations recruited by these stimuli (Seitz et al., 2005; Been et al., 2011). 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Neverthe- less, we demonstrated that people’s ability to introspect their own imagery strength does improve with training, which sug- gests distinct mechanisms underlying imagery and metacognition. Being able to improve metacognition by means of practice can have important implications for real-life situations. It would be interesting to know if training metacognition could help people improve certain cognitive functions, such as decision-making or Our study demonstrated improvements of the metacogni- tion of imagery, whereas no changes in imagery strength itself were observed. This dissociation suggests distinct brain mech- anisms underlying metacognition and visual imagery respec- tively. Similar distinctions have been made regarding metacogni- tion of perception: Neuroanatomical substrates of introspective ability are distinct from those supporting primary perception July 2012 | Volume 3 | Article 224 | 9 www.frontiersin.org www.frontiersin.org The training of imagery and metacognition Rademaker and Pearson ACKNOWLEDGMENTS ACKNOWLEDGMENTS planning actions. If so,this may prove especially helpful for specific patient populations. Finally, future investigations of prolonged training of imagery can prove advantageous in outlining the over- lap between mechanisms of perception and imagery. Imagery as defined here is a highly voluntary process that allows introspec- tion in the absence of direct perceptual input. As such, imagery can provide a unique gateway to understanding how perceptual and introspective processes are represented in the brain. We thank Frank Tong for discussions and helpful input with design and execution of this work. We also thank Jan Brascamp, Elias Cohen, Janneke Jehee, Jan Schepers, and Jascha Swisher for helpful comments and discussions regarding this research. This work was funded by grants from the National Health and Medical Research Council of Australia CJ Martin Fellowship 457146 and project grant APP1024800 to Joel Pearson. REFERENCES in the theory of signal detectability: discrimination between correct and incorrect decisions. Psychon. Bull. Rev. 10, 843–876. REFERENCES C., and Passingham, R. E. (2006).Relativeblindsightinnormal observers and the neural correlate of visual consciousness. Proc. Natl. Acad. Sci. U.S.A. 103, 18763–18768. Kamphuisen, A., Van Wezel, R., and VanEe,R.(2007).Inter-oculartrans- fer of stimulus cueing in dom- inance selection at the onset of binocular rivalry. Vision Res. 47, 1142–1144. Fine, I., and Jacobs, R. A. (2002). Com- paring perceptual learning tasks: a review. J. Vis. 2, 190–203. Chong, S., and Blake, R. (2006). 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July 2012 | Volume 3 | Article 224 | 11 REFERENCES Received: 27 April 2012; accepted: 16 June 2012; published online: 10 July 2012. Seitz, A. R., and Watanabe, T. (2003). Psychophysics: is subliminal learn- ing really passive? Nature 422, 36. Received: 27 April 2012; accepted: 16 June 2012; published online: 10 July 2012. Citation: Rademaker RL and Pearson J (2012)Training visual imagery: improve- ments of metacognition, but not imagery strength. Front. Psychology 3:224. doi: 10.3389/fpsyg.2012.00224 Citation: Rademaker RL and Pearson J (2012)Training visual imagery: improve- ments of metacognition, but not imagery strength. Front. Psychology 3:224. doi: 10.3389/fpsyg.2012.00224 Seitz, A. R., Yamagishi, N., Werner, B., Goda, N., Kawato, M., and Watanabe, T. (2005). Task-specific disruption of perceptual learning. Proc. Natl. Acad. Sci. U.S.A. 102, 14895–14900. Pearson, J., Rademaker, R. L., and Tong, F. (2011). Evaluating the mind’s eye: the metacognition of visual imagery. Psychol. 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(1910). An experimental study of imagination?. Am. J. Psychol. 21, 422–452. Weiss, T., Hansen, E., Rost, R., Beyer, L., Merten, F., Nichelmann, C., and Zippel, C. (1994). Mental practice of motor skills used in poststroke reha- bilitation has own effects on central nervous activation. Int. J. Neurosci. 78, 157–166. Shibata, K., Watanabe, T., Sasaki,Y., and Kawato,M. (2011). Perceptual learn- ing incepted by decoded fMRI neu- rofeedbackwithoutstimuluspresen- tation. Science 334, 1413–1415. Pylyshyn, Z. (2003). Return of the men- tal image: are there really pictures in the brain? Trends Cogn. Sci. (Regul. Ed.) 7, 113–118. July 2012 | Volume 3 | Article 224 | 11 www.frontiersin.org www.frontiersin.org
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Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks
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Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks CECILIA PASQUALE , (Member, IEEE), ENRICO SIRI , (Member, IEEE), SILVIA SIRI , (Member, IEEE), AND SIMONA SACONE, (Member, IEEE) Department of Informatics, Bioengineering, Robotics and Systems Engineering, University of Genoa, 16145 Genoa, Italy Corresponding author: Cecilia Pasquale (cecilia.pasquale@edu.unige.it) CECILIA PASQUALE , (Member, IEEE), ENRICO SIRI , (Member, IEEE), SILVIA SIRI , (Member, IEEE), AND SIMONA SACONE, (Member, IEEE) Department of Informatics, Bioengineering, Robotics and Systems Engineering, University of Genoa, 16145 Genoa, Italy Corresponding author: Cecilia Pasquale (cecilia.pasquale@edu.unige.it) This work was supported by the European Union via the Structural Reform Support Program and implemented by the Centro Italiano di Eccellenza sulla Logistica i Trasporti e le Infrastrutture (CIELI)—University of Genoa (UniGe), in cooperation with the European Commission, under Project ‘‘New Economic Regulation for Transport in Case of Emergency Events.’’ ABSTRACT In this paper, a two-stage modeling framework is proposed to represent the route choices and dynamics of two classes of traffic flows, namely passengers and freight, in a large-scale intermodal transportation network. More in details, the transport modes considered in this model are road transport, represented through a highway network, and rail transport. The methodology adopted in this work relies on a multi-class intermodal assignment model combined with a multi-class intermodal dynamic model. The proposed modeling scheme can be adopted for different purposes, such as to support decision makers who intend to utilize the full mobility capacity of a geographical area by allocating the mobility demand on different modes or by suggesting intermodal itineraries also when the network is affected by disruptive events. The proposed framework has been tested on a benchmark network with the aim of showing the mutual relations that occur between different modes of transport in case a connection of the intermodal network fails. Specifically, the analysis reported in the paper shows that the collapse of a railway arc can cause a high increase in travel times for some highway arcs, which result 30-40% higher than in the pre- disruption scenario, and an even greater growth in the average occupancy of many arcs. INDEX TERMS Assignment model, dynamic discrete-time model, intermodal rail-road transport network, freight transport. INDEX TERMS Assignment model, dynamic discrete-time model, intermodal rail-road transport network, freight transport. is work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ VOLUME 10, 2022 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons Received 28 June 2022, accepted 6 July 2022, date of publication 11 July 2022, date of current version 18 July 2022. Received 28 June 2022, accepted 6 July 2022, date of publication 11 July 2022, date of current version 18 July 2022. Digital Object Identifier 10.1109/ACCESS.2022.3189775 I. INTRODUCTION of the infrastructure network layout due to the verification of disruptive events. The need to move passengers and freight is becoming increas- ingly pervasive in conducting daily human activities and more generally in the social and economic advancement of countries. As a result, the availability and accessibility of transport infrastructures and mobility services allow this need to be met and, at the same time, increase the attractiveness of an area. In this regard, it is of utmost importance to quantify the capacity necessary to satisfy the transport demand of a geographical area and to try to predict how the transport sys- tems included in it will react to unforeseen events. These latter may appear in different forms as changes in demand, e.g. the transfer of passengers from public to private transport that have occurred due to the ongoing pandemic, or modification Model-based approaches are the foremost methodologies for evaluating the performance of transportation networks because they allow: • to quantify the efficiency of transport networks subject to different scenarios through the calculation of perfor- mance indexes (e.g., average travel times, fuel consump- tion, pollutant emissions, etc.); • to evaluate the effects produced by the occurrence of critical events (e.g., infrastructure collapse, natural dis- asters limiting the functionality of transport systems, terrorist attacks, etc.); • to evaluate the effects produced by the introduction of new systems (e.g., new infrastructure, modification of existing layout, etc.); The associate editor coordinating the review of this manuscript and approving it for publication was Wen-Sheng Zhao . • to develop or test regulation policies. 73583 C. Pasquale et al.: Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks dynamic model is improved. As a consequence, the results reported and discussed in this paper are completely new. Our work is a model-based approach which relies on the combination of a traffic assignment model with a discrete-time dynamic model used for simulation. In par- ticular, the assignment model is adopted to represent the route choices of users and to evaluate, in a static way, the distribution of flows on the network. The simulative model, instead, is used to represent the impact of the route choices on the transport network by describing the flow evolution over time. I. INTRODUCTION By analyzing the literature on these topics, it is possible to observe that most of the researches’ efforts have been focused so far on the definition of assignment and sim- ulative models on mono-modal transport networks, i.e. road networks or railway networks only (see for instance [1]–[3]). However, if the goal is to analyze the accessibility of a given geographic area or to give better information to users, it is necessary to adopt more extensive models which consider the intermodal transport network as a whole, by properly modeling the different modes of transport and the possibility of transfer among them. To summarize, the modeling framework proposed in this work is composed of two stages (see Fig. 1): • a multi-class intermodal rail-road assignment model; • a multi-class intermodal rail-road assignment model; • a multi-class intermodal rail-road discrete-time dynamic model; and has the following goals: • to represent the route choices and the dynamic behavior of traffic flows in an intermodal rail-road network; • to explicitly capture the behavior of passengers and freight in the network; • to allow performance analysis of the intermodal network in different scenarios. The present paper is organized as follows. A literature review on intermodal transport network models is presented in Section II. In Section III the general features of the pro- posed modeling framework and the basic notation are intro- duced. Then, the proposed two-stage modeling approach is presented: the assignment model is outlined in Section IV, while the discrete-time dynamic model is described in Section V. The application of the proposed methodology to a test network is shown in Section VI, while some conclusive remarks are gathered in Section VII. Another aspect that cannot be overlooked in the analysis of transport networks is that, as in all critical infrastruc- ture systems, the interdependencies increase the potential for cascading failures [4]. Referring specifically to intermodal transport networks [5], the interdependence among different transport modes is particularly relevant. It can be related to pursue the transport activity itself, in this case several transport services operate synergistically to allow the sat- isfaction of the mobility demand of users and the efficient distribution of goods and services. Interdependence can be related to physical reasons such as the overlap of different routes, even belonging to different transport modes, through bridges or tunnels, or to hidden relationships, such as those analyzed in [6]. I. INTRODUCTION Regardless of how these interdependencies occur, they imply an increased vulnerability of the transport network as a whole. In fact, it is possible that critical events affecting even one mode of transport could cause a ripple effect involving other transport modalities or, in the worst case, the entire transport network. For these reasons, the development of intermodal models represents an important step in the analysis of complex and highly interdependent systems such as transport networks. II. LITERATURE REVIEW II. LITERATURE REVIEW Sketch of the two-stage modeling framework. the network, the time-dependent transport times on roads, and the time schedules for trains and barges. When dealing with freight intermodal networks, it is worth noting that the system performance must include different factors, such as costs, times, flexibility, reliability, quality and sustainability [32] and, then, multi-criteria analysis can be effectively applied in this context (see e.g. [33], [34]). freight flows. In particular, intermodal traffic assignment has been studied for some decades [20]–[23], by considering the so-called hypernetworks, which allow to include both links representing a portion of the real intermodal transport network (road, rail, private or public transport) and links associated with users’ decisions. Intermodal traffic assign- ment approaches have also been studied more recently, e.g. in [24] the intermodal transport network of an urban area is represented as a graph composed of a number of sub- networks, each of which is associated with a transportation mode. In that work, the intermodal network is translated into an augmented-state network in which transfer rules and transfer probabilities between the different transport modes are defined. In [25], a stochastic model is proposed in order to account for the reliability of the chosen transport modes and paths, while in [26] different modes are considered, only- driving, carpooling, ride-hailing, public transit and park-and- ride, for a urban transportation system including private cars, freight trucks, buses, and so on. freight flows. In particular, intermodal traffic assignment has been studied for some decades [20]–[23], by considering the so-called hypernetworks, which allow to include both links representing a portion of the real intermodal transport network (road, rail, private or public transport) and links associated with users’ decisions. Intermodal traffic assign- ment approaches have also been studied more recently, e.g. in [24] the intermodal transport network of an urban area is represented as a graph composed of a number of sub- networks, each of which is associated with a transportation mode. In that work, the intermodal network is translated into an augmented-state network in which transfer rules and transfer probabilities between the different transport modes are defined. In [25], a stochastic model is proposed in order to account for the reliability of the chosen transport modes and paths, while in [26] different modes are considered, only- driving, carpooling, ride-hailing, public transit and park-and- ride, for a urban transportation system including private cars, freight trucks, buses, and so on. II. LITERATURE REVIEW On the basis of the related literature review reported above, we can highlight that the main novelties and contributions of this work stand in: • combining a traffic assignment model with a simulative model; • capturing the travel choice and dynamic behavior of passengers and freight jointly in an intermodal network. • representing a large-scale transport network covering a regional territory. III. GENERAL FEATURES AND BASIC NOTATION As shown in Fig. 1, the proposed modeling scheme consists of two stages: an assignment model defined to allocate the demand of passengers and freight on a intermodal transport network, and a discrete-time dynamic model that allows to replicate the evolution of the system over time. It is worth noting that the discrete-time dynamic model receives as input the mobility demand and the path choices (route and modal choices) defined through the intermodal assignment model. Simulation-based approaches are either referred to passen- gers or freight separately. For instance, [27]–[29] provide simulative approaches for representing the choices of passen- gers in urban intermodal networks. In [27], three transport modes are considered, i.e. private vehicle transport, public vehicle transport and pedestrian transport, all represented through a three-dimensional macroscopic fundamental dia- gram (MFD) that allows to evaluate the accumulation of private and public vehicles according to different traffic scenarios. In [28], the considered transport modes are private vehicle transport and public road transport: private trans- portation is represented through the MFD, while constant transfer speeds are considered for public transportation. The derived intermodal model is used for the definition of adap- tive pricing policies. The work presented in [29] proposes an agent-based simulation tool to test innovative planning methods involving pedestrians and autonomous shuttles in large urban areas. Regarding instead freight logistics, [30] reports a very detailed survey about simulation approaches for intermodal freight transportation systems, used to repre- sent stakeholders, decision makers, operations, and planning activities. In [31], a dynamic model for an intermodal freight transport network is proposed to represent the mode changes at intermodal terminals, the physical capacity constraints of Both the assignment model and the dynamic model are based on a regional intermodal transport network in which the considered transport modes are road transport, represented by a highway network, and rail transport. The two modes of transport are connected with some intermodal arcs distin- guished depending on the flow class, i.e., passengers or cargo units, which they can receive. The rail-road intermodal trans- port network is represented by means of an oriented graph, as depicted in Fig. 2, denoted with G = (N, A), in which N indicates the set of nodes, whereas A = AH ∪AR∪AIp∪AIf represents the set of arcs. II. LITERATURE REVIEW Intermodal transport networks have been studied extensively by operations researchers with the purpose of taking deci- sions referred to freight transport operations. The related planning problems are normally distinguished in strategic, tactical and operational ones, according to the length of the planning horizon (more details and classifications can be found in the review papers [9]–[11]). Location decisions are probably the most representative examples of strategic plan- ning problems [12] and regard the definition of the optimal positioning of nodes of the intermodal network (see e.g. [13]–[15]). Tactical and operational decisions in intermodal networks regard medium-short term horizons and address different types of problems, such as, for example, the ser- vice network design [16], [17] or the selection of the best routes [18], [19]. This work, based on preliminary papers [7] and [8], falls in this field of research by representing large-scale transporta- tion networks in which the considered transport modes are road and rail connected to each other through appropriate intermodal connections. Another peculiarity of this modeling framework, both in the assignment and in the dynamic model, is that the user demand is multi-class, hence it is distinguished into passengers and cargo units (e.g., containers). This dis- tinction is of particular importance because passenger and freight flows may be characterized by different behaviors and may be subject to different restrictions, such as on route choices. Note that, compared with [7] and [8], in the present work the assignment model is multi-class (while in the previ- ous versions there was no distinction of user classes) and the Differently from these latter papers, in which the goal is to design and organize activities in an intermodal freight net- work, in this work we aim at defining a modeling framework for representing the spontaneous behavior of both freight and passengers in an intermodal network, not only in terms of alternative path choices but also including their dynamics. Hence, the scientific literature considered as a reference for this work is associated, firstly, with traffic assignment models and, secondly, with simulation-based approaches for inter- modal networks. The majority of the works addressing assignment mod- els for intermodal transport networks are referred to urban areas and consider the choices of passengers rather than 73584 VOLUME 10, 2022 VOLUME 10, 2022 C. Pasquale et al.: Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks FIGURE 1. Sketch of the two-stage modeling framework. FIGURE 1. IV. THE INTERMODAL ASSIGNMENT MODEL FOR PASSENGER AND FREIGHT FLOWS The purpose of the intermodal assignment model is to rep- resent the spontaneous decisions of users about their routes. The purpose of the intermodal assignment model is to rep- resent the spontaneous decisions of users about their routes. Before providing the details of the proposed approach, it is important to emphasize that the assignment model is a static approach in which all the mobility demand, estimated for a specific time window, is assumed to use the transport network simultaneously. As shown in Fig. 1, the result of this model is the redistribution of flows on the paths, i.e., f od,1 l for passen- gers and f od,2 l for freight, which are obtained by the solution of some optimization problems. The flows thus obtained will be suitably transformed into splitting rates, see Section V, and used as input data for the dynamic model. FIGURE 2. Sketch of the intermodal transport network. Intermodal arcs are distinguished for passengers and for freight because modal shifts for passengers and freight typi- cally occur at different locations and in different ways. Let P(i) indicate the set of nodes preceding node i and S(i) the set of nodes succeeding node i, i ∈N. The network is defined in an origin-destination-oriented mode in which JO ⊆N represents the set of all possible origin nodes, JD ⊆N represents the set of all possible destination nodes. The nodes which are not origins nor destinations simply allow the transit between successive arcs. The intermodal arcs, for both flow classes, are considered as fictitious arcs that allow modal transfers between road transport and rail transport and vice versa. For this reason, an origin node o ∈JO cannot be followed by intermodal arcs, and similarly, a destination node d ∈JD cannot be preceded by intermodal arcs. The passenger and freight assignment models are treated separately, firstly introducing the intermodal assignment model for passengers and, then, applying the intermodal assignment model for freight, since the latter uses the results of the passenger assignment to define the freight route choices. In particular, for the passenger assignment procedure we assume that the marginal impact on the network due to the presence of freight is sufficiently small so that it does not decisively influence the behavior of passengers. IV. THE INTERMODAL ASSIGNMENT MODEL FOR PASSENGER AND FREIGHT FLOWS This assumption is quite reasonable since, referring to the overall flows on a transport network, the freight component typically constitutes a rather low percentage compared with the flow of passengers. In the proposed approach the flows of users are distin- guished in passengers and freight, with superscript c denoting the class of users. In particular we define with c = 1 the passenger flow and with c = 2 the freight flow. Then, Dod,c indicates the demand of class c that originates at node o ∈ JO and has destination at node d ∈JD: this is the total demand associated with the od pair for the whole simulation horizon. This demand defined for all od pairs represents the so-called ‘‘origin-destination matrix’’. Let us also specify that the passenger demand, i.e., the demand for class c = 1, is expressed in number of passengers, while the demand of freight, corresponding to c = 2, is expressed in number of cargo units. The assignment procedure presented below is conducted in an iterative manner in which at the first step the inter- modal assignment model for passenger flows described in Section IV-A is run. Then the results of this assignment are used to estimate the average passenger travel times on the network using the dynamic model described in Section V. Finally, the intermodal freight assignment model, presented in Section IV-B, is run using the average travel times defined in the previous step. The main parameters and variables of the proposed approach are summarized in Tables 1-3. Specifically, Table 1 collects all the parameters that are used in both the assignment model and the dynamic simulation model. Table 2 collects the parameters and variables used in the assignment model only, while Table 3 reports parameters and variables used in the dynamic simulation model only. III. GENERAL FEATURES AND BASIC NOTATION Each subset of arcs is defined as follows: • AH is the set of highway connections; • AR is the set of railway connections; • AIp is the set of intermodal arcs for passengers; If • AIf is the set of intermodal arcs for freight. 73585 73585 VOLUME 10, 2022 C. Pasquale et al.: Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks FIGURE 2. Sketch of the intermodal transport network. (i, j) ∈AH ∪AR, the following condition: (i, j) ∈AH ∪AR, the following condition: (i, j) ∈AH ∪AR, the following condition: T ≤min 1i,j vH i,j , 1i,j vR i,j  (1) (1) A. THE INTERMODAL ASSIGNMENT MODEL FOR PASSENGER FLOWS Modeling the behavior and therefore the mobility choices of users is a challenging problem for which several approaches have been developed by researchers. One of the most accepted, and widely adopted, methodologies concerns the use of traffic assignment models. Given an origin-destination matrix, representing the users’ demand, and knowing the functional characteristics of the network infrastructure, a traf- fic assignment model allows to estimate how users will be dispersed in the network, taking into account the rela- tion between the infrastructure supply and the interaction of users mutual choices. The criteria underlying the mathemat- ical representation of such behaviors can vary considerably As it will be described in Section V, the dynamic behavior of the whole network is represented with a discrete-time model in which the time horizon is divided in K time steps, where k = 1, . . . , K indicates the temporal stage, and T [h] represents the sample time interval. In order to ensure a cor- rect time discretization, the length of the time interval T must allow a proper dynamic evolution of the system, therefore the length of the time step is chosen in order to fulfil, for any arc 73586 VOLUME 10, 2022 VOLUME 10, 2022 C. Pasquale et al.: Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks TABLE 1. Parameters common to the two models in the two-stage modeling framework. TABLE 1. Parameters common to the two models in the two-stage modeling framework. TABLE 2. Parameters and variables present only in the assignment model described in Section IV. TABLE 2. Parameters and variables present only in the assignment model described in Section IV. depending on the assignment model used. A comprehensive review of traffic assignment models is given in [35]. at a given time minimizes their own travel time’’. A way to compute the arc flows corresponding to such equilibrium involves finding the optimum solution of the Beckmann’s Transformation [37]. The N-Path Restricted variant of the aforementioned model [38] is obtained by constraining the assignment to a priori defined sets of admissible paths, which do not necessarily include all the possible ones for each origin-destination pair. This allows to avoid all the paths that are theoretically possible but quite implausible in practice. In this paper, all the paths involving more than one modal depending on the assignment model used. A comprehensive review of traffic assignment models is given in [35]. A. THE INTERMODAL ASSIGNMENT MODEL FOR PASSENGER FLOWS In the present work, the N-Path Restricted User- Equilibrium traffic assignment model has been applied. Generally speaking, the User Equilibrium traffic assignment model aims to estimate the network equilibrium state such that no user has unilaterally any interest in choosing an alternative path, since no other path would guarantee lower travel times. Such equilibrium is achieved if Wardrop’s first principle [36] is met, whereby ‘‘users choose the path that In the present work, the N-Path Restricted User- Equilibrium traffic assignment model has been applied. Generally speaking, the User Equilibrium traffic assignment model aims to estimate the network equilibrium state such that no user has unilaterally any interest in choosing an alternative path, since no other path would guarantee lower travel times. Such equilibrium is achieved if Wardrop’s first principle [36] is met, whereby ‘‘users choose the path that VOLUME 10, 2022 73587 C. Pasquale et al.: Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks ABLE 3. Parameters and variables present only in the dynamic model described in Section V. TABLE 3. Parameters and variables present only in the dynamic model described in Section V. flows. Moreover, constraints (5) define the relation among f od,1 l and x1 i,j by means of the path-arc incidence matrix whose elements are defined as follows: shift have been excluded. Then, denoted with l, l = 1, . . . , L, a generic path existing in the network and with Lod the set of all possible paths connecting the od pair, let Pod ⊆Lod be the set of admissible paths from origin node o to destination node d. The resulting optimization problem for the traffic assignment of passengers demand in the intermodal transport network is the following. δod,1 i,j,l = ( 1 if (i, j) belongs to path l from o to d 0 otherwise (6) δod,1 i,j,l = ( 1 if (i, j) belongs to path l from o to d 0 otherwise (6) (6) In (2), the terms τ 1 i,j(·) are the performance functions of arcs related to class c = 1, i.e., passengers. for (i, j) ∈AR. for (i, j) ∈AR. for (i, j) ∈AR. min y(x) = X (i,j)∈A x2 i,j · ci,j(x2 i,j) (13) (13) Finally, as mentioned above, the performance functions of intermodal arcs are constant functions, but it has been necessary to make them strictly increasing by introducing a (although very small) relation of direct proportionality between travel time and number of users on the arc in order to guarantee the uniqueness of the solution of the optimization problem, as detailed further on. subject to subject to jX l∈Pod f od,2 l = Dod,2 o ∈JO, d ∈JD (14) f od,2 l ≥0 o ∈JO, d ∈JD, l ∈Pod (15) x2 i,j = X o∈JO X d∈JD X l∈Pod,2 f od,2 l · δod,2 i,j,l (i, j) ∈A (16) (14) The performance functions for intermodal arcs are then defined as follows τ 1 i,j(x1 i,j) = αi,j · T + 1 M x1 i,j (9) (9) where f od,2 l and x2 i,j are, respectively, the flow of class c = 2 of od pair using path l and the total flow of freight on arc (i, j). Constraints (14)-(16) are analogous to constraints (3)-(5) included in Problem 1, and where δod,2 i,j,l is defined as follows for (i, j) ∈AIp, where αi,j ≥1 and M is a positive coefficient sufficiently big such that the impact of the number of users on the performance of an intermodal arc is negligible compared to the other types of arcs. δod,2 i,j,l = ( 1 if (i, j) belongs to path l from o to d 0 otherwise (17) By defining the performance functions of the arcs as above, it can be proven that Problem 1 is strictly convex on a convex domain and therefore admits a unique optimal solution with respect to variables x1 i,j but not for variables f od,1 l . Since f od,1 l , properly converted into splitting rates, represent the input of the dynamic model, their uniqueness is an essen- tial requirement in this work. Several methodologies have been developed to overcome this issue associated with the User-Equilibrium model [39]. A. THE INTERMODAL ASSIGNMENT MODEL FOR PASSENGER FLOWS Problem 2: Problem 2: min h(f ) = X o∈JO X d∈JD X l∈Pod f od,1 l · ln(f od,1 l ) (10) subject to X l∈Pod f od,1 l = Dod,1 o ∈JO, d ∈JD (11) x1,UE i,j = X o∈JO X d∈JD X l∈Pod f od,1 l · δod,1 i,j,l (i, j) ∈A (12) min h(f ) = X o∈JO X d∈JD X l∈Pod f od,1 l · ln(f od,1 l ) (10) (10) In order to reduce the computational effort, appropriate linear versions of the same functions have been used within the assignment process. In the case of highway arcs, the resulting linear functions are obtained by interpolating two points: the first is obtained using the free-flow travel time corresponding to an arc completely empty, while the other uses the value assumed by the hyperbolic function when the number of users is equal to φ · nmax i,j , where φ ∈[0, 1). The linear performance functions in the highway case are therefore defined as follows subject to subject to jX l∈Pod f od,1 l = Dod,1 o ∈JO, d ∈JD (11) x1,UE i,j = X o∈JO X d∈JD X l∈Pod f od,1 l · δod,1 i,j,l (i, j) ∈A (12) (11) Equations (11) – (12) convey the same constraints of Problem 1 with the only but fundamental difference that the flows on the arcs are now fixed and equal to x1,UE i,j , obtained as solution of Problem 1. Also, the non-negativity constraint (4) is now implicit in the fact that f od,1 l appear in (10) as the argument of a logarithm. τ 1 i,j(x1 i,j) = 1i,j ωi,jnmax i,j (1 −φ) · x1 i,j 1 η + 1i,j vH i,j (7) (7) for (i, j) ∈AH. Similarly, for the railway case, the second interpolating point is associated with the value assumed by the hyperbolic function when the number of users reaches the technical limit Cp1i,j smin i,j (see (39)). The following linear performance function for the railway case is therefore obtained: B. THE INTERMODAL ASSIGNMENT MODEL FOR FREIGHT FLOWS In this section, the intermodal assignment model for freight flows is presented. The purpose of this assignment model is to replicate the average freight route choices by considering as objective the minimization of the total travel costs needed to satisfy a given demand. Given these premises, the intermodal assignment model for freight is given as follows τ 1 i,j(x1 i,j) = hi,jsmin i,j (smin i,j −L) · x1 i,j Cp + 1i,j vR i,j (8) (8) g g g Problem 3: Problem 3: Problem 3: A. THE INTERMODAL ASSIGNMENT MODEL FOR PASSENGER FLOWS These functions, representative of the functional characteristics of the network arcs, express the relation between the travel time spent by passengers traveling through an arc and the amount of con- gestion on the same arc, which is expected to perform worse (and thus resulting in increased travel times) if the number of users traveling on it increases. The performance functions adopted for this model are derived from (37), with c = 1, introduced in Section V, considering the definitions provided in (38) and (39). For highway and railway arcs, this equation computes the transit time as a function of the estimated speed. The average speed on these arcs, in turn, depends on the number of users who are using those arcs. Regarding intermodal passenger arcs, on the other hand, the transfer time is considered constant and independent of the number of users present on it. For further information the reader is referred to Problem 1: Problem 1: min z(x) = X (i,j)∈A x1 i,j Z 0 τ 1 i,j(ω)dω (2) (2) subject to subject to jX l∈Pod f od,1 l = Dod,1 o ∈JO, d ∈JD (3) f od,1 l ≥0 o ∈JO, d ∈JD, l ∈Pod (4) x1 i,j = X o∈JO X d∈JD X l∈Pod f od,1 l · δod,1 i,j,l (i, j) ∈A (5) (3) where f od,1 l and x1 i,j are, respectively, the flow of class 1 of od pair using path l and the total flow of class 1 on arc (i, j). Con- straints (3) imply passengers’ demand satisfaction for each od pair, while (4) are non-negativity constraints regarding traffic 73588 VOLUME 10, 2022 VOLUME 10, 2022 C. Pasquale et al.: Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks at the equilibrium, maximizing an entropy function and for this reason more likely to occur [40]. the description of equations (37), (38) and (39) in Section V. It is worth noting that, with the exception of the intermodal case, the link performance functions are strictly increasing hyperbolic functions and therefore diverging in proximity to the arc theoretical maximum capacity. The resulting optimization problem is as follows. V. THE INTERMODAL DYNAMIC MODEL FOR PASSENGER AND FREIGHT FLOWS The complete list of parameters and variables adopted in the dynamic model is given in Tables 1 and 3; for further clarity, the main aggregate variables used to describe the dynamics of the intermodal road-rail system are recalled below: ,j The performance functions for freight intermodal arcs (i, j) ∈AIf are defined as follows: τ 2 i,j(x2 i,j) = γi,j · T + 1 M x2 i,j (20) • nod,c i,j (k) is the number of units of class c in arc (i, j) associated with the od pair at time step k; od c (20) where γi,j ≥1 and M is a positive coefficient large enough so that the impact due to the presence of cargo units on the arc is insignificant. Given the performance functions for the freight transport of each arc, the cost function in (13) associated with each arc of the network is defined as • Iod,c i,j (k) is the number of units of class c entering arc (i, j) associated with the od pair at time step k; od c • Ood,c i,j (k) is the number of units of class c exiting arc (i, j) associated with the od pair at time step k; d • βod,c i,j (k) are the splitting rates of class c in arc (i, j) associated with the od pair at time step k; note that the condition P j∈S(i) βod,c i,j (k) = 1 must be verified ∀i, ∀o, ∀d, ∀c, ∀k. ci,j(x2 i,j) = τ 2 i,j(x2 i,j)Ctime i,j + 1i,jCspace i,j + Cfix i,j (21) (21) where Ctime i,j , Cspace i,j and Cfix i,j are the cost per time unit, cost per space unit, and the fixed cost of arc (i, j), respectively. Depending on the type of arc, the contribution made by each of the three parameters can change significantly. For example, in the case of an intermodal freight arc, it is reasonable to assume Cspace i,j = 0. It is worth clarifying that different units are adopted in the model, depending on the arc type and the flow class. V. THE INTERMODAL DYNAMIC MODEL FOR PASSENGER AND FREIGHT FLOWS The dynamic model adopted in this work is used to cap- ture the dynamic features of the overall system through a set of discrete-time equations. Aggregate discrete-time mod- els have already been used for performance evaluation and optimization of specific intermodal transportation processes. In [41], [42], for instance, discrete-time models for freight movements by rail in maritime terminals are described. The model presented in this section is much more extensive considering the movement not only of freight but also of passengers and considering a more general applicative con- text, that is an intermodal rail-road multi-class transport net- work. Specifically, this dynamic model allows to evaluate the impact of the users’ route choices and to analyze the behavior of the intermodal system at a more detailed level than the one provided by the assignment model. p i,j g p g In the case of railway arcs, a similar approach is adopted and the relative performance function is defined as described below τ 2 i,j(x2 i,j) = hi,jsmin i,j (smin i,j −L) · x2 i,j Cf + t1 i,j (19) (19) which estimates the marginal contribution to the travel time of the arc due to the presence of freight, summed to the travel time due exclusively to the presence of passengers t1 i,j. The variable x2 i,j expresses the amount of cargo units present on the arc. Therefore, it follows that x2 i,j Cf represents the equivalent number of freight trains. The free-flow travel time 1i,j vR i,j is implicitly contained in t1 i,j. The system evolution in time and in space is described by means of aggregate variables defined for each class c = 1, 2, for each arc (i, j) ∈A, for each od pair, with o ∈JO, d ∈JD, and for each time step k, k = 0, . . . , K. for (i, j) ∈AR. A possible solution is looking for a pattern of flows f , coherent with the distribution of users (17) As done for the passenger assignment, the performance functions that estimate the level of congestion and the travel time on arcs are defined by linearizing (37), with (38) and (39), where c = 2. Therefore, for highway arcs, the perfor- mance function for freight is defined as follows τ 2 i,j(x2 i,j) = 1i,j ωi,jnmax i,j (1 −φ) · ςx2 i,j + t1 i,j (18) (18) 73589 VOLUME 10, 2022 C. Pasquale et al.: Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks the routes most consistent with the routing problem described in Problem 3. where ς is the conversion factor adopted to express the number of trucks x2 i,j in the arcs (i, j) ∈ AH in terms of passenger car equivalents. Equation (18) estimates the marginal contribution on travel times due to the presence of freight vehicles, which is summed to t1 i,j, that is the average travel time over arc (i, j) due exclusively to the presence of passengers, as calculated by the dynamic model. The term 1i,j vH i,j does not appear explicitly, since the free-flow travel time is contained in t1 i,j. In fact, it is assumed that the maximum speed vH i,j is the same for both freight and passenger vehicles. V. THE INTERMODAL DYNAMIC MODEL FOR PASSENGER AND FREIGHT FLOWS These splitting rates βod,c i,j (k), for each user class c, are obtained from the flows f od,c l resulting from the application of the intermodal and multi-class assignment procedure i.e., ϵ1 n,i =    1 if (n, i) ∈AH ∪AR η if (n, i) ∈AIp and (i, j) ∈AR 1 η if (n, i) ∈AIp and (i, j) ∈AH (25) (25) βod,c i,j (k) = P l∈Pod,c f od,c l · δod,c i,j,l P p∈P(i) P l∈Pod,c f od,c l · δod,c p,i,l (22) where η is the average number of passengers per car, used to translate the number of vehicles in passengers and viceversa. (22) p g The conversion factor ξ1 i,j is instead defined considering that the passenger demand is given in terms of number of passengers and that an origin node cannot be followed by an intermodal arc, therefore for all k, with k = 0 . . . , K. Note that βod,c i,j (k) are constant along the simulation horizon since, in this work, Dod,c is the total demand of the whole horizon. In case the simulation horizon is divided in different time intervals, each one char- acterized by a different demand, the intermodal assignment model described in Section IV is applied for each time inter- val, resulting in different splitting rates βod,c i,j (k). ξ1 i,j =    1 if (i, j) ∈AR 1 η if (i, j) ∈AH (26) (26) As for class c = 2, i.e., the class referring to freight, both conversion factors ϵ2 n,i and ξ2 i,j are set equal to 1 because, as mentioned above, a cargo unit is assumed to correspond to one truck and to one rail wagon. i,j Virtual queues at the origin nodes are considered in order to model the presence of flows that have to wait before entering the network. At this purpose, for each time step k, k = 0, . . . , K, the following variables are introduced: Note that the flows that actually enter an arc (i, j) depend on the capability of the arc to receive flows, i.e., the residual capacity qres i,j (k), calculated based on the total number of units in the arc (i, j) at the time step k. 1) ENTERING FLOWS N tot i,j (k) = X o∈JO X d∈JD nod,1 i,j (k) Cp + X o∈JO X d∈JD nod,2 i,j (k) Cf (28) ) The entering flows Iod,c i,j used in (23) are given by (28) Iod,c i,j =βod,c i,j (k)  X n∈P(i) ϵc n,i · Ood,c n,i (k)+ξc i,j·qod,c(k)  (24) (24) for all (i, j) ∈AR. The total number of vehicles and the total number of trains present in each arc at each time step must ensure the following conditions: 0 ≤ntot i,j (k) ≤nmax i,j and 0 ≤ N tot i,j (k) ≤N max i,j . for all (i, j) ∈AR. The total number of vehicles and the total number of trains present in each arc at each time step must ensure the following conditions: 0 ≤ntot i,j (k) ≤nmax i,j and 0 ≤ N tot i,j (k) ≤N max i,j . meaning that the flows of each user class c that enter a generic arc (i, j) of the network are the flows that actually succeed in exiting from the previous arcs plus the flows that actually manage to enter from node i if this is also an origin node o. These flows are then multiplied for βod,c i,j (k), which indicates the portion that decides to use arc (i, j) to reach destination d. j j The residual capacity of highway and railway arcs qres i,j (k) is given by qres i,j (k) = ( nmax i,j −ntot i,j (k) if (i, j) ∈AH N max i,j −N tot i,j (k) if (i, j) ∈AR (29) (29) Since we are describing the behavior of two class of users in an intermodal transport network, some parameters neces- sary to quantify the effective traffic load in the network have to be introduced, i.e., the two conversion factors ϵc n,i and ξc i,j used in (24). Starting by class c = 1, i.e., passengers, and considering that the transition between two different modes of transport can only occur in an intermodal arc, the conversion As for the intermodal arcs (i, j) ∈AIp∪AIf , being fictitious arcs, we chose to not impose bounds on the capacity. As for the intermodal arcs (i, j) ∈AIp∪AIf , being fictitious arcs, we chose to not impose bounds on the capacity. V. THE INTERMODAL DYNAMIC MODEL FOR PASSENGER AND FREIGHT FLOWS To this end let us define with ntot i,j (k) the total number of vehicles (expressed in terms of passenger car equivalents) present in arc (i, j) ∈AH at time step k and with N tot i,j (k) the total number of trains in arc (i, j) ∈AR at time step k, which are computed as follows • qod,c(k) is the number of units of class c associated with the od pair, that can actually enter the network from node o ∈JO; • qod,c(k) is the number of units of class c associated with the od pair, that can actually enter the network from node o ∈JO; d • lod,c(k) is the queue length of class c, associated with the od pair, which waits at the origin node o ∈JO. • lod,c(k) is the queue length of class c, associated with the od pair, which waits at the origin node o ∈JO. The dynamic evolution of the system is described, for each class c and for each k, with k = 0, . . . , K, by the following dynamic equation nod,c i,j (k + 1) = nod,c i,j (k) + Iod,c i,j (k) −Ood,c i,j (k) (23) (23) ntot i,j (k) = X o∈JO X d∈JD nod,1 i,j (k) + X o∈JO X d∈JD ςnod,2 i,j (k) (27) (27) for all (i, j) ∈ A, o ∈ JO, d ∈ JD. Let us now describe separately the entering flows Iod,c i,j (k) and the exiting flows Ood,c i,j (k). for all (i, j) ∈AH, where ς is a coefficient introduced in order to translate the trucks in an equivalent number of cars, and V. THE INTERMODAL DYNAMIC MODEL FOR PASSENGER AND FREIGHT FLOWS Specifically: • for class c = 1, i.e., passengers, the unit considered in railway arcs is the number of passengers, while in high- way arcs is the number of vehicles; in intermodal arcs the units can be either passengers or vehicles depending on the type of arc preceding the intermodal one; ,j Being the performance functions (18)-(20) monotonically increasing with respect to the freight flows and being the cost function (21) linear with respect to the travel times, it fol- lows that also the cost function of the arcs is monotonically increasing with respect to the freight flows. This makes the function y(·) strictly convex and defined on a convex set (14)-(16) admitting a single optimal solution with respect to the variables x2 i,j. However, also for the route choices for freight flows, the uniqueness of the solution is not guaranteed with respect to the flows on paths f od,2 l . • for class c = 2, i.e., freight, the unit considered in railway arcs is the number of railway wagons, while in highway arcs is the number of trucks; in intermodal arcs, again, the units depend on the type of preceding arc. In this paper a single cargo unit corresponds to one rail wagon and to one truck. Extending this model for considering different load capacities in road and rail modes is straightforward and omitted here only for the sake of simplicity. To overcome this issue, a problem analogous to Problem 2 can be formulated to obtain the pattern of freight flows on 73590 73590 VOLUME 10, 2022 VOLUME 10, 2022 C. Pasquale et al.: Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks factor ϵ1 n,i is given by ϵ1 n,i =    1 if (n, i) ∈AH ∪AR η if (n, i) ∈AIp and (i, j) ∈AR 1 η if (n, i) ∈AIp and (i, j) ∈AH (25) factor ϵ1 n,i is given by As mentioned in Section III, the dynamic model receives as inputs the route choices of passengers and freight, i.e., the splitting rates βod,c i,j (k). 1) ENTERING FLOWS The potential outflow Sod,c i,j (k) is computed as W tot i,j (k)=    X o∈JO X d∈JD wod,1 i,j (k)+ςwod,2 i,j (k) if (i, j)∈AH X o∈JO X d∈JD wod,1 i,j (k) Cp + wod,2 i,j (k) Cf if (i, j)∈AR Sod,c i,j (k) = T ti,j(k)nod,c i,j (k) (36) (36) (31) (31) where ti,j(k) is the transfer time required to cover arc (i, j) defined according to the following relation Hence, thanks to the residual capacity defined in (29) we can determine the percentage of excess units πi,j(k) that cannot enter at a given time step k in the arc (i, j) ti,j(k) =    1i,j Vi,j(ntot i,j (k)) if (i, j) ∈AH 1i,j Vi,j(N tot i,j (k)) if (i, j) ∈AR αi,j · T if (i, j) ∈AIp γi,j · T if (i, j) ∈AIf (37) πi,j(k) =    max{0, W tot i,j (k) −qres(k)} W tot i,j (k) if W tot i,j (k) > 0 0 otherwise (37) otherwise (32)  (32) where πi,j(k) ∈[0, 1]. Specifically, πi,j(k) is equal to 0 when W tot i,j < qres(k), i.e., when the residual capacity is sufficient to host all units that want to enter arc (i, j), while πi,j(k) is equal to 1 when qres(k) = 0, i.e., when the residual capacity is zero and all units that want to enter arc (i, j) cannot do so. For intermodal connections allowing the modal change from rail to road, the transfer time ti,j(k) is considered con- stant and equal to αi,j · T, with αi,j ≥1 for each (i, j) ∈AIp and equal to γi,j · T, with γi,j ≥1 for each arc (i, j) ∈AIf . Instead, in intermodal connections allowing the road-to-rail modal change, the transfer time is not fixed and depends on the possibility to fill trains, i.e., on (35). Having said that, the units that can effectively enter from an origin node is computed as follow qod,c(k) = Dod,c K +lod,c(k)  X n∈S(o) βod,c o,n (k) · (1 −πo,n(k)) With regard to highway and railway arcs, it should be noted that, for both types of arc, the transfer time is estimated as a function of the total number of vehicles or trains present in the connection. 1) ENTERING FLOWS Now let us analyze the receptive capacity of an arc accord- ing to the load conditions it is experiencing in a given time step. Then, let us define with wod,c i,j (k) the amount of users of VOLUME 10, 2022 73591 73591 C. Pasquale et al.: Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks class c related to the od pair who want to enter arc (i, j) in time step k only enter the rail network if they are enough to fully load at least a freight train with capacity Cf . Since the transfer of cargo units from road to rail is realized through a freight intermodal arc, we consider that this arc behaves as a buffer where cargo units wait until their number is sufficient to fill at least one train and then leave the intermodal arc. The number of cargo units possibly leaving a road-to-rail intermodal arc (i, j) ∈AIf is then given by wod,c i,j (k) = βod,c i,j (k)  X n∈P(i) ϵc n,i · Sod,c n,i (k) + ξc i,j · Dod,c K + lod,c(k)  (30) + ξc i,j · Dod,c K + lod,c(k)  (30) (30) (30) In (30), wod,c i,j (k) includes the potential outflow from the previous arcs Sod,c n,i (k), better detailed in Section V-2, and the demand associated with the pair od and the eventual queue length at the node i if it coincides with the origin o. Sod,2 i,j (k) = nod,2 i,j (k) Cf  Cf (35) (35) g g Therefore, the total amount of units that potentially enter arc (i, j) at time step k is given by: Now let us discuss the potential outflow for all classes c for the arcs (i, j) ∈AH ∪AR, for passengers in arcs (i, j) ∈AIp and for freight in rail-to-road intermodal arcs (i, j) ∈AIf . 1) ENTERING FLOWS More in details, for each highway arc (i, j) ∈AH, the transfer time ti,j(k) is computed according to the current traffic conditions through the steady-state relation between speed and number of vehicles given by (33) whereas the relative queue of units at the origin node o is given by lod,c(k + 1) = lod,c(k) + Dod,c K −qod,c(k) (34) (34) VI. RESULTS AND DISCUSSION The focus of this section is to show the potential benefits that can be obtained by adopting the proposed multi-class intermodal modeling scheme, simulating a perturbation on the network and then analyzing how this event can affect the rest of the network. Specifically, the considered perturbation is the failure of a connection in the intermodal network. The experimental tests have been performed in two distinct scenarios: • Scenario pre-disruption: network at the equilibrium before the advent of the disruption; TABLE 5. Main parameters referred to railway arcs. TABLE 5. Main parameters referred to railway arcs. • Scenario post-disruption: network once a new equilib- rium is reached some time after the initial perturbation. The results have been obtained by adopting a test network derived from the well-known Nguyen-Dupuis network prop- erly modified to consider the intermodal case (for further information see [44]). This network is composed of 14 nodes and 21 arcs, as depicted in Fig. 3. The critical event is simu- lated considering the total loss of functionality of the railway arc 12-8. simulation has been set equal to one hour, that corresponds to K = 60 time steps. The transportation demand is expressed with two origin-destination matrices, one referring to passen- gers, as shown in Table 6, and one referring to freight, as indi- cated in Table 7. The mobility demand of both passengers and freight is assumed to be perfectly inelastic and therefore it is the same in the two tested scenarios. The main parameters of the highway and railway arcs are reported in Table 4 and Table 5, respectively. The other parameters have been set as follows: the conversion factor η, representing the average number of passengers per car, is equal to 1.45, the congestion wave speed ωi,j is equal to 30 [km/h], ∀(i, j) ∈AH, the average time headway hi,j is 15 minutes and the minimum average space headway smin i,j is equal to 2 [km], ∀(i, j) ∈AR, while the constant parameter αi,j has been set equal to 15, ∀(i, j) ∈AIp and γi,j equal to 30, ∀(i, j) ∈AIf . The capacity of a freight train Cf is 25 rail wagons, while the capacity of a passenger train Cp is chosen equal to 700 passengers. 2) EXITING FLOWS TABLE 4. Main parameters referred to highway arcs. TABLE 5 Main parameters referred to railway arcs Ood,c i,j (k) = Sod,c i,j (k) X n∈S(j) βod,c j,n (k) 1 −πj,n(k)  (40) (40) VI. RESULTS AND DISCUSSION Note finally that, in both scenarios described below, the splitting rates βod,c i,j (k) computed by the intermodal traffic assignment are constant throughout the simulation period, and, then, k is omitted for the sake of simplicity, i.e., βod,c i,j (k) = βod,c i,j . 2) EXITING FLOWS Vi,j(ntot i,j (k)) = min  vH i,j, wi,j ntot i,j (k)1i,j nmax i,j 1i,j − ntot i,j (k) 1i,j  (38) Even with respect to outflows from an arc, these are calcu- lated based on the residual capacity of the arcs they wish to enter. For this reason, we distinguish Ood,c i,j (k), i.e., the units actually exiting arc (i, j), from Sod,c i,j (k), i.e., the units that would like to exit arc (i, j) at time step k. (38) Relation (38) has been derived from a triangular funda- mental diagram, as the one proposed in [43], and expressed in terms of number of vehicles. With regard to railway arcs, the steady-state speed relation Vi,j(N tot i,j (k)) for all (i, j) ∈AR Let us start by describing the relation that defines the potential outflow from an intermodal freight arc (i, j) ∈AIf preceded by a highway arc. We assume that cargo units can 73592 73592 VOLUME 10, 2022 C. Pasquale et al.: Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks FIGURE 3. Sketch of the test intermodal transport network. may be formulated as Vi,j(N tot i,j (k)) =    vR i,j if N tot i,j (k) 1i,j ≤ 1 hi,jvR i,j + L 1 hi,j  1i,j N tot i,j (k) −L  if 1 hi,jvR i,j + L < N tot i,j (k) 1i,j ≤ 1 smin i,j (39) may be formulated as may be formulated as Vi,j(N tot i,j (k)) =    vR i,j if N tot i,j (k) 1i,j ≤ 1 hi,jvR i,j + L 1 hi,j  1i,j N tot i,j (k) −L  if 1 hi,jvR i,j + L < N tot i,j (k) 1i,j ≤ 1 smin i,j (39) (39) It is worth noting that, for each highway or railway arc, condition (1) with (38) and (39) implies that the transfer time ti,j(k) is never lower than T, ensuring the validity of the conservation equations. For further details concerning the development of this relationship, please refer to Appendix. d p p p pp Finally, given Sod,c i,j (k) and πi,j(k), we can compute the outflow Ood,c i,j (k) which represent the units of class c referred to the pair od that actually exit the arc (i, j) as FIGURE 3. Sketch of the test intermodal transport network. TABLE 4. Main parameters referred to highway arcs. A. PERFORMANCE INDICATORS Let us begin with the Total Travel Time, that is an indicator that computes the total time spent by each unit, both of passengers and freight type, in a connection, considering that an arc can belong to multiple paths at the same time. This indicator refers to each arc (i, j) ∈A and is calculated as follows: • od pair 1-2: β12,1 1,12 = 1, β12,1 5,6 = 1, β12,1 6,7 = 1,β12,1 7,11 = 1, β12,1 8,2 = 1, β12,1 11,2 = 1, β12,1 12,8 = 1; • od pair 1-3: β13,1 1,5 = 0.92, β13,1 1,12 = 0.92, β13,1 5,6 = 0.36, β13,1 5,9 = 0.64, β13,1 6,7 = 1, β13,1 7,11 = 1, β13,1 9,10 = 0.12,β13,1 9,14 = 1, β13,1 10,11 = 1, β13,1 11,3 = 1, β13,1 12,6 = 1, β13,1 13,3 = 1, β13,1 14,3 = 1; • od pair 4-2: β42,1 4,5 = 0.62, β42,1 4,9 = 0.38, β42,1 5,6 = 0.75, β42,1 5,9 = 0.25, β42,1 6,7 = 0.67, β42,1 6,10 = 0.33, β42,1 7,11 = 1, β42,1 9,10 = 1, β42,1 10,11 = 1, β42,1 11,2 = 1; • od pair 4-3: β43,1 4,5 = 0.18, β43,1 4,9 = 0.82, β43,1 5,6 = 1, β43,1 6,7 = 1, β43,1 7,11 = 1, β43,1 9,10 = 0.28, β43,1 9,14 = 0.72, β43,1 10,11 = 1, β43,1 11,3 = 1, β43,1 13,3 = 1, β43,1 14,13 = 1. TTTi,j = T K X k=1 2 X c=1 X o∈JO X d∈JD nod,c i,j (k) (41) (41) • od pair 4-2: β42,1 4,5 = 0.62, β42,1 4,9 = 0.38, β42,1 5,6 = 0.75, β42,1 5,9 = 0.25, β42,1 6,7 = 0.67, β42,1 6,10 = 0.33, β42,1 7,11 = 1, β42,1 9,10 = 1, β42,1 10,11 = 1, β42,1 11,2 = 1; • od pair 4-3: β43,1 4,5 = 0.18, β43,1 4,9 = 0.82, β43,1 5,6 = 1, β43,1 6,7 = 1, β43,1 7,11 = 1, β43,1 9,10 = 0.28, β43,1 9,14 = 0.72, β43,1 10,11 = 1, β43,1 11,3 = 1, β43,1 13,3 = 1, β43,1 14,13 = 1. B. SCENARIO PRE-DISRUPTION It should be noted that among these routes, only one is of intermodal type, with a change from road to rail, one route is entirely by railway, while the remaining ones involve only the use of highway arcs. The methodology presented in Section IV has been used to allocate the passengers demand on possible routes and the resulting assignment is shown in Fig. 4. It is worth reminding that the feasible paths are those that have at most one modal shift. The paths used at the equilibrium, before the disruption, are reported in Table 8: one path adopts only the rail mode, five are highway routes, while the remaining eight require the use of both modes of transport. A. PERFORMANCE INDICATORS The Mean Arc Occupancy is an indicator for assessing the average occupancy of an arc, either road or rail, given the total number of vehicles or trains occupying it on average, therefore the Mean Arc Occupancy is given by MAOi,j =    1 K XK k=1 ntot i,j (k) if (i, j) ∈AH 1 K XK k=1 N tot i,j (k) if (i, j) ∈AR (42) (42) , , , , The freight flows are fixed and their distribution is shown again in Fig. 4, while the paths are reported in Table 9. The corresponding non-zero splitting rates, again considered constant throughout the simulation, are the following: Finally, the Mean Arc Saturation relates the Mean Arc Occupancy to the capacity of an arc, i.e. Finally, the Mean Arc Saturation relates the Mean Arc Occupancy to the capacity of an arc, i.e. • od pair 1-2: β12,2 1,12 = 1, β12,2 8,2 = 1, β12,2 12,8 = 1; • od pair 1-3: β13,2 1,5 = 1, β13,2 5,9 = 1, β13,2 9,14 = 1, β13,2 13,3 = 1, β13,2 14,13 = 1; MASi,j =    MAOi,j nmax i,j · 100 if (i, j) ∈AH MAOi,j N max i,j · 100 if (i, j) ∈AR (43) (43) • od pair 4-2: β42,2 4,5 = 0.75, β42,2 4,9 = 0.24 β42,2 5,6 = 0.17, β42,2 5,9 = 0.82 β42,2 6,7 = 1, β42,2 7,11 = 1, β42,2 11,2 = 1, β42,2 9,10 = 1, β42,2 10,11 = 1; Note that the Mean Arc Occupancy and Mean Arc Satura- tion indicators are calculated for highway and rail arcs only, as for intermodal arcs we assume no capacity restrictions. , • od pair 4-3: β43,2 4,9 = 0.21, β43,2 4,5 = 0.78, β43,2 5,9 = 1, β43,2 9,14 = 1, β43,2 13,3 = 1, β43,2 14,13 = 1. A. PERFORMANCE INDICATORS • od pair 1-2: β12,1 1,12 = 1, β12,1 5,6 = 1, β12,1 6,7 = 1,β12,1 7,11 = 1, β12,1 8,2 = 1, β12,1 11,2 = 1, β12,1 12,8 = 1; • od pair 1-3: β13,1 1,5 = 0.92, β13,1 1,12 = 0.92, β13,1 5,6 = 0.36, β13,1 5,9 = 0.64, β13,1 6,7 = 1, β13,1 7,11 = 1, β13,1 9,10 = 0.12,β13,1 9,14 = 1, β13,1 10,11 = 1, β13,1 11,3 = 1, β13,1 12,6 = 1, β13,1 13,3 = 1, β13,1 14,3 = 1; • od pair 4-2: β42,1 4,5 = 0.62, β42,1 4,9 = 0.38, β42,1 5,6 = 0.75, β42,1 5,9 = 0.25, β42,1 6,7 = 0.67, β42,1 6,10 = 0.33, β42,1 7,11 = 1, β42,1 9,10 = 1, β42,1 10,11 = 1, β42,1 11,2 = 1; • od pair 4-3: β43,1 4,5 = 0.18, β43,1 4,9 = 0.82, β43,1 5,6 = 1, β43,1 6,7 = 1, β43,1 7,11 = 1, β43,1 9,10 = 0.28, β43,1 9,14 = 0.72, β43,1 10,11 = 1, β43,1 11,3 = 1, β43,1 13,3 = 1, β43,1 14,13 = 1. TABLE 6. Passengers origin-destination matrix. TABLE 7. Freight origin-destination matrix. TABLE 8. Paths used by passengers in the pre-disruption scenario. TABLE 8. Paths used by passengers in the pre-disruption scenario. TABLE 9 Paths used by freight in the pre disruption scenario TABLE 6. Passengers origin-destination matrix. TABLE 7. Freight origin-destination matrix. TABLE 6. Passengers origin-destination matrix. TABLE 6. Passengers origin-destination matrix. TABLE 7. Freight origin-destination matrix. TABLE 9. Paths used by freight in the pre-disruption scenario. have been developed using some of the dynamic variables introduced in Section V to describe the behavior of the system over time and space. Specifically, the proposed indicators are the Total Travel Time, the Mean Arc Occupancy, and the Mean Arc Saturation. Let us begin with the Total Travel Time, that is an indicator that computes the total time spent by each unit, both of passengers and freight type, in a connection, considering that an arc can belong to multiple paths at the same time. This indicator refers to each arc (i, j) ∈A and is calculated as follows: have been developed using some of the dynamic variables introduced in Section V to describe the behavior of the system over time and space. Specifically, the proposed indicators are the Total Travel Time, the Mean Arc Occupancy, and the Mean Arc Saturation. A. PERFORMANCE INDICATORS As for the dynamic model, a sample time T equal to one minute has been adopted, while the time horizon of the In order to evaluate the effects of the mobility demand on the intermodal transport network, some performance indicators VOLUME 10, 2022 73593 73593 C. Pasquale et al.: Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks TABLE 8. Paths used by passengers in the pre-disruption scenario. TABLE 9. Paths used by freight in the pre-disruption scenario. • od pair 1-2: β12,1 1,12 = 1, β12,1 5,6 = 1, β12,1 6,7 = 1,β12,1 7,11 = 1, β12,1 8,2 = 1, β12,1 11,2 = 1, β12,1 12,8 = 1; • od pair 1-3: β13,1 1,5 = 0.92, β13,1 1,12 = 0.92, β13,1 5,6 = 0.36, β13,1 5,9 = 0.64, β13,1 6,7 = 1, β13,1 7,11 = 1, β13,1 9,10 = 0.12,β13,1 9,14 = 1, β13,1 10,11 = 1, β13,1 11,3 = 1, β13,1 12,6 = 1, β13,1 13,3 = 1, β13,1 14,3 = 1; • od pair 4-2: β42,1 4,5 = 0.62, β42,1 4,9 = 0.38, β42,1 5,6 = 0.75, β42,1 5,9 = 0.25, β42,1 6,7 = 0.67, β42,1 6,10 = 0.33, β42,1 7,11 = 1, β42,1 9,10 = 1, β42,1 10,11 = 1, β42,1 11,2 = 1; • od pair 4-3: β43,1 4,5 = 0.18, β43,1 4,9 = 0.82, β43,1 5,6 = 1, β43,1 6,7 = 1, β43,1 7,11 = 1, β43,1 9,10 = 0.28, β43,1 9,14 = 0.72, β43,1 10 11 = 1, β43,1 11 3 = 1, β43,1 13 3 = 1, β43,1 14 13 = 1. TABLE 8. Paths used by passengers in the pre-disruption scenario. TABLE 9. Paths used by freight in the pre-disruption scenario. C. SCENARIO POST-DISRUPTION As mentioned earlier, the disruption is represented in this example by removing the railway arc 12-8. Regarding passen- gers, the new path configuration for each origin-destination The non-zero splitting rates are in this case: 73594 VOLUME 10, 2022 C. Pasquale et al.: Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks FIGURE 4. Pre-disruption equilibrium. TABLE 10. Paths used by passengers after the disruption. FIGURE 4. Pre-disruption equilibrium. TABLE 10. Paths used by passengers after the disruption. TABLE 11. Paths used by freight after the disruption. FIGURE 5. Post-disruption equilibrium. TABLE 11. Paths used by freight after the disruption. FIGURE 5. Post-disruption equilibrium. FIGURE 4. Pre-disruption equilibrium. FIGURE 4. Pre-disruption equilibrium. FIGURE 4. Pre-disruption equilibrium. TABLE 10. Paths used by passengers after the disruption. TABLE 10. Paths used by passengers after the disruption. FIGURE 5. Post-disruption equilibrium. pair is shown in Table 10. As can be seen, the railway path [1-12-8-2] no longer appears since it includes the damaged arc. As shown in Fig. 5, there is a shift of od pair 1-2 flows and, as a consequence, a greater load on the central arcs of the network, in particular on path [1-12-6-7-8-2]. On the other hand, not surprisingly, the southern paths in the network for pair 4-3 are the least perturbed as testified by the fact that the flows do not undergo excessive redistribution, meaning that for those users the most attractive routes have remained the same. The splitting rates for passenger flows are the following: As far as freight flows are concerned, similar considera- tions can be made, since the railway path can no longer be used and the freight flow of od pair 1-2 is reassigned to the remaining path, as reported in Table 11 with the resulting assignment shown in Fig. 5. C. SCENARIO POST-DISRUPTION The corresponding non-zero splitting rates are as follows: • od pair 1-2: β12,2 1,12 = 1, β12,2 6,7 = 0.83, β12,2 6,10 = 0.16, β12,2 6,7 = 1, β12,2 10,11 = 1, β12,2 11,2 = 1, β12,2 12,6 = 1; • od pair 1-3: β13,2 1,5 = 1, β13,2 5,9 = 1, β13,2 9,14 = 1, β13,2 13,3 = 1, β13,2 14,13 = 1; • od pair 4-2: β42,2 4,5 = 0.65, β42,2 4,9 = 0.35, β42,2 5,9 = 1, β42,2 6,7 = 1,β42,2 7,11 = 1 β42,2 9,10 = 1, β42,2 10,11 = 1, β42,2 11,2 = 1; • od pair 4-3: β43,2 4,5 = 0.89, β43,2 4,9 = 0.11, β43,2 5,9 = 1, • od pair 1-2: β12,2 1,12 = 1, β12,2 6,7 = 0.83, β12,2 6,10 = 0.16, β12,2 6,7 = 1, β12,2 10,11 = 1, β12,2 11,2 = 1, β12,2 12,6 = 1; • od pair 1-3: β13,2 1,5 = 1, β13,2 5,9 = 1, β13,2 9,14 = 1, β13,2 13,3 = 1, β13,2 14,13 = 1; • od pair 4-2: β42,2 4 5 = 0 65 β42,2 4 9 = 0 35 β42,2 5 9 = 1 • od pair 1-2: β12,2 1,12 = 1, β12,2 6,7 = 0.83, β12,2 6,10 = 0.16, β12,2 6,7 = 1, β12,2 10,11 = 1, β12,2 11,2 = 1, β12,2 12,6 = 1; 13 2 13 2 13 2 13 2 • od pair 1-3: β13,2 1,5 = 1, β13,2 5,9 = 1, β13,2 9,14 = 1, β13,2 13,3 = 1, β13,2 14,13 = 1; g • od pair 1-2: β12,1 1,5 = 0.41, β12,1 1,12 = 0.59, β12,1 5,6 = 0.85, β12,1 5,9 = 0.15, β12,1 6,7 = 0.69, β12,1 6,10 = 0.31, β12,1 7,8 = 0.27, β12,1 7,11 = 0.73, β12,1 8,2 = 1, β12,1 9,10 = 1, β12,1 10,11 = 1, β12,1 11,2 = 1, β12,1 12,6 = 1; • od pair 1-3: β13,1 1,5 = 0.52, β13,1 1,12 = 0.48, β13,1 5,6 = 0.1, β13,1 5,9 = 0.9, β13,1 6,7 = 0.65, β13,1 6,10 = 0.35, β13,1 7,11 = 1, β13,1 9,14 = 1, β13,1 10,11 = 1, β13,1 11,3 = 1, β13,1 12,6 = 1, β13,1 13,3 = 1, β13,1 14,3 = 1; 42 1 42 1 42 1 • od pair 1-2: β12,1 1,5 = 0.41, β12,1 1,12 = 0.59, β12,1 5,6 = 0.85, β12,1 5,9 = 0.15, β12,1 6,7 = 0.69, β12,1 6,10 = 0.31, β12,1 7,8 = 0.27, β12,1 7,11 = 0.73, β12,1 8,2 = 1, β12,1 9,10 = 1, β12,1 10,11 = 1, β12,1 11,2 = 1, β12,1 12,6 = 1; • od pair 1-3: β13,1 1,5 = 0.52, β13,1 1,12 = 0.48, β13,1 5,6 = 0.1, β13,1 5,9 = 0.9, β13,1 6,7 = 0.65, β13,1 6,10 = 0.35, β13,1 7,11 = 1, β13,1 9,14 = 1, β13,1 10,11 = 1, β13,1 11,3 = 1, β13,1 12,6 = 1, β13,1 13,3 = 1, β13,1 14 3 = 1; • od pair 1-2: β12,1 1,5 = 0.41, β12,1 1,12 = 0.59, β12,1 5,6 = 0.85, β12,1 5,9 = 0.15, β12,1 6,7 = 0.69, β12,1 6,10 = 0.31, β12,1 7,8 = 0.27, β12,1 7,11 = 0.73, β12,1 8,2 = 1, β12,1 9,10 = 1, β12,1 10,11 = 1, β12,1 11,2 = 1, β12,1 12,6 = 1; 13 1 13 1 13 1 • od pair 4-2: β42,2 4,5 = 0.65, β42,2 4,9 = 0.35, β42,2 5,9 = 1, β42,2 6,7 = 1,β42,2 7,11 = 1 β42,2 9,10 = 1, β42,2 10,11 = 1, β42,2 11,2 = 1; • od pair 4-3: β43,2 4,5 = 0.89, β43,2 4,9 = 0.11, β43,2 5,9 = 1, β43,2 9,14 = 1, β43,2 13,3 = 1, β43,2 14,13 = 1. VOLUME 10, 2022 C. SCENARIO POST-DISRUPTION The deviation from the unperturbed case is not reported for this indicator because, being derived from the Mean Arc Occupancy, this indicator has the same variations. As can be seen the disruption of 12-8 arc implies that TABLE 13. The Mean Arc Occupancy for highway and railway arcs. FIGURE 7. Mean Arc Occupancy for some of the most affected highway arcs. Time, by means of the dynamic model presented in this pap allows therefore to identify the elements of the network th will be more stressed after the perturbation. Analyzing Tables 13 and 14, it is possible to observe th the disruptive event has effects on Mean Arc Occupancy an FIGURE 6. Total Travel Time absolute variations. FIGURE 6. Total Travel Time absolute variations. TABLE 13. The Mean Arc Occupancy for highway and railway arcs. FIGURE 6. Total Travel Time absolute variations. TABLE 12. The Total Travel Time for each arc. TABLE 12. The Total Travel Time for each arc. TABLE 12. The Total Travel Time for each arc. FIGURE 7. Mean Arc Occupancy for some of the most affected highway arcs. the values of the Mean Arc Saturation for each roadway and railway arc, in the pre-disruption scenario, i.e., MASE i,j, and in the post-disruption scenario, i.e., MASD i,j. The deviation from the unperturbed case is not reported for this indicator because, being derived from the Mean Arc Occupancy, this indicator has the same variations. Time, by means of the dynamic model presented in this paper, allows therefore to identify the elements of the network that will be more stressed after the perturbation. Analyzing Tables 13 and 14, it is possible to observe that the disruptive event has effects on Mean Arc Occupancy and Mean Arc Saturation even for arcs that are not connected with the collapsed one. In addition, it can be seen that the disturbance affects not only the railway arcs but also the high- way arcs, as highlighted in Fig. 7. Referring to the highway arcs, the most significant change in the average occupancy evaluated in a time interval of one minute is experienced for arc 6-10 which approximately doubles this indicator. As can be seen, the disruption of 12-8 arc implies that freight and passenger flows, which previously used the peripheral rail route, are re-assigned in more internal routes, particularly intermodal routes. C. SCENARIO POST-DISRUPTION Table 12 shows, for each arc, TTT E i,j, i.e., the Total Travel Table 12 shows, for each arc, TTT E i,j, i.e., the Total Travel Time computed in the pre-disruption scenario, TTT D i,j, i.e., the Total Travel Time in the post-disruption scenario, and 1TTTi,j, i.e., the absolute deviations of the same metric. Analogously, Fig. 6 shows, for each arc, the Total Travel Time percent variation by exhibiting the effects of disruption more clearly. Table 13 shows instead the values of the Mean Arc Occupancy for each roadway and railway arc, specifically MAOE i,j is the Mean Arc Occupancy computed in the pre- disruption scenario, MAOD i,j is the Mean Arc Occupancy in the post-disruption scenario, and 1MAOi,j is the variation of MAOD i,j with respect to MAOE i,j. Fig. 7 depicts the Mean Arc Occupancy in pre- and post-disruptive scenarios for some of the most affected highway arcs. Finally, Table 14 displays • od pair 1-3: β13,1 1,5 = 0.52, β13,1 1,12 = 0.48, β13,1 5,6 = 0.1, β13,1 5,9 = 0.9, β13,1 6,7 = 0.65, β13,1 6,10 = 0.35, β13,1 7,11 = 1, β13,1 9,14 = 1, β13,1 10,11 = 1, β13,1 11,3 = 1, β13,1 12,6 = 1, β13,1 13,3 = 1, β13,1 14,3 = 1; , • od pair 4-2: β42,1 4,5 = 0.57, β42,1 4,9 = 0.43, β42,1 5,6 = 0.8, β42,1 5,9 = 0.2, β42,1 6,7 = 0.78, β42,1 6,10 = 0.22, β42,1 7,8 = 0.54, β42,1 7,11 = 0.46, β42,1 8,2 = 1, β42,1 9,10 = 1, β42,1 10,11 = 1, β42,1 11,2 = 1; , • od pair 4-3: β43,1 4,5 = 0.19, β43,1 4,9 = 0.81, β43,1 5,9 = 1, β43,1 6,10 = 1, β43,1 9,10 = 0.22, β43,1 9,14 = 0.78, β43,1 10,11 = 1, β43,1 11,3 = 1, β43,1 13,3 = 1, β43,1 14,13 = 1. β43,1 11,3 = 1, β43,1 13,3 = 1, β43,1 14,13 = 1. 73595 C. Pasquale et al.: Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks FIGURE 6. Total Travel Time absolute variations. TABLE 12. The Total Travel Time for each arc. the values of the Mean Arc Saturation for each roadway and railway arc, in the pre-disruption scenario, i.e., MASE i,j, and in the post-disruption scenario, i.e., MASD i,j. VII. CONCLUSION In this paper, a two-stage modeling approach is proposed to represent passenger and freight flows on a intermodal trans- portation network, i.e., a network in which there are road- ways, railways, and connections that allow the modal shifts. The modeling scheme consists of an assignment model and a discrete-time dynamic model. The assignment model allows to represent the choices of the users, both passengers and freight, in terms of routes and transport modes. These route choices provide the input to the dynamic model that allows to represent the evolution in time and space of user flows, allowing to evaluate some dynamic characteristics such as speed and travel time on the network arcs. In order to provide some insights about the proposed approach, this concluding section has been structured into four parts, in order to further discuss the results produced in this paper, to illustrate some possible applications of the proposed modeling framework for scenario evaluation, on the one hand, and for testing regulatory policies and control actions, on the other hand, and, finally, to argue about the possible weaknesses of this methodology. Scenarios of particular relevance are those concerning the occurrence of critical events. Indeed, large transport networks are anyway susceptible to critical events that may have severe implications on the whole activity system of a territory. These disruptive events may be caused by natural phenomena (such as floods, earthquakes, pandemics, etc.) or anthropogenic causes (such as terrorist attacks, infrastructure failures, but also planned maintenance works): whatever the cause, they may affect the transport network as a variation of mobility demand or as events that partially or totally deteriorate the capacity of a transport network. In this regard, a topic that is attracting particular attention in scientific research is the eval- uation of the resilience of a transport network, i.e., its ability to resist, adapt or change in order to maintain acceptable per- formance in case of critical events. Although the concept of resilience has been initially introduced to describe a property of natural systems [45], recently it has been applied to trans- port networks and in particular to road networks [46]–[48] and railway networks [49]–[51]. However, as shown in the case study presented in this paper, transport networks are complex and highly interdependent systems and a critical event affecting one mode of transport can have an impact on other modes giving rise to a chain effect. VII. CONCLUSION What is lacking in the literature, and what this paper has aimed to address, is the possibility of using a tool that allows to quantify the effects of these interdependencies and to evaluate the ability of a transport network to maintain acceptable performance even when it is affected by disruptions. C. SCENARIO POST-DISRUPTION analysis than the one which can be conducted using modeling frameworks that contemplate a single mode of transport only. analysis than the one which can be conducted using modeling frameworks that contemplate a single mode of transport only. 1) DISCUSSION OF THE PROPOSED TEST CASE The objective of the proposed case study has been to illustrate the benefits of the proposed intermodal modeling scheme by analyzing the behavior of a network subject to a disruptive event, specifically the loss of functionality of a railway arc. This application revealed the ability of the intermodal model to capture the ripple effects of such events that cannot be gained if analyzed with models representing a single trans- portation mode only. 3) POSSIBLE APPLICATIONS FOR DEVELOPMENT AND TESTING OF REGULATORY POLICIES AND CONTROL ACTIONS C. SCENARIO POST-DISRUPTION Consistently with an overall load increase on the central arcs of the network, it is possible to observe a shift, though small, of flows of the 4-3 pair in favor of the outermost intermodal route [4-9-14-13-3]. Not surprisingly, the arcs relatively most affected by the pertur- bation are those in proximity to the disrupted arc, such as arcs 12-6, 6-7 and 7-8. However, it can be seen that even the central arc 6-10 experiences a 30% increase in Total Travel Time. The computation of a metric such as the Total Travel From the analysis of these results, it can then be concluded that the adoption of a modeling framework of this type per- mits to grasp the interdependencies that exist between the road and rail modes of transport, enabling a more accurate 73596 VOLUME 10, 2022 VOLUME 10, 2022 C. Pasquale et al.: Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks TABLE 14. The Mean Arc Saturation for highway and railway arcs. analysis than the one which can be conducted using modeling frameworks that contemplate a single mode of transport only. TABLE 14. The Mean Arc Saturation for highway and railway arcs. collapsed arc. Specifically, some arcs, both of road and rail type, have almost doubled in volume, making them obvi- ously more critical to any further disruption. In addition, the post-disruption scenario showed a significant increase in the use of intermodal arcs, both for passengers and freight, suggesting to a possible decision maker that the adoption of more connections of this type can make the transport network more robust to the occurrence of unexpected events. 2) POSSIBLE APPLICATIONS FOR SCENARIO EVALUATION The proposed model can be adopted to evaluate the effects of the decisions of the users on a transport network. These effects can be quantified through the adoption of specific per- formance indexes, such as those introduced above, or through the development of other indicators. For example, through the evaluation of the Mean Arc Saturation index, a preliminary analysis can be conducted to identify, under specific mobility demand conditions, which arcs are closest to saturation and assess the effects of their failure. Furthermore, this model can also be used to assess the sustainability of user choices by integrating this modeling scheme with models that estimate emissions or energy consumptions. 3) POSSIBLE APPLICATIONS FOR DEVELOPMENT AND TESTING OF REGULATORY POLICIES AND CONTROL ACTIONS The analysis showed that the collapse of a railway arc can bring a substantial increase in travel time for some highway arcs, which can reach a 30-40% increase, and an even greater growth in the average occupancy of highway and railway arcs that are not directly connected with the The modeling framework proposed in this paper may consti- tute the basis for regulation and control approaches finalized VOLUME 10, 2022 73597 C. Pasquale et al.: Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks FIGURE 8. Triangular fundamental diagram for railway traffic. at defining routing and modal indications to be provided to the users. First of all, this model can be used to provide more detailed information to users about travel times or route choices for improving sustainability. Moreover, specific routing instructions can be defined for the users and, since the modeling scheme is multi-class, such instructions can be suitably defined for each class of users. The proposed two-stage modeling framework may be adopted to test control policies that aim at fully exploiting the mobility capacity of a large-scale intermodal transport network by suggesting routes that may include one or more transport modes. This can be done both in nominal conditions of the network or when the transport system is affected by an event which changes its structure or the mobility demand. FIGURE 8. Triangular fundamental diagram for railway traffic. g y Finally, this approach can be used to develop online jour- ney re-planning strategies such as the one developed in [52] for the re-routing of freight during disruptive events. FIGURE 9. Steady-state speed-headway functions for railway traffic. 4) POSSIBLE WEAKNESSES OF THE PROPOSED METHODOLOGY The major weakness concerning the proposed methodology is related to the collection and elaboration of data that must describe both the transport supply, i.e., the infrastructural and technical characteristics of the intermodal network, and the mobility demand that must be expressed both in terms of origin/destination matrix, to represent the willingness of freight and passengers to move, and punctual measure- ments on the network for the validation of the modeling scheme. This phase is particularly challenging because, since a large-scale intermodal network is considered, the involve- ment of different actors over a large territory is required and, then, it is necessary to integrate data from different, and pos- sibly heterogeneous, data sources, as thoroughly discussed in [53]. FIGURE 9. Steady-state speed-headway functions for railway traffic. fundamental diagram in the railway context can be defined as follows REFERENCES [1] E. Cascetta, Transportation Systems Analysis: Models and Applications. Cham, Switzerland: Springer, 2009. [26] X. Pi, W. Ma, and Z. Qian, ‘‘A general formulation for multi-modal dynamic traffic assignment considering multi-class vehicles, public transit and parking,’’ Transp. Res. Proc., vol. 38, pp. 914–934, Jan. 2019. [2] S. P. Hoogendoorn and P. H. L. Bovy, ‘‘State-of-the-art of vehicular traffic flow modeling,’’ Proc. Inst. Mech. Eng., I, J. Syst. Control Eng., vol. 215, 283–303, Jun. 2001. [27] A. Loder, L. Ambühl, M. Menendez, and K. W. Axhausen, ‘‘Empirics of multi-modal traffic networks—Using the 3D macroscopic fundamental diagram,’’ Transp. Res. C, Emerg. Technol., vol. 82, pp. 88–101, Sep. 2017. [3] A. Gille, M. Klemenz, and T. Siefer, ‘‘Applying multiscaling analysis to detect capacity resources in railway networks,’’ WIT Trans. 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Thomas, ‘‘Modelling a rail/road intermodal transportation system,’’ Transp. Res. E, Logistics Transp. Rev., vol. 40, no. 3, pp. 255–270, May 2004. [39] M. Borchers, P. Breeuwsma, W. Kern, J. Slootbeek, G. Still, and W. Tibben, ‘‘Traffic user equilibrium and proportionality,’’ Transp. Res. B, Methodol., vol. 79, pp. 149–160, Sep. 2015. [14] K. Sörensen, C. Vanovermeire, and S. Busschaert, ‘‘Efficient metaheuris- tics to solve the intermodal terminal location problem,’’ Comput. Oper. Res., vol. 39, no. 9, pp. 2079–2090, Sep. 2012. [40] T. F. Rossi, S. McNeil, and C. REFERENCES Hendrickson, ‘‘Entropy model for consistent impact-fee assessment,’’ J. Urban Planning Develop., vol. 115, no. 2, pp. 51–63, Sep. 1989. [15] M. Oudani, ‘‘A simulated annealing algorithm for intermodal trans- portation on incomplete networks,’’ Appl. Sci., vol. 11, no. 10, p. 4467, May 2021. [41] C. Caballini, C. Pasquale, S. Sacone, and S. Siri, ‘‘An event-triggered receding-horizon scheme for planning rail operations in maritime ter- minals,’’ IEEE Trans. Intell. Transp. Syst., vol. 15, no. 1, pp. 365–375, Feb. 2014. [16] E. Demir, M. Hrusovsky, W. Jammernegg, and T. Van Woensel, ‘‘Green intermodal freight transportation: Bi-objective modelling and analysis,’’ Int. J. Prod. Res., vol. 57, pp. 6162–6180, Oct. 2019. [42] C. Caballini, S. Fioribello, S. Sacone, and S. Siri, ‘‘An MILP optimization problem for sizing port rail networks and planning shunting operations in container terminals,’’ IEEE Trans. Autom. Sci. Eng., vol. 13, no. 4, pp. 1492–1503, Oct. 2016. [17] I. C. Bilegan, T. G. Crainic, and Y. Wang, ‘‘Scheduled service network design with revenue management considerations and an intermodal barge transportation illustration,’’ Eur. J. Oper. Res., vol. 300, no. 1, pp. 164–177, Jul. 2022. [43] C. F. Daganzo, ‘‘The cell transmission model: A dynamic representation of highway traffic consistent with the hydrodynamic theory,’’ Transp. Res. B, Methodol., vol. 28, no. 4, pp. 269–287, Aug. 1994. [18] T.-S. Chang, ‘‘Best routes selection in international intermodal networks,’’ Comput. Oper. Res., vol. 35, no. 9, pp. 2877–2891, Sep. 2008. [44] S. Nguyen and C. Dupuis, ‘‘An efficient method for computing traffic equilibria in networks with asymmetric transportation costs,’’ Transp. Sci., vol. 18, no. 2, pp. 185–202, May 1984. [19] C. Shao, H. Wang, and M. Yu, ‘‘Multi-objective optimization of customer- centered intermodal freight routing problem based on the combination of DRSA and NSGA-III,’’ Sustainability, vol. 14, no. 5, p. 2985, Mar. 2022. [45] C. S. Holling, ‘‘Resilience and stability of ecological systems,’’ Annu. Rev. Ecol. Systematics, vol. 4, no. 1, pp. 1–23, Nov. 1973. [20] S. C. Dafermos, ‘‘Integrated equilibrium flow models for transportation planning,’’ in Traffic Equilibrium Methods. Berlin, Germany: Springer, 1976, pp. 106–118. [46] P. Gauthier, A. Furno, and N.-E. El Faouzi, ‘‘Road network resilience: How to identify critical links subject to day-to-day disruptions,’’ Transp. Res. Rec., J. Transp. Res. Board, vol. 2672, no. 1, pp. 54–65, Dec. 2018. [21] M. Florian, ‘‘A traffic equilibrium model of travel by car and public transit modes,’’ Transp. Sci., vol. 11, no. 2, pp. ACKNOWLEDGMENT [23] Y. Sheffiand C. Daganzo, ‘‘Hypernetworks and supply-demand equi- librium obtained with disaggregate demand models,’’ Transp. Res. Rec., vol. 673, pp. 113–121, Jan. 1978. In this article, the opinions expressed are those of the authors and do not represent the official position of the European Commission. [24] H. K. Lo, C. W. Yip, and K. H. Wan, ‘‘Modeling transfer and non-linear fare structure in multi-modal network,’’ Transp. Res. B, Methodol., vol. 37, no. 2, pp. 149–170, Feb. 2003. [25] X. Fu, W. H. K. Lam, and B. Y. Chen, ‘‘A reliability-based traffic assign- ment model for multi-modal transport network under demand uncertainty,’’ J. Adv. Transp., vol. 48, no. 1, pp. 66–85, Jan. 2014. APPENDIX In the following, the main steps that have led to the definition of the function (39) expressing the steady-state speed with respect to the number of trains are briefly sketched. Let us start by considering a generic graphical train timetable and let us define with 1X the spatial interval, 1T the time interval and with N the number of trains present in that frame, hence the average space headway ¯s can be defined as ¯s = 1X N while the average time headway ¯h is given by ¯h = 1T N . Let us also define the relation between the average space headway and the average time headway that is given by ¯s = ¯h·v+L, where v is the train speed and L is the average length of trains. Q 1 ¯s  =    1 ¯s · vmax if 1 ¯s ≤ 1 ¯h · vmax + L 1 ¯h 1 −L ¯s  if 1 ¯h · vmax + L < 1 ¯s ≤ 1 smin (44) (44) where Q 1 ¯s  is the ‘‘flow of trains’’ in function of the aver- age space headway. The corresponding steady-state speed- headway relation V 1 ¯s  is given by V 1 ¯s  =    vmax if 1 ¯s ≤ 1 ¯h · vmax + L 1 ¯h(¯s −L) if 1 ¯h · vmax + L < 1 ¯s ≤ 1 smin (45) Inspired by the traffic fundamental diagram, we can assume that, for a given average time headway, the maximum flow of trains in an arc corresponds to an average space headway equal to ¯s = ¯h·vmax+L, where vmax is the maximum speed allowed. At the same time, it has to be remembered that trains have to maintain a minimum distance smin that allows them to stop safely; consequently, as the number of trains on an arc increases, they have to reduce their speed in order to guarantee this safety distance. Then, a triangular traffic (45) Thus, equation (39), has been derived on the basis of (45) and by considering that the inverse of the average space headway has an equivalent meaning of ‘‘density of trains’’ in an arc that is N tot i,j (k) 1i,j . 73598 VOLUME 10, 2022 C. Pasquale et al.: Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks REFERENCES 166–179, May 1977. [47] E. Siri, S. Siri, and S. Sacone, ‘‘A progressive traffic assignment procedure on networks affected by disruptive events,’’ in Proc. Eur. Control Conf. (ECC), May 2020, pp. 130–135. [22] M. Florian and S. Nguyen, ‘‘A combined trip distribution modal split and trip assignment model,’’ Transp. Res., vol. 12, no. 4, pp. 241–246, Aug. 1978. 73599 VOLUME 10, 2022 C. Pasquale et al.: Two-Stage Multiclass Modeling Approach for Intermodal Rail-Road Transport Networks SILVIA SIRI (Member, IEEE) received the M.Sc. degree in management engineering and the Ph.D. degree in information and communication tech- nologies from the University of Genoa, Italy, in 2002 and 2006, respectively. She is currently an Associate Professor of automatic control. She is a coauthor of more than 120 papers in international journals, chapters, and conference proceedings. Her research interests include control of freeway traffic systems, planning of logistics systems and intermodal terminals, and optimal management of electric mobility systems. From 2019 to 2021, she was a member of the Board of Governors of the IEEE Intelligent Transportation Systems Society. Currently, she is a Senior Editor of the IEEE TRANSACTIONS ON INTELLIGENT TRANSPORTATION SYSTEMS, an Associate Editor of the IEEE TRANSACTIONS ON AUTOMATION SCIENCE AND ENGINEERING, and an Editor of the IEEE INTELLIGENT TRANSPORTATION SYSTEMS SOCIETY NEWSLETTER. [48] E. Siri, S. Siri, and S. Sacone, ‘‘Network performance evaluation under disruptive events through a progressive traffic assignment model,’’ IFAC World Congr., vol. 53, no. 2, pp. 15017–15022, 2020. [49] K. Adjetey-Bahun, B. Birregah, E. Châtelet, and J.-L. Planchet, ‘‘A model to quantify the resilience of mass railway transportation systems,’’ Rel. Eng. Syst. Saf., vol. 153, pp. 1–14, Sep. 2016. [50] R. Dorbritz, ‘‘Assessing the resilience of transportation systems in case of large-scale disastrous events,’’ in Proc. Int. Conf. Environ. Eng., vol. 8, 2011, p. 1070. [51] N. Bešinović, ‘‘Resilience in railway transport systems: A literature review and research agenda,’’ Transp. Rev., vol. 40, no. 4, pp. 457–478, Jul. 2020. [52] M. Hrušovský, E. Demir, W. Jammernegg, and T. Van Woensel, ‘‘Real-time disruption management approach for intermodal freight transportation,’’ J. Cleaner Prod., vol. 280, Jan. 2021, Art. no. 124826. [53] C. Ferrari, A. Bottasso, M. Conti, E. Musso, C. Pasquale, P. P. Puliafito, S. Sacone, M. Santagata, S. Siri, and A. Tei. (2022). New Economic Reg- ulation for Transport in Case of Emergency Events. Handbook. [Online]. REFERENCES Available: https://cieli.unige.it/SRSP CECILIA PASQUALE (Member, IEEE) received the bachelor’s degree in civil engineering from the Polytechnic University of Turin, Italy, in 2009, and the master’s degree in transports and logistics engineering and the Ph.D. degree in monitoring of systems and environmental risk management from the University of Genoa, Italy, in 2012 and 2016, respectively. Currently, she is a Postdoctoral Research Fellow with the University of Genoa. Her research interests include modeling, optimization, and control methods applied to the field of transportation systems. Currently, she serves as an Associate Editor for the IEEE OPEN JOURNAL OF INTELLIGENT TRANSPORTATION SYSTEMS and IEEE TRANSACTIONS ON INTELLIGENT VEHICLES. SIMONA SACONE (Member, IEEE) received the master’s degree in electronic engineering and the Ph.D. degree in electronic engineering and computer science, in 1992 and 1997, respec- tively. Currently, she is an Associate Professor of automatic control with the Department of Infor- matics, Bioengineering, Robotics and Systems Engineering, University of Genoa, Italy, where she acts as the Coordinator of the Ph.D. course on systems engineering. She has authored and coau- thored more than 150 papers published on international journals, interna- tional books, and international conference proceedings. Since 2019, she has been a member of the Board of Governors of the IEEE Intelligent Transportation Systems Society. She is the Chair of the Technical Committee on Planning and Control of Transportation Networks of the IEEE Intelligent Transportation Systems Society. Currently, she serves as an Senior Editor for the IEEE TRANSACTIONS ON INTELLIGENT TRANSPORTATION SYSTEMS, an Associate Editor for the IEEE Control Systems Magazine, and an Associate Editor for the IEEE TRANSACTIONS ON CONTROL SYSTEMS TECHNOLOGY. ENRICO SIRI (Member, IEEE) received the B.Sc. degree in industrial engineering and the M.Sc. degree in management engineering from the University of Genoa, in 2016 and 2018, respectively, where he is currently pursuing the Ph.D. degree with the Department of Informat- ics, Bioengineering, Robotics and Systems Engi- neering. His research interests include systems engineering, traffic systems modeling, assignment models, and optimization and control. 73600 VOLUME 10, 2022
https://openalex.org/W3174314089
https://journal.ipb.ac.id/index.php/jabm/article/download/32520/21751
Indonesian
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FORMULASI STRATEGI UNIT BISNIS LAUNDRY SEPATU (STUDI KASUS DARMAWAN WASH SHOE BOGOR)
Jurnal Aplikasi Bisnis dan Manajemen
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1 Corresponding author: Email: danangwicak72@gmaiI.com Jurnal Aplikasi Manajemen dan Bisnis, Vol. 7 No. 2, Mei 2021 Permalink/DOI: http://dx.doi.org/10.17358/jabm.7.2.356 Tersedia online http://journal.ipb.ac.id/index.php/jabm Jurnal Aplikasi Manajemen dan Bisnis, Vol. 7 No. 2, Mei 2021 Permalink/DOI: http://dx.doi.org/10.17358/jabm.7.2.356 Tersedia online http://journal.ipb.ac.id/index.php/jabm TERAKREDITASI PERINGKAT 2 Direktur Jenderal Penguatan Riset dan Pengembangan, Kemenrisek DIKTI No. 30/E/KPT/2018 Kata kunci: EFE, formuIasi strategi, IFE, QSPM, SWOT Danang Wicaksono*)1, Lukman M. Baga**), dan Tanti Novianti*** Danang Wicaksono*)1, Lukman M. Baga**), dan Tanti Novianti***) *) Sekolah Bisnis, IPB University Jl. Raya Pajajaran, Bogor 16151, Indonesia **) Departemen Agribisnis, Fakultas Ekonomi dan Manajemen, IPB University Jl. Agatis Kampus IPB Dramaga, Bogor 16680, Indonesia **) Departemen Ilmu Ekonomi, Fakultas Ekonomi dan Manajemen, IPB University Jl. Agatis Kampus IPB Dramaga, Bogor 16680, Indonesia *) Sekolah Bisnis, IPB University Jl. Raya Pajajaran, Bogor 16151, Indonesia **) Departemen Agribisnis, Fakultas Ekonomi dan Manajemen, IPB University Jl. Agatis Kampus IPB Dramaga, Bogor 16680, Indonesia **) Departemen Ilmu Ekonomi, Fakultas Ekonomi dan Manajemen, IPB University Jl. Agatis Kampus IPB Dramaga, Bogor 16680, Indonesia Abstract: Shoes are not only used for daily needs now, but people rather consider that shoes are an important fashion item for their appereance. Despite the growing culture of Indonesian sneakerhead, Darmawan Wash Shoe had recently experiencing decreased sales from 2017 until 2019. This research aimed to 1) identifying internal and external factors which are strengths, weaknesses, opportunities and threats that influences the sales of Darmawan Wash Shoe; 2) formulate alternative strategies that can be implemented to increase sales based on the influences of each internal and external factors on Darmawan Wash Shoe; 3) determine the priority from those alternative strategies to be implemented to achieve sales targets in Darmawan Wash Shoe. The methods used in this study are: 1) identification of Darmawan Wash Shoe internal factors using the Internal Factor Evaluation (IFE) method and identification of Darmawan Wash Shoe external factors using the External Factor Evaluation (EFE) method, positioning Darmawan Wash Shoe business using the Internal External (IE) matrix; 2) aIternative strategy formulations using the SWOT matrix method; 4) formulating priority strategies with the Quantitative Stratategy Planning Matrix (QSPM) method. The results of internal and external analysis found that the position of Darmawan Wash Shoe is in quadrant V, the strategy that must be done is market penetration and development product in the QSPM analysis, the main priority strategy is to conduct onIine discount promotions. Keywords: EFE, IFE, QSPM, Startegy Formulation, SWOT Keywords: EFE, IFE, QSPM, Startegy Formulation, SWOT Mengacu pada data yang berasal dari Kementerian Koperasi dan UMKM RepubIik Indonesia mengenai Perkembangan Usaha Mikro, Kecil, Menengah (UMKM) dan Usaha Besar (UB) Tahun 2012-2018, UMKM di Indonesia pada tahun 2018 berjumlah sebanyak 64,2 juta unit serta diharuskan mencukupi tenaga kerja sebesar 97 % (116,9 juta) dilihat pada nilai masukan pada Produk Domestik Bruto (PDB) nasional sebesar 61,07 % (8,5 miliar rupiah). Peningkatan jumlah UMKM daIam kurun waktu 2012 hingga 2018 dengan tingkat perkembangan sebesar 15,5 % (8,9 juta unit) terIihat cukup pesat. Sehingga dapat diklasifikasikan dari total unit usaha yang ada menjadi 99,99% UMKM, 99,99% usaha mikro dan 1,22% usaha kecil. Hal tersebut mengartikan bahwa UMKM memiIiki peran yang cukup strategis daIam kemajuan ekonomi di Indonesia. Pengusaha laundry sepatu memikat calon pelanggan dengan cara inovasi dan pengembangan yang berkelanjutan. Nuriyawan (2011) menyatakan bahwa harga, fasilitas dan kuaIitas pelayanan adalah faktor utama kepuasan konsumen Iaundry sepatu. Penelitian yang dilakukan Asmawati (2018) mengklasifikasikan beberapa faktor internal yang harus menjadi concern pada usaha bisnis Iaundry sepatu, yaitu harga, kualitas produk, pelayanan one day service, diversifikasi produk, komunikasi internal unit bisnis, jumlah dan kinerja tenaga kerja dan quality control. Faktor-faktor strategis baik internal maupun eksternal yang digunakan dalam menyusun alternatif strategi pada penelitian terhadap DWS, diperoleh langsung dari kondisi real yang dihadapi perushaan. Penelitian ini menggunakan 3 tahap perumusan strategi, yaitu input, matching dan decision. Penyebab muncuInya jenis usaha baru yang salah satunya adalah bisnis laundry sepatu disebabkan oleh peningkatan UMKM dan industri sepatu di Indonesia. Pada tahun 2013, laundry sepatu di Indonesia ramai diperbincangkan seiring dengan perkembangan industri sepatu itu sendiri. Industri pencucian sepatu atau Iaundry sepatu sudah merada pada semua kota besar di Indonesia. Bisnis ini tidak terlepas dari rasa ingin yang tinggi untuk membeli oIeh masyarakat terutama pada model sepatu jenis kats dan sneakers. Rahaju dan Sumarlan (2013) menyatakan bahwa penyebab pengaruh motivasi konsumen mengggunakan jasa Iaundry adalah faktor ekonomi, gaya hidup dan faktor alam. Dapat dilihat pada Gambar 1, penjualan DWS cenderung menurun dari tahun 2017 hingga 2019. Menurut data yang diperoleh, DWS memiliki penjualan tertinggi pada triwulan II tahun 2017 sebanyak 1.969 pasang sepatu. SeteIah itu penjualan DWS berfluktuasi tidak menentu, namun hingga triwulan IV tahun 2019 tidak pernah mencapai titik penjualan tertinggi pada triwulan II tahun 2017. Sebaliknya, penjualan terendah terjadi pada triwulan II 2019 sebanyak 806 pasang sepatu. Pada Tahun 2019 adalah titik terendah DWS dicerminkan dengan total penjualan yang sangat rendah. Keywords: EFE, IFE, QSPM, Startegy Formulation, SWOT Abstrak: Sepatu saat ini tidak hanya dipakai untuk kebutuhan sehari-hari, sebagian besar masyarakat menganggap sepatu merupakan icon penting daIam kehidupan. Darmawan Wash Shoe memiIiki penjualan cenderung menurun dari tahun 2017 hingga 2019. Penelitian ini memiliki tujuan sebagai berikut, yaitu 1) mengidentifikasi faktor internal dan eksternal yang menjadi kekuatan, kelemahan, peluang dan ancaman dalam memengaruhi penjuaIan jasa Darmawan Wash Shoe; 2) merumuskan alternatif strategi yang dapat dilakukan untuk meningkatkan penjuaIan berdasarkan pengaruh masing-masing faktor internal dan eksternal pada Darmawan Wash Shoe; 3) memilih prioritas strategi yang dapat digunakan Darmawan Wash Shoe. Penelitian ini menggunakan metode, yaitu 1) identifikasi faktor internaI Darmawan Wash Shoe menggunakan metode InternaI Factor EvaIuation (IFE) dan identifikasi faktor eksternaI Darmawan Wash Shoe menggunakan metode ExternaI Factor EvaIuation (EFE), penentuan posisi bisnis Darmawan Wash Shoe dengan menggunakan matriks InternaI ExternaI (IE); 2) formulasi alternatif strategi menggunakan metode matriks SWOT; 3) perumusan strategi prioritas dengan metode Quantitative Stratategy PIanning Matrix (QSPM). Hasil analisis internal dan eksternal didapatkan posisi Darmawan Wash Shoe berada pada kuadran V yaitu pada posisi sedang, sehingga grand strategy yang tepat adalah penetrasi pasar dan perkembangan produk. Hasil dari alternatif strategi dengan QSPM, didapatkan melakukan promosi diskon secara daring sebagai prioritas strategi. 356 Copyright © 2020, ISSN: 2528-5149/EISSN: 2460-7819 Jurnal Aplikasi Bisnis dan Manajemen (JABM), Vol. 7 No. 2, Mei 2021 Jurnal Aplikasi Bisnis dan Manajemen (JABM), Vol. 7 No. 2, Mei 2021 Jurnal Aplikasi Bisnis dan Manajemen (JABM), Vol. 7 No. 2, Mei 2021 PENDAHUIUAN Salah satu faktor penyebab industri laundry sepatu berkembang adalah kota-kota besar. Jumlah penduduk Kota Bogor yang mencapai 1.081.009 juta jiwa dengan laju pertumbuhan 1,53 % pertahunnya (Badan Pusat Stastistik, 2018) menjadi bidang strategis yang menguntungkan bagi para pelaku usaha laundry sepatu. Banyaknya industri sejenis yang tersebar di daerah usaha DWS, membuat persaingan laundry sepatu di Kota Bogor semakin ketat dan membawa dampak kurang baik bagi kelangsungan bisnis DWS. Penentuan strategi yang sesuai bagi perusahaan berasal dari pemahaman yang baik dilihat pada aspek kondisi internal dan eksternal (Chan, 2011). Komaryatin (2007) dan Kurniawati dan Sari (2009) menyatakan bahwa untuk memprediksi persaingan yang semakin ketat, setiap perusahaan harus mampu merumuskan strategi secara visioner. Perkembangan Usaha Mikro Kecil dan Menengah (UMKM) menjadi saIah satu sektor vital guna memajukan perekonomian di era industri 4.0, khususnya di Indonesia. Keywords: EFE, IFE, QSPM, Startegy Formulation, SWOT DWS dapat meningkatkan volume penjualan dan bertahan pada persaingan industri laundry sepatu apabila memanfaatkan kelebihan dan meminimalisasi kekurangan yang dimiliki. Seiring dengan perkembangan industri sepatu, khususnya laundry sepatu, Darmawan Wash Shoe atau DWS berdiri. DWS adalah salah satu UMKM di bidang laundry sepatu yang berdiri sejak tahun 2016. DWS memiIiki segmentasi pasar baru dan luas berdasarkan dengan perkembangan industri sepatu di Indonesia, dan perubahan gaya hidup konsumen di Indonesia seperti sneakerhead atau kolektor sepatu sneakers yang menjadikan ragam jenis sepatu sebagai tanda status sosial. Philip (2019), menyatakan perubahan gaya hidup yang konsumtif adalah variabel yang berpengaruh positif signifikan terhadap keputusan pembeIian sepatu oIeh konsumen. 357 Jurnal Aplikasi Bisnis dan Manajemen (JABM), Vol. 7 No. 2, Mei 2021 Jurnal Aplikasi Bisnis dan Manajemen (JABM), Vol. 7 No. 2, Mei 2021 Jurnal Aplikasi Bisnis dan Manajemen (JABM), Vol. 7 No. 2, Mei 2021 07-5434 E-ISSN: 2407-7321 d by Ministry of RTHE Number 32a/E/KPT/2017 Jurnal Aplikasi Bisnis dan Manajemen (JABM), Vol. 7 No. 2, Mei 2021 Gambar 1. Penjualan Darmawan Wash Shoe Gambar 1. Penjualan Darmawan Wash Shoe Gambar 1. Penjualan Darmawan Wash Shoe Uraian permasalahan menunjukkan perlunya langkah strategis yang tepat melalui formulasi strategi. Oleh karena itu, perlu dilakukan analisis lingkungan internal dan eksternal pada DWS untuk mendapatkan alternatif dan strategi prioritas yang dapat diterapkan dalam persaingan untuk meningkatkan pendapatan penjualan dan bertahan di industri laundry sepatu. Tujuan penelitian ini adalah mengidentifikasi faktor internal dan eksternal yang memengaruhi penjualan jasa DWS; Merumuskan alternatif strategi DWS; Menentukan strategi penjualan yang menjadi prioritas pada DWS. Penelitian ini mencakup ruang lingkup terbatas meliputi melakukan pengamatan kondisi internal dan eksternal DWS, memformulasi alternatif strategi penjualan dan memprioritaskan strategi aIternatif berdasarkan data penjualan DWS pada tahun 2017-2019. Uraian permasalahan menunjukkan perlunya langkah strategis yang tepat melalui formulasi strategi. Oleh karena itu, perlu dilakukan analisis lingkungan internal dan eksternal pada DWS untuk mendapatkan alternatif dan strategi prioritas yang dapat diterapkan dalam persaingan untuk meningkatkan pendapatan penjualan dan bertahan di industri laundry sepatu. Tujuan penelitian ini adalah mengidentifikasi faktor internal dan eksternal yang memengaruhi penjualan jasa DWS; Merumuskan alternatif strategi DWS; Menentukan strategi penjualan yang menjadi prioritas pada DWS. Penelitian ini mencakup ruang lingkup terbatas meliputi melakukan pengamatan kondisi internal dan eksternal DWS, memformulasi alternatif strategi penjualan dan memprioritaskan strategi aIternatif berdasarkan data penjualan DWS pada tahun 2017-2019. Penelitian ini menggunakan jenis dan sumber berupa data primer dan sekunder. Data primer, diperoleh langsung dari responden hasil wawancara. Keywords: EFE, IFE, QSPM, Startegy Formulation, SWOT Responden dalam penelitian ini berjumlah 2 orang internal perusahan yaitu pemiIik unit bisnis dan manajer dan 120 konsumen DWS. Rumusan masalah dan perumusan pengembangan bisnis dijawab dengan melihat data primer penelitian yang dikaitkan dengan tujuan spesifik penelitian (Sarwono, 2006). Data sekunder, diperoleh dalam bentuk dokumen dan dapat berupa berbagai sumber rujukan atau literatur berupa dokumen-dokumen yang berhubungan dengan topik penelitian ini. Membaca, melihat dan mendengarkan oleh peneliti adalah data sekunder yang sudah tersedia (Sarwono, 2006). Penentuan responden menggunakan teknik purposive sampling (penentuan responden secara sengaja), yaitu pihak yang menjawab berbagai pertanyaan dari kuisioner untuk kepentingan penelitian. Responden dipilih berdasarkan kompetensi keahian dalam pemberian penilaian strategi pengembangan bisnis pada DWS dikarenakan mengetahui kondisi lapangan perusahaan. Teknik purposive sampIing mempertimbangkan responden yang dipilih memiIiki pengetahuan, keahlian dan pengalaman dalam bidang yang di teliti (Sugiyono, 2013). METODE PENELITIAN Data penelitian berasal dari salah satu UMKM yang bergerak di bidang jasa laundry sepatu yaitu DWS yang berada di JaIan KoI. Ahmad Syam, Bogor. Penelitian ini dilakukan pada bulan Maret-Juni 2020. Data penelitian dikumpulkan dalam beberapa tahap, yaitu tahap pertama pengumpulan data melalui wawancara dengan stakeholders yang terkait dengan dunia usaha. Pada tahap pertama, tujuannya adalah untuk menyaring persepsi dan informasi dari sumber DWS untuk mengetahui faktor internal yaitu kelebihan dan kekurangan, dan faktor eksternal yaitu peIuang dan ancaman DWS. Tahap selanjutnya adalah dengan memberikan kuesioner kepada 120 konsumen yang diIakukan untuk mengidentifikasi alasan konsumen memilih Iayanan DWS. AnaIisis IFE (Internal Factor Evaluation) digunakan untuk mengidentifikasi faktor-faktor strategis berupa kekuatan dan kelemahan internal. Sedangkan, EFE (External Factor Evaluation) digunakan untuk menganalisis faktor strategis berupa peIuang dan ancaman perusahaan. Masalah ini menjadi sangat penting karena kinerja perusahaan akan dipengaruhi secara langsung maupun tidak langsung oleh faktor 358 Jurnal Aplikasi Bisnis dan Manajemen (JABM), Vol. 7 No. 2, Mei 2021 7-7321 E Number 32a/E/KPT/2017 Jurnal Aplikasi Bisnis dan Manajemen (JABM), Vol. 7 No. 2, Mei 2021 Jurnal Aplikasi Bisnis dan Manajemen (JABM), Vol. 7 No. 2, Mei 2021 Jurnal Aplikasi Bisnis dan Manajemen (JABM), Vol. 7 No. 2, Mei 2021 internal dan eksternal. Selain itu, hasil pembobotan IFE dan EFE akan dimasukkan ke daIam kuadran atau matriks berbentuk segiempat IE. AnaIisis IE merupakan kombinasi dari faktor internal dan eksternal. Parameter yang digunakan meliputi faktor internal dan eksternal yang dihadapi perusahaan. Menurut Raymond et aI. (2012) matriks IE adalah alat perumusan strategi yang menguraikan dan mengevaluasi kekuatan dan kelemahan utama area fungsional bisnis, dan juga berfungsi sebagai dasar untuk mengidentifikasi dan mengevaluasi hubungan antara area tersebut. menentukan daya tarik relatif (reIative attractiveness) dari strategi alternatif yang dipilih, untuk menentukan strategi mana yang dianggap paIing tepat untuk diimplementasikan (David dan David, 2011). AnaIisis QSPM digunakan untuk menentukan strategi yang terbaik dalam pemutusan keputusan yang akan dijalankan oleh perusahaan dalam jangka pendek maupun panjang (Isnandar et al. 2016). DWS adalah UMKM yang bergerak di bidang jasa laundry sepatu di Kota Bogor. Penurunan penjualan dan tidak stabilnya penjualan terjadi pada Darmawan Wash Shoe. Menganalisis faktor eksternal dan internal Darmawan Wash Shoe untuk mendapatkan faktor strategis penting untuk merumuskan alternatif strategi DWS. Faktor-faktor tersebut kemudian diidentifikasi dan dipiIih mana yang menjadi kekuatan, kelemahan, peIuang dan ancaman dengan mengunakan analisis SWOT dan IE. Identifikasi Faktor Internal HASIL HASIL Analisis internal merupakan analisis yang digunakan untuk mengidentifikasi kekuatan dan kelemahan suatu perusahaan atas kualitas sumber daya dan kemampuan yang dimiliki (Capps dan GIissmeyer, 2012). Kekuatan merupakan faktor internal yang memberi perusahaan keunggulan komparatif di pasar (Kotler dan Keller, 2016). Pada penelitian Asmawati (2018), harga yang terjangkau adalah salah satu kekuatan yang harus dimiliki unit usaha laundry. Penelitian yang diIakukan Umar et aI. (2016), kuaIitas pelayanan yang baik merupakan salah satu kekuatan yang harus dimiliki unit usaha laundry. Kekuatan yang diidentifikasi pada DWS yaitu harga yang ditawarkan terjangkau konsumen, kuaIitas pelayanan yang baik, terdapatnya Iayanan fast cIeaning, banyaknya varian parfum, komunikasi yang baik terhadap konsumen, terdapat garansi/cuci uIang. Kelemahan yang diidentifikasi yaitu belum menggunakan teknologi pengering sepatu, manajemen keuangan yang belum baik, SOP lapangan yang suIit direalisasikan, komunikasi yang kurang baik pada internal unit usaha, administrasi yang belum baik dan modal promosi yang terbatas. Skor rata-rata keseluruhan faktor internal adalah 2,30 yang menunjukkan bahwa DWS memiliki kondisi internal sedang. Hasil matriks IFE selengkapnya pada TabeI 1. Gambaran Umum Unit Bisnsi Darmawan Wash Shoe Darmawan Wash Shoe merupakan salah satu unit usaha yang berdiri pada tahun 2016 bergerak daIam bidang jasa industri yaitu washing shoes oIeh Vicky Darmawan. DWS melihat pangsa pasar baru dan terbilang Luas dengan melihat perkembangan industri sepatu dan perubahan gaya hidup konsumen di Indonesia seperti sneakerhead atau kolektor sepatu sneaker yang menjadikan koleksi sepatu sebagai tanda status sosial. DWS mendirikan outlet di Jalan Kol. Ahmad Syam, Bogor dimana lokasi tersebut dapat dikatakan sebagai Iokasi yang strategis untuk melakukan kegiatan bisnis. Motivasi Vicky sebagai pemilik bisnis dalam mendirikan bisnis laundry sepatu yaitu karena dirinya adalah salah satu sneakerhead atau koIektor sepatu sneaker. Vicky mendapat peluang bisnis yang baru, setiap jenis sepatu terbuat dari berbagai jenis dan bahan yang berbeda. Perbedaan jenis setiap bahan sepatu tentu memiIiki perawatan yang berbeda. DWS menyediakan layanan laundry sepatu, perusahaan sudah menentukan setiap jenis bahan sepatu dan jenis perawatan yang sesuai. DWS tidak hanya menerima laundry sepatu, tetapi juga menerima berbagai perawatan sepatu Iainnya. TabeI 1. METODE PENELITIAN Kemudian diIakukan formulasi dalam penentuan alternatif strategi dari berbagai faktor yang menjadi kekuatan, kelemahan, peluang dan ancaman. Analisis QSPM digunakan untuk merumuskan strategi apa yang akan menjadi prioritas dalam pencapaian target penjualan pada DWS. Adapun kerangka pemikiran penelitian dapat dilihat pada Gambar 2. Matriks SWOT digunakan sebagai alat untuk mengembangkan strategi alternatif. Matriks SWOT secara jeIas menggambarkan beberapa peIuang dan ancaman eksternal yang dihadapi perusahaan berdasarkan kekuatan dan kelemahannya. Langkah seIanjutnya adalah mengembangkan opsi strategis meIaIui perbandingan berpasangan. Matriks SWOT dapat membuat perbandingan berpasangan antara kekuatan dan peluang (SO), kekuatan dan ancaman (ST), peluang dan kelemahan (WO), serta kelemahan dan ancaman (WT). SeteIah didapatkan alternatif strategi analisis SWOT, maka penentuan prioritas strategi akan menggunakan Quantitative Strategic Planning Matrix (QSPM). QSPM digunakan untuk Gambar 2. Kerangka pemikiran penelitian Gambar 2. Kerangka pemikiran penelitian 359 Jurnal Aplikasi Bisnis dan Manajemen (JABM), Vol. 7 No. 2, Mei 2021 Identifikasi Faktor Eksternal yang dikatakan menjadi peluang DWS adalah Iokasi yang strategis dengan skor 0,61. Sedangkan faktor yang menjadi ancaman utama DWS adalah munculnya pesaing di area unit bisnis dengan skor 0,23. total skor rata-rata dari faktor eksternal adalah 2,92 seIengkapnya pada Tabel 2. AnaIisis faktor eksternal perusahaan meliputi lingkungan industri dari berbagai faktor yang akan menyebabkan perusahaan menghadapi peIuang dan ancaman. Penelitian yang dilakukan Rofik (2017) mengidentifikasi beberapa faktor peluang yang diidentifikasi, yaitu pelanggan tetap, perkembangan penduduk sekitar usaha, bahan baku yang murah dan relatif melimpah, harga layanan yang relatif murah dibanding kompetitor, lokasi strategis dan modal usaha yang relatif tidak membutuhkan modal besar. Ancaman yang diidentifikasi pada penelitian Eko (2013) adalah Banyaknya pesaing di perusahaan sejenis, kenaikan harga bahan bakar yang relatif tinggi, juga kenaikan harga dasar listrik, serta perusahaan sejenis atau pesaing dengan dana besar dan cuaca yang tidak menentu. Identifikasi Faktor Internal HasiI matriks IFE Bobot Peringkat Skor Ranking Kekuatan Harga yang ditetapkan terjangkau 0,06 3 0,18 6 KuaIitas peIayanan yang baik 0,09 4 0,36 2 MemiIiki peIayanan Fast cIeaning/One day service 0,08 3 0,24 4 Banyak piIihan varian parfum 0,07 3 0,21 5 MemiIiki hubungan dan komuniasi yang baik 0,10 4 0,40 1 Konsumen mendapatkan garansi/cuci uIang 0,08 4 0,32 3 KeIemahan BeIum menggunakan teknoIogi pengering sepatu 0,05 2 0,10 4 SOP Iapangan suIit direaIisasikan 0,09 1 0,09 5 Komunikasi pada internaI bisnis kurang 0,09 1 0,09 3 Administrasi beIum baik 0,08 2 0,16 6 Manajemen keuangan yang beIum baik 0,08 1 0,08 2 ModaI promosi terbatas 0,07 1 0,07 1 TotaI Skor Faktor Internal 2,30 TabeI 1. HasiI matriks IFE TabeI 1. HasiI matriks IFE 360 Jurnal Aplikasi Bisnis dan Manajemen (JABM), Vol. 7 No. 2, Mei 2021 Jurnal Aplikasi Bisnis dan Manajemen (JABM), Vol. 7 No. 2, Mei 2021 Jurnal Aplikasi Bisnis dan Manajemen (JABM), Vol. 7 No. 2, Mei 2021 Matriks Internal-Eksternal (IE) Dwiastuti (2008), menyatakan bahwa kuadran IE berdasar pada nilai total matriks IFE berbobot sumbu X dan nilai total matriks EFE berbobot sumbu Y. Analisis matriks IE adalah strategi yang meringkas dan mengevaluasi keunggulan dan kelemahan utama pada kuadran bisnis, serta menjadi landasan untuk mengklasifikasikan dan mengidentifikasi hubungan area tersebut (Raymond et aI. 2012). Hasil penggunaan matriks IFE untuk menganalisis faktor internal mendapatkan skor 2,30, dan hasil penggunaan matriks EFE untuk analisis eksternal mendapatkan skor 2,92. Dengan demikian terlihat bahwa posisi DWS saat ini berada pada kuadran V yang digambarkan pada Gambar 3. Dilihat dari matriks IE, maka posisi perusahaan berada pada kuadran V dengan posisi dalam kategori sedang yang dapat diIihat pada Gambar 3. HaI ini menandakan grand strategy yang paling tepat diIakukan oIeh DWS adalah hold and maintain strategy. Menurut David (2016) strategi yang dapat dikembangkan adalah market penetration dan product development. Peluang yang diidentifikasi pada DWS yaitu perkembangan jumIah penduduk, perubahan gaya hidup masyarakat, segmentasi relatif luas, kemajuan teknoIogi internet/sosial media, lokasi strategis dan melakukan diversifikasi produk. Ancaman yang diidentifikasi pada DWS yaitu munculnya pesaing baru sejenis, harga sewa Iahan yang selalu naik, musim hujan. Penelitian yang diIakukan Siregar (2019) mengidentifikasi beberapa faktor peIuang yang diidentifikasi, yaitu pertumbuhan jumlah penduduk yang mengimplikasinya pertumbuhan pangsa pasar ikut meningkat dan fasilitas laundry yang memadai. Berdasarkan hasil analisis tersebut faktor TabeI 2. HasiI matriks EFE Bobot Peringkat Skor Ranking PeIuang Perkembangan jumIah penduduk 0,06 3 0,20 6 Perubahan gaya hidup masyarakat 0,11 4 0,47 2 Segmentasi pasar reIatif Iuas 0,10 4 0,43 3 Kemajuan teknoIogi internet/sociaI media 0,12 3 0,38 4 Iokasi strategis 0,15 4 0,61 1 MeIakukan diversifikasi produk 0,09 3 0,29 5 Ancaman MuncuInya pesaing di area unit bisnis 0,11 2 0,23 1 Biaya sewa Iahan yang seIaIu naik 0,12 1 0,12 3 Musim hujan 0,07 2 0,15 2 TotaI Skor Faktor EnternaI 2,92 TabeI 2. HasiI matriks EFE Strategi S-O Perumusan tersebut didasarkan pada ide korporat untuk memanfaatkan peluang dengan menggunakan semua kelebihannya. Strategi S-O digunakan untuk melihat kekuatan internal perusahaan sebagaimana dimanfaatkan daIam melihat peluang eksternal. Dalam matrik SWOT dirumuskan strategi SO, yaitu promosi diskon online diIakukan untuk meningkatkan jumIah pelanggan. Strategi promosi diskon secara daring untuk meningkatkan jumlah kosumen sejaIan dengan penelitian yang dilakukan Varatisha (2017). HaI ini dengan menggunakan kekuatan DWS seperti S1: harga yang ditetapkan terjangkau konsumen, untuk merebut dan memanfaatkan peluang yaitu O1: perkembangan jumIah penduduk, O2: gaya hidup masyarakat, O4: kemajuan teknologi internet dan sosial media. Selanjutnya, melakukan reminder wash shoe secara berkala dengan memanfaatkan sosial media, hal ini dengan menggunakan kekuatan DWS  seperti S5: Menjaga komunikasi yang baik dengan konsumen untuk merebut dan memanfaatkan peluang, yaitu O4: kemajuan teknoIogi internet dan sosial media. PeIuang 361 -ISSN: 2407-5434 E-ISSN: 2407-7321 Accredited by Ministry of RTHE Number 32a/E/KPT/2017 Jurnal Aplikasi Bisnis dan Manajemen (JABM), Vol. 7 No. 2, Mei 2021 Jurnal Aplikasi Bisnis dan Manajemen (JABM), Vol. 7 No. 2, Mei 2021 Gambar 3. Matriks IE Darmawan Wash Shoe Gambar 3. Matriks IE Darmawan Wash Shoe Strategi W-O Strategi W-O diterapkan dengan tujuan meminimaIisir kelemahan DWS dan meIihat keberuntungan atau peluang yang ada, Strategi W-O. Pada matriks SWOT terdapat strategi mengadakan agenda gathering atau berwisata ke tempat-tempat yang sedang trend secara berkala dengan memanfaatkan kelemahan W2: SOP lapangan yang sulit direaIisasikan, W3: kurangnya hubungan dan komunikasi yang baik pada internal bisnis, W4: administrasi yang belum baik, W5: manajemen keuangan yang belum baik, untuk meraih peluang O2: gaya hidup masyarakat yang konsumtif. Strategi ini ditujukan kepada bagian internal bisnis. Strategi SWOT DWS seperti S2: kualitas pelayanan yang baik, S3: terdapatnya pelayanan Fast CIeaning/One Day Service yang diminati konsumen, S4: banyaknya pilihan varian parfum, S5: memiliki hubungan dan komunikasi yang baik dengan konsumen, S6: terdapat garansi/cuci uIang, untuk mengatasi ancaman, yaitu T2: harga sewa Iahan yang selalu naik. Selanjutnya, meningkatkan daya saing dengan peningkatan kualitas pelayanan, haI ini dengan menggunakan kekuatan DWS seperti S1: harga yang ditetapkan terjangkau konsumen, S2: kualitas pelayanan yang baik, S4: banyaknya pilihan varian parfum, S5: memiliki hubungan dan komunikasi yang baik dengan konsumen, S6: terdapat garansi cuci uIang, untuk mengatasi ancaman, yaitu T1: munculnya pesaing baru di area unit bisnis. Strategi tersebut konsisten serta tepat, yaitu memiliki daya saing dan kualitas pelayanan yang terus meningkat, DWS akan memiliki brand awareness yang baik bagi konsumen dan dapat menjadi laundry sepatu yang unggul dari competitor (Dewi, 2018). Strategi S-T Perumusan disusun berdasarkan keunggulan korporat dalam mencegah terjadinya ancaman. Strategi S-T melihat keunggulan internal perusahaan untuk mengatasi ancaman eksternal. DaIam matriks SWOT, strategi pemberian hak istimewa dirumuskan kepada pemiIik lahan berupa layanan gratis yang terdapat pada DWS, haI ini dengan menggunakan kekuatan 362 Jurnal Aplikasi Bisnis dan Manajemen (JABM), Vol. 7 No. 2, Mei 2021 Jurnal Aplikasi Bisnis dan Manajemen (JABM), Vol. 7 No. 2, Mei 2021 Jurnal Aplikasi Bisnis dan Manajemen (JABM), Vol. 7 No. 2, Mei 2021 Selanjutnya, memanfaatkan investor untuk penambahan modal promosi daIam upaya meningkatkan daya saing dan pangsa pasar dengan memanfaatkan kelemahan, yaitu W6: modal promosi yang terbatas, untuk meraih peluang O1: perkembangan jumIah penduduk, O2: gaya hidup masyarakat, O3: segmentasi pasar relatif Luas. DWS dapat mempertimbangkan untuk melakukan penambahan modal baik mandiri ataupun dari pihak investor, dimana diharapkan dengan modal yang cukup maka promosi akan diIakukan lebih maksimal sehingga penetrasi pasar akan berjaIan dengan baik. Selanjutnya, memanfaatkan investor untuk penambahan modal promosi daIam upaya meningkatkan daya saing dan pangsa pasar dengan memanfaatkan kelemahan, yaitu W6: modal promosi yang terbatas, untuk meraih peluang O1: perkembangan jumIah penduduk, O2: gaya hidup masyarakat, O3: segmentasi pasar relatif Luas. DWS dapat mempertimbangkan untuk melakukan penambahan modal baik mandiri ataupun dari pihak investor, dimana diharapkan dengan modal yang cukup maka promosi akan diIakukan lebih maksimal sehingga penetrasi pasar akan berjaIan dengan baik. (TAS) tertinggi hingga terendah, semua alternatif strategi yang dianilisis dengan menggunakan QSPM pada Darmawan Wash Shoe selengkapnya pada Tabel 6. Tabel 6. Alternatif strategi hasil analisis QSPM NO Alternatif strategi TAS 1 Melakukan promosi diskon secara daring 6,15 2 Mengadakan kerja sama dengan beberapa unit bisnis seperti coffee shop dan barbershop 6,07 3 Melakukan reminder wash shoe secara berkala 5,80 4 Meningkatkan daya saing dengan peningkatan kualitas layanan 5,64 5 Memberikan privilege kepada pemilik lahan berupa layanan gratis 5,56 6 Mengadakan agenda gathering dan training dengan ke tempat-tempat yang sedang trend 5,11 7 Menggunakan alat berteknologi pengering sepatu 4,86 8 Memanfaatkan investor untuk penambahan modal dan biaya promosi 4,78 Tabel 6. ImpIikasi ManajeriaI Prioritas strategis utama yang direkomendasikan untuk DWS adalah meIakukan promosi diskon secara daring untuk meningkatkan meningkatkan daya saing dan jumIah kosumen. Perusahaan harus dapat mempertahankan dan meningkatkan jumIah konsumen baru. DWS dapat membuat potongan harga berupa voucher dan didistribusikan kepada setiap konsumen dengan memanfaatkan media sosiaI onIine seperti Instagram, Youtube, Facbook, Iine dan Iain sebagainya. Prioritas strategi kedua yang disarankan adalah mengadakan kerja sama dan kemitraan dengan beberapa unit bisnis seperti coffee shop dan barbershop daIam upaya melakukan promosi dan meningkatkan daya saing. Perkembangan zaman dan perkembangan gaya hidup masyarakat sangat berarti bagi upaya perusahaan untuk menjalin kerja sama dengan departemen bisnis jenis lain, sehingga perusahaan dapat memasuki pasar yang lebih luas. DWS dapat mengerjakan sesuatu haI yang unik seperti membuat suatu event seperti expo dan menjadikan mitra sebagai drop zone sepatu dengan bekerja sama atau bermitra dengan jenis unit usaha Strategi S-T Alternatif strategi hasil analisis QSPM NO Alternatif strategi TAS 1 Melakukan promosi diskon secara daring 6,15 2 Mengadakan kerja sama dengan beberapa unit bisnis seperti coffee shop dan barbershop 6,07 3 Melakukan reminder wash shoe secara berkala 5,80 4 Meningkatkan daya saing dengan peningkatan kualitas layanan 5,64 5 Memberikan privilege kepada pemilik lahan berupa layanan gratis 5,56 6 Mengadakan agenda gathering dan training dengan ke tempat-tempat yang sedang trend 5,11 7 Menggunakan alat berteknologi pengering sepatu 4,86 8 Memanfaatkan investor untuk penambahan modal dan biaya promosi 4,78 Strategi W-T Strategi ini bersifat defensif dengan meminimaIisir kelemahan dan mengkaji ulang ancaman terhadap DWS. Strategi W-T bertujuan meminimaIisir kelemahan internal dengan mengkaji ulang ancaman eksternal. DaIam matriks SWOT, strategi W-T dirumuskan, yaitu menggunakan aIat berteknoIogi sebagai pengering sepatu berdasarkan jenis dan bahan sepatu  dalam upaya mengontrol perubahan cuaca yang memengaruhi kuaIitas pelayanan pencucian sepatu dengan meminimalkan kelemahan W1: belum menggunakan teknologi mesin pengering, untuk menghindari ancaman T3: musim hujan. Strategi ini merupakan strategi difensif yang sangat baik untuk menghindari ancaman penurunan penjualan akibat musim hujan. SeIanjutnya, mengadakan kerja sama dan kemitraan dengan beberapa unit bisnis dengan meminimalkan kelemahan W6: modal promosi yang terbatas, untuk menghindari ancaman T1: munculnya pesaing baru di area unit bisnis. Penelitian yang dilakukan Hutahaean (2017) menghasilkan strategi yang serupa, yaitu mengadakan kerja sama dengan bisnis lain. Strategi ini merupakan bentuk difensif atau bertahan yang sangat baik jika dilakukan oIeh DWS. daIam kerja sama akan memperoIeh keuntungan. DAFTAR PUSTAKA Asmawati H. 2018. Strategi pengembangan usaha dengan metode anaIisa SWOT pada usaha Istiqomah Samarinda. JournaI of administration business 6(1): 65-76. [BPS] Badan Pusat Statistik. 2018. Kota Bogor daIam angka 2017. https://bogorkota.bps.go.id/ statictabIe/2018/10/01/183/jumIah-penduduk- dan-Iaju-pertumbuhan-penduduk-menurut- kecamatan-di-kota-bogor-2010-2016-dan-2017. htmI [2 Mar 2020]. Saran Strategi prioritas ketiga yang disarankan adalah meIakukan reminder wash shoe secara berkala dengan memanfaatkan media sosial online. Strategi ini bertujuan menjadi pengingat kepada setiap konsumen dan bahkan seluruh penduduk Kota Bogor yang memiliki sepatu untuk membersihkan sepatu yang sudah kotor. Strategi tersebut adalah hal lumrah yang diIakukan oIeh perusahaan besar, dengan tujuan menjaga performa dan kualitas produk. Hasil penelitian ini dibatasi hingga perumusan prioritas strategi pada bisnis laundry sepatu DWS, sehingga penelitian seIanjutnya disarankan untuk meIakukan analisis terhadap implementasi strategi yang sudah dihasilkan dan melakukan evaluasi terhadap pelaksanaan strategi. Penelitian selanjutnya disarankan untuk mencari pembanding terhadap shoes laundry lain sebagai penentu faktor strategis. Penelitian selanjutnya disarankan untuk melakukan identifikasi ulang terhadap persaingan industri selama pandemic Covid-19. Strategi prioritas keempat yang disarankan adalah meningkatkan daya saing dengan peningkatan kuaIitas layanan. Memiliki daya saing dan kualitas pelayanan yang unggul, membuat konsumen cenderung akan Iebih memilih suatu merek. Strategi prioritas selanjutnya adalah menggunakan alat berteknologi pengering sepatu dalam upaya mengontrol musim hujan yang memengaruhi volume penjualan dan kualitas pelayanan laundry sepatu. Perusahaan harus segera membuat perencanaan pembelian alat teknologi pengering sepatu, dengan mengimplementasikan strategi kelima, diharapkan perusahaan dapat menghindari ancaman penurunan penjualan akibat musim hujan. KESIMPULAN DAN SARAN Capps CJ, GIissmeyer MD. 2012. Extending the competitive profiIe matrix using internal factor evaIuation and externaI factor evaIuation matrix concepts. JournaI of AppIied Business Research. 28(5):1059-1062. AnaIisis Matriks QSPM MeIihat analisis lingkungan internal dan eksternal dengan matriks IFE dan EFE serta dipadankan dengan matriks IE dan SWOT. Selanjutnya, adalah tahap pengambiIan putusan prioritas strategis dengan QSPM. AnaIisis SWOT dan Quantitative Strategic Planning Matrix (QSPM) adalah analisis tingkat tinggi yang biasa digunakan untuk menentukan strategi manajemen (Setyorini, 2017). Berdasarkan hasil analisis QSPM, strategi diperoIeh nilai Total Attractiveness Score 363 Jurnal Aplikasi Bisnis dan Manajemen (JABM), Vol. 7 No. 2, Mei 2021 Jurnal Aplikasi Bisnis dan Manajemen (JABM), Vol. 7 No. 2, Mei 2021 berteknologi pengering sepatu. Strategi prioritas yang dapat diusulkan adalah meIakukan promosi diskon secara daring untuk meningkatkan jumIah kosumen dengan skor TAS 6,15. lainnya. Dua strategi di atas diutamakan karena strategi melakukan promosi dan bekerja sama dengan unit usaha lain akan dengan cepat meningkatkan daya saing atau peningkatan pangsa pasar sehingga perusahaan dapat menyiapkan strategi lain. KesimpuIan Hasil analisis internal dengan menggunakan matriks IFE adalah 2,30, dan hasil analisis eksternal dengan menggunakan matriks EFE adalah 2,92. Posisi perusahaan pada matriks IE berada pada kondisi perusahaan yang sedang. Strategi yang dapat dirumuskan adalah market penetration dan product development. Analisis SWOT DWS menghasilkan 8 alternatif strategi ,yaitu melakukan promosi diskon secara daring, mengadakan kerja sama dan kemitraan dengan beberapa unit, meningkatkan daya saing dengan peningkatan kualitas layanan, melakukan reminder wash shoe secara, memberikan privilege kepada pemiIik lahan berupa layanan gratis, mengadakan agenda training dalam bentuk gathering, memanfaatkan investor untuk penambahan modal dan biaya promosi daIam upaya meningkatkan segmentasi pasar, menggunakan alat Chan X. 2011. A SWOT study of the development strategy of haier group as one of the most successfuI chinese enterprises. InternationaI JurnaI of Business and SociaI Science 2(11): 147–153. David FR. 2016. Manajemen Strategik Suatu Pendekatan KeungguIan Bersaing. Puspasari, Novita, penerjemah; Dedy A, editor. Jakarta: SaIemba Empat. Terjemahan dari: Strategic Management. A Competitive Advantage Approach, Concepts amd Case. David ME, David FR. 2011. The quantitative strategic pIanning matrix (QSPM) appIied to a retaiI computer store. The CoastaI Business JournaI. 8(1):42-52. 364 Jurnal Aplikasi Bisnis dan Manajemen (JABM), Vol. 7 No. 2, Mei 2021 Jurnal Aplikasi Bisnis dan Manajemen (JABM), Vol. 7 No. 2, Mei 2021 branded oIeh generasi z di Surabaya. JurnaI Manajemen Bisnis 7(2): 4-5. Dewi NR. 2018. Formulasi strategi pengembangan bisnis salon xyz. Jurnal ApIikasi Manajemen dan Bisnis 4(3):372. Rahaju EE, Sumarlan. 2013. Identifikasi variabeI yang memotivasi konsumen menggunakan jasa laundry. JurnaI business review 2(2):55-56. Dwiastuti I. 2008. Analisis manajemen strategi industri aIternatif (Studi Kasus Biofuel). Jurnal Ekonomi dan Pembangunan 16(1):21-33. Raymond MIB, Ine M, Iwang G. 2012. AnaIisis pengembangan usaha pemindangan ikan di Kecamatan bekasi Barat. JurnaI Perikanan dan KeIautan 3(1): 17–24. Eko JAS. 2013. Strategi keunggulan bersaing pada Diva Iaundry daIam menghadapi persaingan antar usaha jasa di Mojokerto. Jurnal IImiah Universitas Brawijaya 2(1):5-7. Rofik A. 2017. The Marketing strategy of Sshoe washing services in Surabaya (case study of farcIean shoes washing in Surabaya). JurnaI Manajemen Kinerja 3(2):7-9. Hutahaean R. 2017. Perumusan strategi bisnis PT XYZ. JurnaI ApIikasi Manajemen dan Bisnis 3(3):454. Sarwono J. 2006. Metode Penelitian Kuantitatif dan KuaIitatif . Yogyakarta: Graha IImu. Isnandar FR, Firdaus M, Maulana A. 2016. Strategi peningkatan aset PT BPR Syariah Harta Insan Karimah (HIK) CiIedug. JurnaI ApIikasi Bisnis dan Manajemen 2(1): 12–22. Setyorini R, Rey RO. 2017. KesimpuIan AnaIisis model bisnis pada eighteen nineteen laundry dengan pendekatan business modeI canvas. Jurnal Sekretariat Administrasi Bisnis 1(1):70-81. Kementerian Koperasi dan UMKM 2020. Perkembangan Data Usaha Mikro, KeciI, Menengah (UMKM) dan Usaha Besar (UB) Tahun 2012-2-18. http:// www.depkop.go.id. [2 Mar 2020]. Siregar IV. 2019. AnaIisis prospek dan strategi pengembangan usaha jasa laundry berbasis syariah di kota Medan. Jurnal Ilmu Manajemen dan Bisnis Islam 5(1):115-117. KotIer P, KeIIer KI. 2016. Marketing Management, 15th. New Jersey: Pearson Pretice HaII, Inc. Sugiyono. 2013. Metode Penelitian Pendidikan Pendekatan Kuantitatif, Kualitatif, dan R&D. Bandung: AIfabeta Kurniawati T, Sari KBDK. 2009. Analisis dan piIihan Strategi: membangun eksistensi perusahaan di masa kritis. JournaI Ekonomi Bisnis 14(3): 179– 190. Umar A, Sasongko AH, Aguzman G, Sugiharto. 2016. Analisa SWOT pada bisnis rumahan studi kasus pada bisnis laundry kiloan. Jurnal Bisnis dan Manajemen 2(2): 91-94. Nuriyawan. 2011. Faktor-faktor yang memengaruhi kepuasan konsumen daIam memakai jasa cuci kiIoan pada D’Iaundry dan dry cIean di Ktintang Baru SeIatan Surabaya [tesis]. Surabaya: Universitas Pembangunan NasionaI. Varatisha AB. 2017. Sosial media sebagai pasar bagi masyarakat modern sebuah kritik terhadap budaya popuIer. 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How to start your monocot CRISPR/Cas project: plasmid design, efficiency detection, and offspring analysis
Rice
2,020
cc-by
14,017
Abstract The breakthrough CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-mediated genome-editing technology has led to great progress in monocot research; however, several factors need to be considered for the efficient implementation of this technology. To generate genome-edited crops, single guide (sg)RNA and Cas9 DNA are delivered into plant cells and expressed, and the predicted position is targeted. Analyses of successful targeted mutations have revealed that the expression levels, expression timing, and variants of both sgRNA and Cas9 need to be sophisticatedly regulated; therefore, the promoters of these genes and the target site positions are the key factors for genome-editing efficiency. Currently, various vectors and online tools are available to aid sgRNA design. Furthermore, to reduce the sequence limitation of the protospacer adjacent motif (PAM) and for other purposes, many Cas protein variants and base editors can be used in plants. Before the stable transformation of a plant, the evaluation of vectors and target sites is therefore very important. Moreover, the delivery of Cas9-sgRNA The breakthrough CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-mediated genome-editing technology has led to great progress in monocot research; however, several factors need to be considered for the efficient implementation of this technology. To generate genome-edited crops, single guide (sg)RNA and Cas9 DNA are delivered into plant cells and expressed, and the predicted position is targeted. Analyses of successful targeted mutations have revealed that the expression levels, expression timing, and variants of both sgRNA and Cas9 need to be sophisticatedly regulated; therefore, the promoters of these genes and the target site positions are the key factors for genome-editing efficiency. Currently, various vectors and online tools are available to aid sgRNA design. Furthermore, to reduce the sequence limitation of the protospacer adjacent motif (PAM) and for other purposes, many Cas protein variants and base editors can be used in plants. Before the stable transformation of a plant, the evaluation of vectors and target sites is therefore very important. Moreover, the delivery of Cas9-sgRNA ribonucleoproteins (RNPs) is one strategy that can be used to prevent transgene issues with the expression of sgRNA and Cas proteins. RNPs can be used to efficiently generate transgene-free genome-edited crops that can reduce transgene issues related to the generation of genetically modified organisms. In this review, we introduce new techniques for genome editing and identifying marker-free genome-edited mutants in monocot crops. Abstract Four topics are covered: the design and construction of plasmids for genome editing in monocots; alternatives to SpCas9; protoplasts and CRISPR; and screening for marker-free CRISPR/Cas9-induced mutants. We have aimed to encompass a full spectrum of information for genome editing in monocot crops. Keywords: Cas12a, Genome editing, Plant transformation, Promoter, Protoplast REVIEW 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. * Correspondence: cslin99@gate.sinica.edu.tw †Jin-Jun Yue, Chwan-Yang Hong, Pengcheng Wei and Yu-Chang Tsai contributed equally to this work. 5Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan Full list of author information is available at the end of the article Yue et al. Rice (2020) 13:9 https://doi.org/10.1186/s12284-019-0354-2 Yue et al. Rice (2020) 13:9 https://doi.org/10.1186/s12284-019-0354-2 Yue et al. Rice (2020) 13:9 https://doi.org/10.1186/s12284-019-0354-2 How to start your monocot CRISPR/Cas project: plasmid design, efficiency detection, and offspring analysis Jin-Jun Yue1†, Chwan-Yang Hong2†, Pengcheng Wei3†, Yu-Chang Tsai4† and Choun-Sea Lin5* Design and Construction of Plasmids for Genome Editing in Monocots double-strand breaks (DSBs), thereby generating a short deletion or insertion. Genome-edited plants can be gen- erated either by stable or transient transformation. For stable transformations, the Agrobacterium-mediated transformation method is typically used to deliver trans- fer DNA (T-DNA) into the plant cell, where it is then inserted into the plant genome (Mikami et al. 2015a; Nandy et al. 2019). For transient transformations, par- ticle bombardment and polyethylene glycol (PEG)-medi- ated methods are used to deliver plasmids or ribonucleoproteins (RNPs) into the plant cells (Woo et al. 2015; Svitashev et al. 2016; Zhang et al. 2016; Lin et al. 2018). Plasmids used for the stable genome editing of plants require a selection cassette, known as a sgRNA cassette, and a clustered regularly interspaced short pal- indromic repeat (CRISPR)/CRISPR-associated protein 9 The CRISPR/Cas9 system has been successfully used for genome editing in a variety of monocots, including rice (Oryza sativa), wheat (Triticum sp.), barley (Hordeum vulgare), and maize (Zea mays) (Feng et al. 2018; Gas- paris et al. 2018; Hu et al. 2018; Kis et al. 2019; Okamoto et al. 2017; Wang et al. 2017; Zhang et al. 2016). To per- form CRISPR/Cas9-mediated genome editing, the Cas9 endonuclease is guided by a single guide RNA (sgRNA) to recognize the complementary sequence and create * Correspondence: cslin99@gate.sinica.edu.tw †Jin-Jun Yue, Chwan-Yang Hong, Pengcheng Wei and Yu-Chang Tsai contributed equally to this work. 5Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan Full list of author information is available at the end of the article Page 2 of 13 Yue et al. Rice Yue et al. Rice (2020) 13:9 (2020) 13:9 Fig. 1 Schematic of the T-DNA region in a binary vector for genome editing in monocots. PPol III: Polymerase III promoter. PPol II: Polymerase II promoter. PUbi1: maize Ubiquitin 1 promoter and the first exon. Hpt: Hygromycin phosphotransferase. (Modified from Howells et al. 2018) (Cas9) cassette in the T-DNA region, while the selection cassette is not necessary for transient transformations (Fig. 1). addition to promoters, the specificity of the sgRNA for its target DNA sequence is another factor that affects the efficiency of genome editing. Several resources available on the web can be used to design highly spe- cific sgRNAs for use with the CRISPR/Cas9 system (Table 1; modified from Zhang lab, https://zlab.bio/ guide-design-resources). Nevertheless, even if these 20-nt sequences perfectly match the target gene, some sgRNAs do not work well. Design and Construction of Plasmids for Genome Editing in Monocots The online tools listed in Table 1 recommend targets with a low risk of an off- target match, but not all predicted target sequences may result in an efficient mutation. In addition, tar- geted DNA sequences with GC contents higher than 50% have higher genome-editing efficiencies (88.5– 89.6%) than those with GC contents lower than 50% (77.2% efficiency) (Ma et al. 2015). Successive Ts in 20-nt target sequence is not good when sgRNA ex- pression is driven by the U3 or U6 promoters (Wu et al. 2014). 1. Selection cassette: In monocots, several genes have served as useful selection markers for the efficient selection of transgenic plants, such as neomycin- phosphotransferase (NPTII), bar, mutated acetolactate synthase (ALS), plant phosphomannose isomerase, and hygromycin phosphotransferase (Hpt) (Miki and McHugh 2004; Hu et al. 2016). Among these genes, Hpt is the most widely used selection marker which confers tolerance to the herbicide hygromycin because several crops have a natural tolerance to kanamycin. Plant phosphomannose isomerase can also be used as a selectable marker for rice transformation (Hu et al. 2016). The expression of Hpt is usually driven by a strong constitutive promoter, such as maize Ubiquitin 1 (ZmUbi1), rice ACTIN 1, or Cauliflower mosaic virus (CaMV) 35S, for the ubiquitous expression of the antibiotic-tolerance gene (Mikami et al. 2015a). 1. Selection cassette: In monocots, several genes have served as useful selection markers for the efficient selection of transgenic plants, such as neomycin- phosphotransferase (NPTII), bar, mutated acetolactate synthase (ALS), plant phosphomannose isomerase, and hygromycin phosphotransferase (Hpt) (Miki and McHugh 2004; Hu et al. 2016). Among these genes, Hpt is the most widely used selection marker which confers tolerance to the herbicide hygromycin because several crops have a natural tolerance to kanamycin. Plant phosphomannose isomerase can also be used as a selectable marker for rice transformation (Hu et al. 2016). The expression of Hpt is usually driven by a strong constitutive promoter, such as maize Ubiquitin 1 (ZmUbi1), rice ACTIN 1, or Cauliflower mosaic virus (CaMV) 35S, for the ubiquitous expression of the antibiotic-tolerance gene (Mikami et al. 2015a). ) . Cas9 cassette: Aspects of the Cas9 cassette that affect the mutation rate during genome editing include the expression level and codon usage of Cas9 (Ma et al. 2015; Xie et al. 2015). Design and Construction of Plasmids for Genome Editing in Monocots Several strategies have been conducted to improve the expression of Cas9, including the use of a strong constitutive promoter, the addition of a translational enhancer, and the addition of nuclear localization signals. Constitutive promoters have been used to direct the expression of Cas9, including the ZmUbi1 and 35S. The Cas9 nucleases from different bacteria may have variations in the protospacer adjacent motif (PAM) sequence that they require for cleavage; therefore, many Cas9 homologs with different PAM requirements have been isolated from different bacteria. These Cas proteins are introduced in ‘Alternatives to SpCas9’ section. Streptococcus pyogenes Cas9 (SpCas9) is the most common Cas used in CRISPR/Cas9-mediated genome editing. It has been codon-optimized for maize (Xing et al. 2014) and rice (Miao et al. 2013) to improve its expression levels in these monocots. 3. 2. sgRNA cassette: sgRNA is a programmable 20- nucleotide (nt) sequence that recognizes the target DNA sequence and an invariant scaffold sequence (Ran et al. 2013), then directs the Cas nuclease to cleave the target sequence. Two factors are important for the function of sgRNA; promoter activity and the specificity of the sgRNA. A mixed dual promoter sys- tem is generally used in CRISPR/Cas9 system. In this system, Cas9 is directed by the RNA Polymerase II (Pol II) promoter while sgRNA expression is regulated by a Pol III promoter such as U6 or U3. To increase the transcription of the sgRNA, several monocot- specific U3 or U6 promoters have been cloned and used to direct the expression of the sgRNA, such as those from rice (Ma et al. 2015), maize (Qi et al. 2018), and wheat (Xing et al. 2014). In rice, the sgRNA driven by the OsU6 promoter produces more tran- scripts than when driven by the OsU3 promoter (Mikami et al. 2015a); however, several promoters, in- cluding OsU3, OsU6a, OsU6b, and OsU6c, have been used to direct the expression of sgRNA, and all of them could effectively direct genome editing with mu- tation rates of 81.4–90.0% (Ma et al. 2015; Shan et al. 2013; Xie and Yang 2013; Zhou et al. 2014). In Strategies for Multiplex Genome Editing in Monocots One advantage of the CRISPR/Cas9 system over other crop-breeding strategies is its flexibility for multiplex Yue et al. Rice (2020) 13:9 Page 3 of 13 Yue et al. Rice (2020) 13:9 Table 1 Web-based tools for sgRNA design Name Website Reference Benchling https://www.benchling.com/crispr/ Benchling, CA Broad Institute GPP https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design Doench et al. 2016 CHOPCHOP http://chopchop.cbu.uib.no/ Labun et al. 2019 CRISPOR http://crispor.tefor.net/ Concordet and Haeussler 2018 CRISPR-P http://cbi.hzau.edu.cn/CRISPR2/. Liu et al. 2017 DeskGen https://www.deskgen.com/landing/#/ Desktop Genetics, MA E-CRISP http://www.e-crisp.org/E-CRISP/designcrispr.html Heigwer et al. 2014 Horizon Discovery https://dharmacon.horizondiscovery.com/gene-editing/crispr-cas9/crispr-design-tool/ Horizon Discovery, UK IDT https://sg.idtdna.com/site/order/designtool/index/CRISPR_CUSTOM Integrated DNA Technologies, IA Off-Spotter https://cm.jefferson.edu/Off-Spotter/ Pliatsika and Rigoutsos 2015 Synthego https://www.synthego.com/products/bioinformatics/crispr-design-tool Synthego, CA ZiFiT http://zifit.partners.org/ZiFiT/ChoiceMenu.aspx Sander et al. 2010 Table 1 Web-based tools for sgRNA design Name Website Reference Benchling https://www.benchling.com/crispr/ Benchling, CA Broad Institute GPP https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design Doench et al. 2016 CHOPCHOP http://chopchop.cbu.uib.no/ Labun et al. 2019 CRISPOR http://crispor.tefor.net/ Concordet and Haeussler 2018 CRISPR-P http://cbi.hzau.edu.cn/CRISPR2/. Liu et al. 2017 DeskGen https://www.deskgen.com/landing/#/ Desktop Genetics, MA E-CRISP http://www.e-crisp.org/E-CRISP/designcrispr.html Heigwer et al. 2014 Horizon Discovery https://dharmacon.horizondiscovery.com/gene-editing/crispr-cas9/crispr-design-tool/ Horizon Discovery, UK IDT https://sg.idtdna.com/site/order/designtool/index/CRISPR_CUSTOM Integrated DNA Technologies, IA Off-Spotter https://cm.jefferson.edu/Off-Spotter/ Pliatsika and Rigoutsos 2015 Synthego https://www.synthego.com/products/bioinformatics/crispr-design-tool Synthego, CA ZiFiT http://zifit.partners.org/ZiFiT/ChoiceMenu.aspx Sander et al. 2010 Table 1 Web-based tools for sgRNA design genome editing (Wang et al. 2017; Wang et al. 2018). The editing of multiple functional genes allows for the rapid improvement of multiple agronomic traits at one time, while editing the cis-acting elements of a promoter affects transcriptional regulation. The deletion of larger fragments between two sgRNA-targeted sites on the same chromosome following the generation of multiple DSBs has been reported in many species. The CRISPR/Cas9 sys- tem has been used to delete DNA fragments ranging from dozens of bases to greater than 1 Mb (Mali et al. 2013; Shan et al. 2013). In addition, targeted deletions of 10 bp to over 200 kb between two target sites have been re- ported in rice (Mikami et al. 2016; Zhou et al. 2014). The elimination of the Tos17 retrotransposon using CRISPR/ genome editing (Wang et al. 2017; Wang et al. 2018). The editing of multiple functional genes allows for the rapid improvement of multiple agronomic traits at one time, while editing the cis-acting elements of a promoter affects transcriptional regulation. The deletion of larger fragments between two sgRNA-targeted sites on the same chromosome following the generation of multiple DSBs has been reported in many species. Strategies for Multiplex Genome Editing in Monocots The CRISPR/Cas9 sys- tem has been used to delete DNA fragments ranging from dozens of bases to greater than 1 Mb (Mali et al. 2013; Shan et al. 2013). In addition, targeted deletions of 10 bp to over 200 kb between two target sites have been re- ported in rice (Mikami et al. 2016; Zhou et al. 2014). The elimination of the Tos17 retrotransposon using CRISPR/ Cas9 was reported in rice, providing a rapid breeding route for making reverting the agronomically important genes that have been inactivated by the insertion of trans- posable elements (Saika et al. 2019). Multiplex genome editing can be achieved by the sim- ultaneous delivery and expression of multiple sgRNAs; however, since most CRISPR/Cas9 components are transferred into plants via Agrobacterium-mediated transformations, an efficient plasmid construction strat- egy is required. Traditionally, multiple sgRNA expression cassettes (including a Pol III promoter, a sgRNA, and a terminator) can be stacked into one T-DNA (Fig. 2a); however, this may increase the cloning difficulties due to the limited restriction sites available, and the fact that Fig. 2 Diagram of a plasmid construct used for multiplex genome editing. a Stacking of multiple sgRNA expression cassettes in one T-DNA. P Pol III: Polymerase III promoter. b Cloning of four tRNA-gRNAs into the CRISPR/Cas9 binary vector using a single Golden Gate ligation. (Modified from Kurata et al. 2018) Fig. 2 Diagram of a plasmid construct used for multiplex genome editing. a Stacking of multiple sgRNA expression cassettes in one T-DNA. P Pol III: Polymerase III promoter. b Cloning of four tRNA-gRNAs into the CRISPR/Cas9 binary vector using a single Golden Gate ligation. (Modified from Kurata et al. 2018) Yue et al. Rice (2020) 13:9 Page 4 of 13 Page 4 of 13 large T-DNAs may decrease the transformation effi- ciency. Alternative strategies have therefore been devel- oped to facilitate multiplexed genome editing in plants. These alternative strategies are based on the expression of multiple sgRNAs as a single transcript, after which multiple functional sgRNAs are generated following the processing of the transcripts by exogenous ribozymes (Gao and Zhao 2014), Csy-type ribonuclease 4 (Csy4) (Cermak et al. 2017), or the plant endogenous transfer RNA (tRNA)-processing system (Xie et al. 2015). The endogenous tRNA-processing system exists in almost all organisms and has been successfully used to perform multiplex genome editing in rice (Xie et al. 2015). Alternatives to SpCas9 p The CRISPR/Cas systems are divided into two classes. The Class 1 systems possess multiple Cas protein sub- units, whereas the Class 2 systems utilize a single, multi- functional protein effector (Shmakov et al. 2015). Class 2 CRISPR systems are further divided into types II, V, and VI. Along with the commonly used type II effector SpCas9, other orthologs with RNA-guided site-specific nuclease (SSN) activity have been engineered for use as tools in the genome editing of eukaryotic cells. These orthologs include proteins from Staphylococcus aureus (SaCas9), Streptococcus thermophilus (St1Cas9), Franci- sella novicida (FnCas9), Neisseria meningitidis (NmCas9), Brevibacillus laterosporus (BlCas9), and Campylobacter jejuni (CjCas9) (Cong et al. 2013; Hou et al. 2013; Sampson et al. 2013; Ran et al. 2015; Karvelis et al. 2015; Kim et al. 2017a; Chatterjee et al. 2018; Edraki et al. 2019). These Cas9s can be a welcome com- plement to the editing ability of SpCas9 in plants. Here, we describe plant genome-editing tools developed from these orthologs and discuss their advantages for research in botany. Reducing off-Target Mutations and Lethality in Monocots Several reports have indicated that constitutively expressed Cas9 produces an excess of sgRNA-Cas9, which may increase the incidence of genome-wide off- target mutations (Hsu et al. 2013; Hu et al. 2018; Patta- nayak et al. 2013; Svitashev et al. 2016). By contrast, con- ditionally or transiently expressing Cas9 significantly reduces the frequency of off-target mutations (Srivastava et al. 2017; Zhang et al. 2014). Increasing evidence has indicated that conditionally expressing Cas9 at the plant regeneration phase can markedly improve genome- editing efficiency, and this kind of conditional targeting could avoid the lethal phenotype caused by the cleavage of genes essential for development (Srivastava et al. 2017; Zhang et al. 2014). The promoter of the gene en- coding heat-shock protein 17.5E (Hsp17.5E) from soy- bean (Glycine max) has been used to direct the expression of Cas9 for genome editing in rice. The mu- tation frequency was 16% and 50–63% among the trans- genic lines before and after a heat treatment, respectively (Nandy et al. 2019). In maize, Cas9 driven by the meiosis-specific Disrupted Meiotic cDNA 1 (ZmDMC1) promoter was able to generate up to 66% homozygous or bi-allelic mutants, and no off-target mu- tations were detected using whole-genome sequencing (Feng et al. 2018). The vector delivery of preassembled Strategies for Multiplex Genome Editing in Monocots In the tRNA-processing system, the Pol III promoter is used to direct the expression of a single synthetic gene contain- ing multiple tRNA-sgRNAs or a polycistronic tRNA- sgRNA (PTG) gene. The PTG gene can be generated using the Golden Gate assembly method (Lowder et al. 2015) (Fig. 2b). After transcribing the PTG, the en- dogenous RNases specific to tRNA would recognize the tRNA components and cleave the individual sgRNAs from the PTG transcript. The resulting sgRNAs would then guide Cas9 to multiple target sites for genome edit- ing. Many toolboxes are currently available in the public database Addgene (Cermak et al. 2017; Castel et al. 2019; Hahn et al. 2019), from where these vectors can be purchased by academic researchers. Cas9-sgRNA RNPs instead of DNA has been reported to significantly reduce the frequency of off-site cleavage in both protoplast and zygote systems (Toda et al. 2019). In maize embryo cells, the delivery of DNA vectors con- taining Cas9 and sgRNA showed a high frequency of off-site mutations (50%) when compared with the Cas9- sgRNA RNP complex (0%) (Svitashev et al. 2016). In most plant species, the isolation, cultivation, and regen- eration of protoplasts remains a challenge; however, re- cent work demonstrated the direct delivery of a Cas9- sgRNA RNP into rice zygotes using the in vitro fertilization of isolated gametes, resulting in a targeted mutation rate of 14–64%. This protoplast-free zygote system makes the RNP-mediated genome-editing system much easier to perform, which could be a potential av- enue for crop improvement in many monocot species (Toda et al. 2019). Type II Cas9 Systems Type II CRISPR systems are abundant in prokaryotic or- ganisms and are the principal resource used to develop gene-editing tools. In addition to the first-reported SpCas9, several studies have described the Cas9 ortholog-mediated genetic engineering of plants. SaCas9 is most frequently used in place of SpCas9 and provides comparable editing efficiency for eukaryotic genomes. Compared with SpCas9, SaCas9 may reduce the delivery barrier of the CRISPR system because of its smaller size (Friedland et al. 2015; Ran et al. 2015). SaCas9 and its engineered variant SaKKH (E782K/N968K/R1015H) (Kleinstiver et al. 2015), which relaxes the canonical Page 5 of 13 Page 5 of 13 Yue et al. Rice (2020) 13:9 Yue et al. Rice (2020) 13:9 Yue et al. Rice NNGRRT PAM of SaCas9 to NNNRRT, have been used to achieve the efficient targeted mutagenesis of Arabi- dopsis thaliana, tobacco (Nicotiana tabacum), rice, and citrus (Citrus sp.) (Kaya et al. 2016; Steinert et al. 2015; Jia et al. 2017; Qin et al. 2019). temperature used in crop culture systems may facilitate a higher editing efficiency when using Cas12a proteins. a higher editing efficiency when using Cas12a proteins. Cas12a proteins not only induce DSBs, but also process their own pre-crRNA for maturation. This RNase activity provides a simple pattern by which single or multiplex crRNA(s) can be expressed from the same transcription unit of Cas12a, facilitating the highly effi- cient genome editing of plants (Wang et al. 2017; Ding et al. 2018; Wang et al. 2018; Xu et al. 2019). In the gen- ome editing mediated by the CRISPR/Cas9 system, RNA could be used as a repair template for homology- directed repair (Butt et al. 2017). The self-processing ac- tivity of Cas12a means that homologous recombination- mediated gene replacements can be generated using the FnCas12a system (Li et al. 2019). In mammalian cells, CBEs were also developed using Cas12a proteins to fa- cilitate precise C-to-T conversions in A/T-rich regions (Li et al. 2018); however, similar systems have not yet been reported in plants. The engineered Alicyclobacillus acidiphilus Cas12b (AaCas12b) has been used to edit mammalian genomes under a wide range of tempera- tures (31–59 °C; Teng et al. 2018), but it is still unclear whether the high-temperature-preferring Cas12b sys- tems function in plants. Type II Cas9 Systems SaCas9 and SaKKH have also been used to develop plant base editors (BEs), including the cytosine BE (CBE) responsible for a C·G to T·A conversion and the adenine BE (ABE) responsible for the reverse substitution of A·T to G·C. Similar to the BEs derived from SpCas9, the SaCas9 BEs have been successfully used to induce spe- cific base conversions in the rice genome (Hua et al. 2018; Hua et al. 2019; Qin et al. 2019). Notably, the edit- ing windows of the SaCas9 BEs are much broader than those of the SpCas9 BEs, possibly due to differences in the formation of the R-loop complex (Kim et al. 2017d). Compared the conventional CRISPR/Cas9-targeted mu- tagenesis system, BEs developed from SpCas9 tend to be less efficient at editing certain targets and less flexible for specific nucleotides. Because of their recognition of different PAMs and the enlargement of the editing win- dow, the SaCas9 BEs have provided alternative tools for precise genome editing in plants. Another Cas9 ortholog, St1Cas9, has been used to in- duce mutations in the Arabidopsis gene ALCOHOL DE- HYDROGENASE 1 (ADH1) (Steinert et al. 2015), while FnCas9 was used to confer molecular immunity against RNA viruses in tobacco and Arabidopsis (Zhang et al. 2018). Type VI Cas Systems Type VI Cas Systems The type VI CRISPR proteins, such as Cas13a, can target and cleave the target RNA under the guidance of crRNA. The heterologous expression of Leptotrichia wadei Cas13a (LwaCas13a) causes the knockdown of target RNAs in mammalian and plant cells (Abudayyeh et al. 2017). Similarly, in both dicot and monocot plants, the transient or stable expression of a Cas13a-crRNA complex results in the targeting and degradation of the RNA transcripts of endogenous genes and foreign re- porters (Aman et al. 2018; Zhang et al. 2019a, 2019b). To precisely edit nucleotides in RNA, the ADAR2 ad- enosine deaminase or an evolved ADAR2 cytidine deam- inase was fused to a catalytically inactive Cas13a (dCas13a), generating C-to-U and A-to-I RNA editors in mammalian cells (Cox et al. 2017; Abudayyeh et al. 2019). RNA editing, especially organellar RNA editing, plays an irreplaceable role in plant growth and develop- ment; therefore, similar Cas13a tools are highly antici- pated to facilitate related research in plants. Type V Cas Systems Type V Cas Systems The CRISPR RNA (crRNA)-guided SSN activity of the type V Cas system, which includes Cas12a and Cas12b, also generates DSBs. Several Cas12a (formerly Cpf1) orthologs, such as F. novicida Cas12a (FnCas12a), Acida- minococcus sp. Cas12a (AsCas12a), and Lachnospiraceae sp. Cas12a (LbCas12a), have been engineered as a class of genome-editing tools distinct from the Cas9 system (Zetsche et al. 2015). The Cas12a proteins induce stag- gered DSBs at sites distal to a 5′ T-rich PAM, generating relatively longer deletions via the non-homologous end joining repair pathway in various plant species, including rice (Endo et al. 2016a; Begemann et al. 2017; Hu et al. 2017; Kim et al. 2017c; Tang et al. 2017; Xu et al. 2017). Similar to the SpCas9 systems, transgene-free Cas12a- mediated mutants can be generated by simply segregat- ing the T-DNA fragment in the transition from the T0 to T1 generations (Xu et al. 2017). In addition, mixing RNP with Cas12a and crRNA has allowed transgene-free genome editing in soybean and tobacco (Kim et al. 2017b). Interestingly, the Cas12a proteins have different sensitivities to temperature in plants (Malzahn et al. 2019); therefore, the optimization of the incubation Single Protoplast Analysis When using protoplasts to validate CRISPR efficiency, more than 100,000 protoplasts are typically used in each transfection experiment. DNA is extracted from the pooled protoplasts to enable the amplification of the tar- get region using PCR. The pooled protoplasts also con- tain unedited DNA, making mutations difficult to detect if the mutagenesis efficiency is low (Lin et al. 2018). Mu- tagenesis efficiency can also be assessed using next- generation sequencing, from which the density ratio of the target fragments or the editing percentage can be de- termined. This method is relatively accurate, but it is ex- pensive and time consuming. Recently, a convenient and reliable protocol for evaluating CRISPR mutagenesis effi- ciency from a single cell was established (Lin et al. 2018). In this approach, single cells can be isolated from various species and subjected to two rounds of PCR amplification and enzyme digestion without DNA purifi- cation to identify successful mutants. The mutated se- quences and the mutation efficiency could thus be analyzed directly, allowing even low-efficiency mutation events to be detected in maize. This single-cell analysis technique could be used to improve the precision and application range of CRISPR gene editing using protoplasts. y ) In comparison with the number of articles on stable rice CRISPR transformations, very few have been pub- lished using protoplasts for validation. One of the rea- sons for this is the efficiency of protoplast transfection and isolation. We have evaluated the different methods of protoplast transfection in rice, including PEG, electro- poration, and liposome delivery, which revealed that the PEG method has highest transfection efficiency (Lin and Hsu, personal comm.). The main bottleneck restricting the application of rice protoplasts in the evaluation of genome-editing reagents is thus considered to be proto- plast isolation. We previously improved the protocol for isolating Arabidopsis protoplasts (Tape Arabidopsis Sandwich; Wu et al. 2009) to facilitate their use for vari- ous purposes, and we are currently trying to develop a convenient rice protoplast isolation method, modifying two of the steps reported in previous rice mesophyll protoplast isolation protocols (Chen et al. 2006; Zhang et al. 2011). First, based on our observations, seedlings cut cross- sectionally retain more cells within the leaf sheath after digestion, meaning the mesophyll cells can be digested but not released. Protoplasts and CRISPR Protoplast Isolation and Validation Many different vectors are available for the expression of Cas proteins using different promoters. Researchers should choose a suitable vector based on their needs, and importantly should evaluate the target sites and con- structs using a transient expression system before per- forming the stable transformation to reduce the time Yue et al. Rice (2020) 13:9 Yue et al. Rice (2020) 13:9 Page 6 of 13 Page 6 of 13 and labor required. Protoplasts are often used for plant science investigations (Marx 2016), and the convenience and speed of their transfection means they are an at- tractive model in which to assess the mutagenesis effi- ciency of a CRISPR/Cas system, including the validation of Cas protein codon optimizations or modifications, sgRNA target sites, the promoters used for sgRNA and Cas9 proteins, and different vector designs (Andersson et al. 2017; Butt et al. 2017; Cermak et al. 2017; Endo et al. 2019; Hsu et al. 2019; Hsu et al. in preparation; Li et al. 2013; Li et al. 2018; Liang et al. 2014; Lowder et al. 2015; Shan et al. 2013; Sun et al. 2015; Zong et al. 2017, 2018). Rice, tobacco, and soybean protoplasts have been used to analyze Cas9 and Cas12a (Kim et al. 2017; Tang et al. 2017), while Cas13a was examined in rice proto- plasts (Abudayyeh et al. 2017). Protoplast transfection can also be used to evaluate the efficiency of the use of RNPs (Andersson et al. 2018; Hsu et al. in preparation; Kim et al. 2017; Malnoy et al. 2016; Woo et al. 2015). (Yakult, Japan) solution typically used for rice (Zhang et al. 2011). This protocol can also be applied to other Poaceae species, including wheat, bamboo (Bambusa oldhamii), millet (Setaria italica), and maize (Lin et al. 2018). We believe this convenient method will be of benefit not only in rice, but also for Poaceae crop re- search in general. We have also established protoplast isolation protocols for use with the Solanaceae and Cru- ciferae (Hsu et al. submitted). The protoplasts isolated using these methods can be used to more rapidly evalu- ate the genome-editing efficiency in crops (Lin et al. 2018). CRISPR-Edited Protoplast Regeneration transfection (Chen et al. 2018); however, the gene- editing/mutation efficiencies of these methods are low (less than 10%). The protoplast transformation strategy had a higher efficiency; for example, over 50% of tobacco protoplasts were mutagenized using this technique, which was similar to the numbers transformed during the efficiency evaluation with- out antibiotic selection. In addition to the validation of transformation efficiency, mutated protoplasts have the advantage of being able to regenerate into entire mutant plants. Protoplasts isolated from meristematic tissues or totipotent cells were first used for plant regeneration in the early 1970s (Takebe et al. 1971), and just a few years later, researchers used protoplasts as materials for plant transformation (Mar- ton et al. 1979). The progeny of N. tabacum regenerated from transformed protoplasts displayed the mutant phenotype, indicating that the transgenic tumor markers (octopine and nopaline) were inherited through meiosis (Wullems et al. 1981a; Wullems et al. 1981b). Monocot protoplast regeneration (Abdullah et al. 1986; Fujimura et al. 1985; Rhodes et al. 1988a) and transformation (Rhodes et al. 1988b; Shimamoto et al. 1989; Toriyama et al. 1988) protocols have also been established. out antibiotic selection. Protoplast transformation can deliver more than one reagent. The current Agrobacterium vectors used for stable transformations are multiplex sgRNAs. The genes encoding Cas proteins are large, and no multiplex Cas protein vectors are currently available. Three different Cas protein vectors (SaCas9, FnCas12a, and nCas9-Target-AID) can be delivered into a single protoplast to edit three dif- ferent target sites without interference (Hsu et al. 2019). The introduction of these three Cas protein vectors requires three subsequent Agrobacterium- mediated transformations, the crossing of individual mutants, or the co-transformation of Agrobacterium harboring independent Cas9s and different selection markers.. In addition to sgRNA and Cas proteins, donor DNA fragments are also required for knock- in genome editing, and must be delivered using Agrobacterium (Endo et al. 2016b; Miki et al. 2018;, Wolter and Puchta 2019). Using these protocols, only a few copies of donor DNA can be used in genome editing, which may be one of the reasons for the low efficiency of knock-in genome editing. Although multiple T-DNAs can be delivered into plant nuclei to increase the editing efficiency, this also increases the difficulty of removing the T- DNAs to produce marker-free plants. In proto- plasts, however, the donor DNA can be delivered in microgram quantities, which may help increase the knock-in efficiency. CRISPR-Edited Protoplast Regeneration Not only can the sgRNAs, Cas proteins, and donor DNAs be more easily delivered into protoplasts, but the application time and amounts of genome-editing reagents can be con- trolled to improve the editing efficiency. 2. p Cas proteins and sgRNAs are sufficient for CRISPR/ Cas genome editing and are no longer required once the genes have been edited. Transient expression or the dir- ect delivery of sgRNAs and Cas proteins into the cells is sufficient for editing; therefore, the DNA encoding the Cas proteins and sgRNAs does not need to be integrated into the genome for their continued expression, making the plants regenerated from these edited cells transgene- free. In 2015, Prof. Jin-Soo Kim’s group published a milestone article using protoplasts (Woo et al. 2015), in which RNP was used as the genome-editing reagent to edit lettuce (Lactuca sativa) protoplasts, which were subsequently regenerated into transgene-free plants. There are several advantages to generating transgene- free edited crops using protoplasts: 1. Protoplast transformation can be applied to edit hybrid and long-juvenile-phase crops, which are typically propagated using vegetative methods. Traditionally, the use of Agrobacterium tumefa- ciens-mediated or other stable transformation tech- niques means the transgene (selection markers, sgRNA, and Cas9) must be integrated into the gen- ome. In inbred crops, such as rice, this transgene can be removed through crossing; however, this causes the segregation of the desired traits in the offspring. This separation issue also occurs in sys- tems with a low editing efficiency, in which the transformants must be crossed in order to obtain the homozygous genotype. By contrast, homozy- gous edited crops can be achieved from heterozy- gous edited protoplasts using a second transfection (Hsu et al. 2019). Several methods are available for achieving this goal, including the delivery of RNP into the callus using a biolistic approach (Liang et al. 2017, 2019) or the transient expression of Cas9 and sgRNA using an Agrobacterium-mediated 1. Protoplast transformation can be applied to edit hybrid and long-juvenile-phase crops, which are typically propagated using vegetative methods. Traditionally, the use of Agrobacterium tumefa- ciens-mediated or other stable transformation tech- niques means the transgene (selection markers, sgRNA, and Cas9) must be integrated into the gen- ome. In inbred crops, such as rice, this transgene can be removed through crossing; however, this causes the segregation of the desired traits in the offspring. Single Protoplast Analysis The veins in rice run parallel to each other; therefore, we changed the cut direction from a random or cross-sectional cut to a longitudinal cut par- allel to the veins, which allows the enzyme solution to more easily access the cells and provides more surface area from which the protoplasts are released. To in- crease the efficiency of this process, multiple blades were fixed by a holder, creating a tool that can increase the speed of cutting (Lin et al. 2018). Second, we also assessed the enzyme components required for the diges- tion solution when using longitudinal cuts, revealing that the less expensive Cellulase R10 (Yakult, Japan) could be used in place of the more expensive Cellulase RS Although these convenient methods could be used for mesophyll isolation to provide the materials for the evaluation of CRISPR editing efficiency and accuracy, it is important to consider the correlation between CRISPR efficiencies using mesophyll protoplasts and stable trans- formation, particularly in rice, for which a callus is typic- ally used as the material for stable transformation (Kaya et al. 2016). Certain target sites were found to have a high CRISPR efficiency in stable transformation experi- ments, but their use did not result in mutations in the mesophyll protoplasts (Toki, Endo, and Lin, personal comm.). We are therefore working on developing a rice protoplast isolation protocol using callus materials, which will enable the assessment of the gene-editing re- lationship between these protoplasts and the stably transformed calli. Page 7 of 13 Yue et al. Rice (2020) 13:9 CRISPR-Edited Protoplast Regeneration This separation issue also occurs in sys- tems with a low editing efficiency, in which the transformants must be crossed in order to obtain the homozygous genotype. By contrast, homozy- gous edited crops can be achieved from heterozy- gous edited protoplasts using a second transfection (Hsu et al. 2019). Several methods are available for achieving this goal, including the delivery of RNP into the callus using a biolistic approach (Liang et al. 2017, 2019) or the transient expression of Cas9 and sgRNA using an Agrobacterium-mediated p g y 3. Using RNPs, the issues of promoter and codon modification in different species can be solved. The original Cas proteins were obtained from microorganisms; therefore, their codons must be modified for vector construction in plant species, and their expression must be driven by plant promoters. These issues have been investigated in Arabidopsis, resulting in the identification of a T- DNA architecture causing homozygous mutations in the first generation after transformation (Castel et al. 2019); however, the transcription and transla- tion of these genes can still be problematic in differ- ent target crops. The resulting amount of Cas 3. Using RNPs, the issues of promoter and codon modification in different species can be solved. The original Cas proteins were obtained from microorganisms; therefore, their codons must be modified for vector construction in plant species, and their expression must be driven by plant promoters. These issues have been investigated in Arabidopsis, resulting in the identification of a T- DNA architecture causing homozygous mutations in the first generation after transformation (Castel et al. 2019); however, the transcription and transla- tion of these genes can still be problematic in differ- ent target crops. The resulting amount of Cas Yue et al. Rice (2020) 13:9 Yue et al. Rice (2020) 13:9 Yue et al. Rice (2020) 13:9 Page 8 of 13 Page 8 of 13 Yue et al. Rice Poaceae species (Rhodes et al. 1988a, 1988b). Recent discoveries have elucidated the mechanisms by which plants can regenerate, which could be applied to improve protoplast regeneration in the future (Lowe et al. 2016). In rice, it is possible to solve the lack of an efficient protoplast regeneration protocol using an alternative method; Cas9-sgRNA RNPs could be directly delivered into rice zygotes, which can then be cultured into mature plants (Toda et al. 2019). CRISPR-Edited Protoplast Regeneration A total of 14–64% of the transgene-free plants obtained using this method were found to contain the target mutations. proteins in the cell can cause a low editing effi- ciency; however, in protoplasts, RNPs can be used to solve this problem and provide a higher delivery efficiency for transfection. 4. With the exception of Arabidopsis, most Agrobacterium-mediated transformation protocols were performed using a tissue culture platform. In dicots, the regenerated plants were derived using organogenesis, meaning they were derived from multiple cells. Many edited transformants are therefore chimeric (Kaya et al2016). To validate their edited sequences, the transformant DNAs were sequenced. Diploids contain two alleles in each cell; therefore, if the transformants contained more than two alleles they were determined to be chimeric (Kaya et al. 2016). If the edited alleles are not present in the reproductive organs, the alleles cannot be passed on to the progeny. In contrast, protoplasts are single cells that are edited before the first cell division is completed. The regenerates are then derived from a single edited protoplast, meaning all cells have same genomic background, which enables the edited alleles to be transmitted to the next generation. In our previous studies, non- chimeric regenerates were derived from protoplasts edited using the Cas proteins Cas9, Cas12a, and Target-AID, and the genotypes were inherited in a Mendelian manner (Hsu et al. 2019). This phenomenon was also reported in lettuce (Woo et al. 2015). g Unexpected mutations can occur during protoplast regeneration. When 15 protoplast potato regenerants were sequenced, they were found to include a variety of mutations, including insertions/ deletions, chromosome rearrangements, and aneuploidy (Fossi et al. 2019). Indeed, somaclonal mutations occur in all tissue culture strategies, including micropropagation and somatic embryogenesis. In our experience using bamboo, somaclonal mutations occurred after a long-term subculture (Lin and Chang 1998; Lin et al. 2006, 2007; Liu et al. 2007). Using a shorter period for the protoplast regeneration or reducing the amount of supplemental plant growth regulators provided may reduce this mutation rate (Lin and Hsu, personal comm.). CRISPR/Cas can also introduce off-target mutations, with the mutation rate dependent on the CRISPR/Cas system; for example, in rice, CBE but not ABE induces genome-wide off-target mutations (Jin et al. 2019). Fortunately, off-target mutations do not have a major impact on crop breeding like they do in medical applications (Tang et al. CRISPR-Edited Protoplast Regeneration 2019); rather, they simply result in more than one edited transformant for each transformation. In addition, mutation breeding is a strategy used in traditional breeding. We can select the transformants with good traits and use these edited lines as parental lines during crop production. In our opinion, al- though mutations can occur during regeneration, the protoplast regeneration techniques currently available are useful tools for transgene-free genome editing. 2. RNPs (Andersson et al. 2018; Woo et al. 2015) and plasmids (Andersson et al. 2017; Zong et al. 2018) have been delivered into lettuce and potato (Solanum tubero- sum) protoplasts, which were subsequently regenerated into transgene-free genome-edited plants. In our lab, we established N. tabacum (Hsu et al. 2019; Lin et al. 2018), rapid cycle brassica (Brassica oleracea), wild tomato (So- lanum peruvianum), and N. benthamiana (Lin et al. in preparation) protoplast regenerations, and used these systems to establish RNP and plasmid DNA gene-editing platforms (Hsu et al. in preparation). Some issues are yet to be resolved in protoplast regeneration during genome editing, however: Screening for Marker-Free CRISPR/Cas9-Induced Mutants 1. The regeneration protocol is difficult to establish. Only a few protocols for genome editing and protoplast regeneration have been developed, all of which were achieved in dicots (Andersson et al. 2017, 2018; Hsu et al. 2019; Jin et al. 2019; Lin et al. 2018; Tuncel et al. 2019; Woo et al. 2015). Many protoplast regeneration protocols are available in other species, including rice (Shimamoto et al. 1989; Toriyama et al. 1988) and other important Genome editing is widely used produce new genetic var- iants in plants. Several approaches for creating genome- edited crops have been developed, including the CRISPR/Cas system, which can be used with various tis- sue types including protoplasts (as described in the pre- vious section), callus, leaf discs, and germline cells. Agrobacterium-mediated, PEG-mediated, particle Yue et al. Rice (2020) 13:9 Page 9 of 13 bombardment, and virus infection transformation methods are commonly used for the delivery of the CRISPR/Cas system into plants, resulting in stable or transient expression patterns. mitochondria and chloroplasts (Oung et al. 2015). These elevated H2O2 levels can be visualized directly using 3, 3’diaminobenzidine (DAB) staining (Wu et al. 2019). This approach can be easily applied to most monocot species. The transient expression of the CRISPR/Cas system can deliver DNA-based sgRNA and Cas9 RNP sequences or the proteins themselves (Chen et al. 2018; Zhang et al. 2016). In a DNA-based CRISPR/Cas9 system, re- combinant DNA can be transferred to the plant using Agrobacterium- or PEG-mediated transformations or particle bombardment, eliminating the need for herbi- cide or antibiotic selection steps. This method allows the expression of the CRISPR/Cas9 plasmid without requir- ing its integration into the plant genome. This approach can reduce regeneration time via tissue culture while producing mutation frequencies similar to the stable ex- pression of the CRISPR/Cas9 vector at the target site. More than 86% of wheat T0 mutants generated using this technique were transgene free (Zhang et al. 2016). In the Transgene-free CRISPR/Cas9 system, RNPs are assembled in vitro and directly delivered into the proto- plasts using a PEG fusion approach. The RNP complex directly targets the recognized sequences and induces DSBs (Park and Choe 2019; Woo et al. 2015). The bind- ing of the RNP complex to the target DNA is tight, and the half-life for dissociation is slow (more than 6 hours in vitro) (Didovyk et al. 2016). Identification of CRISPR/Cas9-Induced Mutations Identification of CRISPR/Cas9-Induced Mutations Following the delivery of the CRISPR/Cas system into monocot calli or other tissues, a T0 generation of plants harboring edited genes is regenerated. Three categories of site-directed nuclease systems (SDN1–3) are employed in genome-editing techniques (Podevin et al. 2013). SDN1 relies on the most common endogenous processes of non-homologous end-joining to repair DSBs in the plant DNA. This process is error prone, and may result in random mutations at the break site (Bor- tesi and Fischer 2015). SDN2 involves homology- directed repair using one or a few nucleotides as a tem- plate (EFSA Panel on Genetically Modified Organisms 2012). SDN3 uses the same repair mechanism as SDN2 but with a longer nucleotide template. Unlike SDN1, the repair processes used by SDN2 and SDN3 is not ran- dom, and does not cause substitutions, insertions, or de- letions at the repair sites. Researchers have developed several methods to in- crease the efficiency of screening large numbers of mu- tants (Table 2). These methods can detect on-target or off-target variants and include the restriction enzyme (RE) cleaved amplified polymorphic sequences (CAPS) assay (Shan et al. 2014), RE site created assays (Hodgens et al. 2017), T7 endonuclease I assays (T7E1) (Vouillot et al. 2015), polyacrylamide gel electrophoresis (PAGE)- based genotyping assay (Zhu et al. 2014), high-resolution melting analysis (HRM) (Thomas et al. 2014), PCR- and labeling-based method (Biswas et al. 2019), and anneal- ing at critical temperature PCR (ACT-PCR) (Hua et al. 2017). The majority of genome-edited plants involve the stable integration of the CRISPR/Cas9 system into the plant genome. CRISPR/Cas9 DNA is delivered by methods similar to those employed for transient expres- sion, followed by a herbicide or antibiotic selection of successful transformants containing a marker gene. Antibiotic and herbicide resistance markers have been widely used in plant biotechnology (Wilmink and Dons 1993); however, genome-integrated CRISPR/Cas9 carries a risk of increased off-target effects and requires re- searchers to follow the current regulations for typical genetically modified crops. To bypass these strict bio- safety regulations, CRISPR/Cas binary vectors containing selection markers or foreign DNA can be segregated in the progeny by self-pollinating or crossing the transfor- mants (Gao et al. 2016). The PCR amplification of vector sequences can be used to verify the presence of foreign DNA in the genome. A rapid method using antibiotics to identify marker-free genome-edited plants was also reported recently (Wu et al. 2019). Screening for Marker-Free CRISPR/Cas9-Induced Mutants After dissociation, the RNP complex is degraded quickly in the cell. Identification of CRISPR/Cas9-Induced Mutations 2014) HRM Homozygous DNA has a unique melting temperature (Tm), while mutated heterozygous DNA has a lower Tm Fast and efficient for detecting SNPs and indels in mutants Requires specific instrumentation and sensitivity is affected by amplicon size (Thomas et al. 2014) ACT-PCR A critical annealing temperature in PCR suppresses the mismatched annealing of the primer to the template, inhibiting the production of amplicons Simple, fast, economical, and can detect homozygous mutants Requires designing specific primers and is time consuming and/or labor intensive (Hua et al. 2017) PCR- and labeling-based assay Simple, effective, and sensitive Not able to reveal the exact nucleotide change in the mutant (Biswas et al. 2019) Whole-genome sequencing Identifies on-target and off-target mutations Costly and time consuming (Tang et al. 2018) Table 2 Advantages and disadvantages of different methods for CRISPR/Cas9-mediated mutant screening. (Adapted from Bao et al. 2019) Table 2 Advantages and disadvantages of different methods for CRISPR/Cas9-mediated mutant screening. (Adapted from Bao et al. 2019) PCR- and labeling-based assay Whole-genome sequencing method allows for the detection of indels with a high sensitivity (down to ±1 bp). to distinguish between homozygous mutants and the wild type. Other PCR-based analyses include PAGE, HRM, ACT- PCR, and PCR−/labeling-based assays. To distinguish genome-edited mutations from wild-type target se- quences, target region amplicons are migrated on a na- tive polyacrylamide gel. Homoduplex DNA migrates faster than heteroduplex DNA. To distinguish homozy- gous mutants from the wild type, unknown amplicons can be pre-mixed with wild-type amplicons before the assay. The migration pattern of the pre-mixed homozy- gous mutant and wild type will be similar to that of the heterozygous mutant. HRM is a fluorescence-based technique for determining the differences in the melting temperatures of heteroduplex and homoduplex DNA fragments. This technique can detect differences as small as 0.1 °C; however, its sensitivity is influenced by the amplicon length and variation of the mutated sequence. ACT-PCR assay can also distinguish homozygous mu- tant sequences at the target site using optimal annealing temperature and specific primers; however, the assay re- quires the design of specific primers and is time con- suming and labor intensive. A simple PCR- and amplicon labeling-based method was recently used to identify the CRISPR/Cas9-generated mutants in rice (Biswas et al. 2019). This approach requires two pairs of primers or a FAM-labeled allele-specific primer. Identification of CRISPR/Cas9-Induced Mutations Leaf sections from the T1 progeny of genome-edited rice were incubated with hygromycin B, an antibiotic commonly used for the positive selection of transgenic plants. In the presence of hygromycin B, the genome-edited rice plants that did not retain CRISPR/Cas vectors in their genome could produce reactive oxygen species (e.g., H2O2) in their The CAPS, indCAPS, and T7E1 cleavage assays for identifying gene-edited mutants are based on an enzym- atic approach. Wild-type and mutant sequences are amplified using PCR then subjected to enzyme digestion. A typical CAPS assay can be used if a RE cutting site is present at the CRISPR target site, which is disrupted once the sequences are mutated; however, RE sites are not always present within target regions. A similar dCAPS assay has been developed for genome regions not possessing different RE sites between the wild type and the mutant. This dCAPS assay introduces or dis- rupts a RE site near the mutation site by amplifying sev- eral mismatched nucleotides, which greatly increases the flexibility for selecting the sgRNA target sites. T7 endo- nuclease 1 can be useful for digesting the mismatched heteroduplexes formed between the wild-type and mu- tated strands; however, this method lacks the sensitivity Page 10 of 13 Yue et al. Rice (2020) 13:9 Yue et al. Rice (2020) 13:9 Yue et al. Rice Table 2 Advantages and disadvantages of different methods for CRISPR/Cas9-mediated mutant screening. (Adapted from Bao et al. 2019) Method Advantages Disadvantages Reference CAPS A RE site within the DNA target site is destroyed by a genome- editing mutation Simple, fast, economical, and can detect homozygous and heterozygous mutants Limited to the original target sequences (Shan et al. 2014) indCAPS A RE site is created using mismatch primers next to a DNA target site More flexibility for different types of indel Requires designing specific primers to distinguish known indel alleles (Hodgens et al. 2017) T7E1 cleavage assay T7 endonuclease 1 digests mismatched heteroduplexes formed between wild-type strands and mutated strands Simple, fast, economical, and can detect heterozygous mutants Cannot detect homozygous mutants (Vouillot et al. 2015) PAGE Homoduplex DNA migrates faster than heteroduplex DNA in native PAGE Simple, fast, economical, and can detect homozygous and heterozygous mutants Time consuming and low throughput (Zhu et al. Acknowledgments Acknowledgments We thank Miranda Loney and Fu-Hui Wu for editing. Authors’ contributions Authors contributions JJY, CYH, PW, YCT, and CSL contributed to the writing. CSL organized and prepared this manuscript. All authors read and approved the final manuscript. Identification of CRISPR/Cas9-Induced Mutations The sensitivity, precision, and reliability of the FAM-labeled Although all these PCR-based analyses enable the ef- fective, accurate, and economical screening of CRISPR/ Cas9-generated mutants, the identification of sequence changes resulting from SDN1-generated mutations re- quires the Sanger sequencing of amplicons generated from the target region. In addition, whole-genome se- quencing is a powerful tool for identifying not only on- target and off-target mutations, but also transgene-free plants produced by genome editing. The only drawback of whole-genome sequencing is its cost and time requirement. Availability of data and materials Not applicable. Funding g This research was supported by Academia Sinica, the Innovative Translational Agricultural Research Administrative Office (AS-KPQ-107-ITAR-10), and the Ministry of Science and Technology (105–2313-B-001 -007 -MY3 and 108– 2313-B-001-11 to CSL; 105-2628-B-036-MY3 and 108-2313-B-002-055-MY3 to CYH; 108-2313-B-002 -051 and107-2313-B-002 -023 to YCT), Taiwan. References Genome Biol 19:1 Bao A, Burritt DJ, Chen H, Zhou X, Cao D, Tran LP (2019) The CRISPR/Cas9 system and its applications in crop genome editing. Crit Rev Biotechnol 39:321–336 Gao Y, Zhao Y (2014) Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing. J Integr Plant Biol 56:343–349 Begemann MB, Gray BN, January E, Gordon GC, He Y, Liu H, Wu X, Brutnell TP, Mockler TC, Oufattole M (2017) Precise insertion and guided editing of higher plant genomes using Cpf1 CRISPR nucleases. 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EFSA J 10:2943 1Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou, China. 2Department of Agricultural Chemistry, College of Bioresources and Agriculture, National Taiwan University, Taipei, Taiwan. 3Key Laboratory of Rice Genetic Breeding of Anhui Province, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei, China. 4Department of Agronomy, National Taiwan University, Taipei, Taiwan. 5Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan. 1Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou, China. 2Department of Agricultural Chemistry, College of Bioresources and Agriculture, National Taiwan University, Taipei, Taiwan. 3Key Laboratory of Rice Genetic Breeding of Anhui Province, Rice Research Endo A, Masafumi M, Kaya H, Toki S (2016a) Efficient targeted mutagenesis of rice and tobacco genomes using Cpf1 from Francisella novicida. 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COMPLEX METHODS OF TREATMENT OF CHILDREN WITH ODONTOGENIC INFLAMMATION
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¬ i W | Yfc`S^dea_Sea^aV[[[ ]cS`[agSi[S^o`nh[dd^XWaUS`[\ ea_•`a_Xc   ɮ ɮ  ɖɹ ɮ ɖɹɮ ɮɹɮ ɮɹ   •  ISSN 2181-0966 Doi Journal 10.26739/2181-0966 ISSN 2181-0966 Doi Journal 10.26739/2181-0966 TОШКЕНТ-2023 TОШКЕНТ-2023 ɖɹ ɮ ɖɹɮ ɮɹɮ ɮɹ¬Yfc`S^dea_Sea^aV[[[]cS`[agSi[S^o`nh[dd^XWaUS`[\  ɮ ɮ  ɖɹ ɮ ɖɹɮ ɮɹɮ ɮɹ №2 (2023) DOI http://dx.doi.org/10.26739/ 2181-0966-2023-2 ɖɹ ɮ ɖɹɮ ɮɹɮ ɮɹ¬Yfc`S^dea_Sea^aV[[[]cS`[agSi[S^o`nh[dd^XWaUS`[\  ɮ ɮ  ɖɹ ɮ ɖɹɮ ɮɹɮ ɮɹ №2 (2023) DOI http://dx.doi.org/10.26739/ 2181-0966-2023-2 ɮ  ɖɹ ɮ ɖɹɮ ɮɹɮ ɮɹ¬Yfc`S^dea_Sea^aV[[[]cS`[agSi[S^o`nh[dd^XWaUS`[\№2 Yfc`S^dea_Sea^aV[[[]cS`[agSi[S^o`nh[dd^XWaUS`[\ №2 (2023) DOI http://dx.doi.org/10.26739/ 2181-0966-2023-2 agSi[S^o`nh[dd^XWaUS`[\№2 | 2023 Si[S^o`nh[dd^XWaUS`[\ 6739/ 2181-0966-2023-2 \]a^^XV[[” Заместитель главного редактора: Юлдашев Абдуазим Абдувалиевич октор медицинских наук, доцент Ташкентского Государственного Стоматологического института, Узбекистан рзахмедов Контакт редакций журналов. www.tadqiqot.uz ООО Tadqiqot город Ташкент, улица Амира Темура пр.1, дом-2. Web: http://www.tadqiqot.uz/; Email: info@tadqiqot.uz Тел: (+998-94) 404-0000 Аветиков Давид Саломонович доктор медицинских наук, профессор, Украина Амхадова Малкан Абдурашидовна доктор медицинских наук, профессор, Россия Копбаева Майра Тайтолеуовна доктор медицинских наук, профессор, Казахстан Грудянов Александр Иванович доктор медицинских наук, профессор, Россия Лосев Фёдор Фёдорович доктор медицинских наук, профессор, Россия Шаковец Наталья Вячеславовна доктор медицинских наук, профессор, Белоруссия Jun-Young Paeng доктор медицинских наук, профессор, Корея Jinichi Sakamoto доктор медицинских наук, профессор, Япония Дустмухамедов Дильшод Махмудович доктор медицинских наук, доцент, Узбекистан Ризаев Элёр Алимджанович доктор медицинских наук, доцент, Узбекистан Камалова Феруза Рахматиллаевна доктор медицинских наук, доцент, Узбекистан Абдувакилов Жахонгир Убайдулла угли доктор медицинских наук, доцент, Узбекистан Зоиров Тулкин Элназарович доктор медицинских наук, доцент, Узбекистан Мамедов Умиджон Суннатович доктор медицинских наук, доцент, Узбекистан Раимкулова Дилноза Фарходдиновна PhD, доцент, Узбекистан Юнусходжаева Мадина Камалитдиновна доцент, Узбекистан Билалов Эркин Назимович доктор медицинских наук, профессор, Узбекистан Билалов Эркин Назимович доктор медицинских наук, профессор, Узбекистан Новиков Вадим Михайлович доктор медицинских наук, профессор, Украина Новиков Вадим Михайлович доктор медицинских наук, профессор, Украина Бекжанова Ольга Есеновна доктор медицинских наук, профессор, Узбекистан Бахритдинова Фазилат Арифовна доктор медицинских наук, профессор, Узбекистан Шомуродов Кахрамон Эркинович доктор медицинских наук, доцент, Узбекистан Шамсиев Жахонгир Фазлиддинович доктор медицинских наук, доцент, Узбекистан Юсупалиходжаева Саодат Хамидуллаевна доктор медицинских наук, доцент, Узбекистан Вахидов Улугбек Нуритдитнович доктор медицинских наук, доцент, Узбекистан Jinichi Sakamoto доктор медицинских наук, профессор, Япония Муртазаев Саидмуродхон Саидаълоевич доктор медицинских наук, доцент, Узбекистан Дустмухамедов Дильшод Махмудович доктор медицинских наук, доцент, Узбекистан Шукурова Умида Абдурасуловна доктор медицинских наук, доцент, Узбекистан Ризаев Элёр Алимджанович доктор медицинских наук, доцент, Узбекистан Хасанова Лола Эмильевна доктор медицинских наук, доцент, Узбекистан Камалова Феруза Рахматиллаевна доктор медицинских наук, доцент, Узбекистан Абдувакилов Жахонгир Убайдулла угли доктор медицинских наук, доцент, Узбекистан Зоиров Тулкин Элназарович доктор медицинских наук, доцент, Узбекистан Мамедов Умиджон Суннатович доктор медицинских наук, доцент, Узбекистан Раимкулова Дилноза Фарходдиновна PhD, доцент, Узбекистан 3 3  ɸɖɮ ɸɮɖ” Fazilat A. Bahritdinova Doctor of Medical Sciences, Professor, Uzbekistan Kakhramon E. Shomurodov Doctor of Medical Sciences, Docent, Uzbekistan Ulugbek N. Vakhidov Doctor of Medical Sciences, Docent, Uzbekistan Jinichi Sakamoto Doctor of Medicine, Professor, Japan Dilshod M. Dustmukhamedov Doctor of Medical Sciences, Docent, Uzbekistan Saidmurodkhon S. Murtazaev Doctor of Medical Sciences, Docent, Uzbekistan Saidmurodkhon S. Murtazaev Doctor of Medical Sciences, Docent, Uzbekistan Rizaev Elyor Alimdjanovich Doctor of Medical Sciences, Docent, Uzbekistan Umida A. Shukurova Doctor of Medical Sciences, Docent, Uzbekistan Umida A. Shukurova Doctor of Medical Sciences, Docent, Uzbekistan Umida A. Shukurova Doctor of Medical Sciences, Docent, Uzbekistan Kamalova Feruza Raxmatillaevna Doctor of Medical Sciences, Docent, Uzbekistan Lola E. Khasanova Doctor of Medical Sciences, Docent, Uzbekistan Lola E. Khasanova Doctor of Medical Sciences, Docent, Uzbekistan Jakhongir U. Abduvakilov Doctor of Medical Sciences, Docent, Uzbekistan Tulkin E. Zoirov Doctor of Medical Sciences, Docent, Uzbekistan 4  ɮ ɮ  ɖɹ ɮ ɖɹɮ ɮɹɮ ɮɹ¬Yfc`S^dea_Sea^aV[[[]cS`[agSi[S^o`nh[dd^XWaUS`[\№2 | 2023 1. Ризаев Жасур Алимджанович, Агзамова Сайёра Саидаминовна, Туляганов Нозим Алишерович РЕЗУЛЬТАТЫ РЕТРОСПЕКТИВНОГО АНАЛИЗА СОЧЕТАННЫХ ТРАВМ СРЕДНЕЙ ЗОНЫ ЛИЦА……………………6 2. Ризаев Элёр Алимджанович, Бузрукзода Жавохирхон Даврон ИЗУЧЕНИЕ МОРФОЛОГИЧЕСКОЙ КАРТИНЫ ПРИ ДЕНТАЛЬНОЙ ИМПЛАНТАЦИИ…………………………………..13 3. Nasretdinova Maxzuna Taxsinovna, Normirova Nargiza Nazarovna, Baxronov Bezod Shavkatovich, Normuradov Nodir Alisherovich MUVOZANAT FUNKSIYASI BUZILGANDA VESTIBULYAR ANALIZATORNI BAHOLASH……………………………..17 4. Исламова Нилуфар Бустановна, Назарова Нодира Шариповна СУРУНКАЛИ ТАРҚАЛГАН ПАРОДОНТИТ БИЛАН КАСАЛЛАНГАН ПОСТМЕНОПАУЗА ДАВРИДАГИ АЁЛЛАРНИНГ ПАРОДОНТ ТЎҚИМАСИНИНГ ДАВОЛАШ САМАРАДОРЛИГИ ОШИРИШ……………20 5. Рахматова Дилнора Саиджоновна БОЛАЛАР ОРАСИДА ТИШ КАРИЕСИ КАСАЛЛИГИ ТЕКШИРИЛАЁТГАН БОЛАЛАРНИНГ КЛИНИК ХУСУСИЯТЛАРИ………………………………………………………………………………………………………25 6. Хамракулова Наргиза Орзуевна СУРУНКАЛИ СИНУСИТНИНГ УЗОҚ МУДДАТЛИ ШАКЛЛАРИ БЎЛГАН БЕМОРЛАРНИ БОШҚАРИШ ХУСУСИЯТЛАРИ………………………………………………………………………………………………….29 7. Насретдинова Махзуна Тахсиновна, Раупова Камола, Лутфуллаева Гульноза, Нормурадов Нодир Алишерович ОЦЕНКА ЭФФЕКТИВНОСТИ ЛЕЧЕНИЯ ПАЦИЕНТОВ С НЕЙРОСЕНСОРНОЙ ТУГОУХОСТЬЮ……………………..31 8. Терехов Алексей Борисович, Нэстасе Корнелиу Иванович СОВРЕМЕННАЯ КОНЦЕПЦИЯ В ДИАГНОСТИКЕ И ЛЕЧЕНИИ КАРИЕСА КОРНЯ………………………………………36 9. Ризаев Жасур Алимджанович, Шодмонов Ахрорбек А, Раджабий Музаянна Азиз кизи ОЦЕНКА ТОЧНОСТИ УСТАНОВКИ ДЕНТАЛЬНЫХ ИМПЛАНТАТОВ…………………………………………………….40 10. Ахмедов Хуршид Камалович ОРТОПЕДИК ДАВОЛАНИШДАН ОЛДИН БЕМОРЛАРДА ОҒИЗ БЎШЛИҒИДАГИ ЦИТОКИН ҲОЛАТИНИНГ ПАРАМЕТРЛАРИ АНИҚЛАШ УСУЛЛАРИ………………………………………………………………….44 11. Қурбонов Дилшод Фарходович, Хабибова Назира Насуллоевна ҲОМИЛАДОР АЁЛЛАР СТОМАТОЛОГИК САЛОМАТЛИГИНИ АНИҚЛАШ КЎРСАТКИЧЛАРИ………………………47 12. Эронов Ёқуб Қуватович ИМКОНИЯТИ ЧЕКЛАНГАН БОЛАЛАРДА ПАРОДОНТ ТЎҚИМАСИНИНГ МИКРОБИОЛОГИК ТАДҚИҚОД НАТИЖАЛАРИ СОЛИШТИРМА ТАҲЛИЛЛАРИ……………………………………………………………….52 13. Атоева Максад Амановна РАСПРОСТРАНЕННОСТЬ, ИНТЕНСИВНОСТЬ И ОСОБЕННОСТИ КЛИНИЧЕСКОГО ТЕЧЕНИЯ ЗАБОЛЕВАНИЙ ПАРОДОНТА У ЛИЦ МОЛОДОГО ВОЗРАСТА…………………………………………………………….55 14. Нурова Шоҳсанам Норпўлотовна АЁЛЛАРДА ЭСТРОГЕН ЕТИШМОВЧИЛИГИ ОҚИБАТИДА ТИШ-ЖАҒ ТИЗИМИДАГИ ЎЗГАРИШЛАР БЎЙИЧА УМУМИЙ ТАВСИФ………………………………………………………………………………….58 15. Идиев Ойбек Элмуродович, Ибрагимова Феруза Икромовна БОШ МИЯ ФАЛАЖИ БИЛАН КАСАЛЛАНГАН БОЛАЛАРДА ЧАЙНОВ МУШАКЛАРИНИНГ ЭЛЕКТРОМИОГРАФИК КЎРСАТКИЧЛАРИНИ АНИҚЛАШ…………………………………………………………………61 16.  ɸɖɮ ɸɮɖ” Астанов Отабек Миржонович ЧАККА-ПАСТКИ ЖАҒ БЎҒИМИ КАСАЛЛИКЛАРИДА КОМПЛЕКС ДАВОЛАШ УСУЛЛАРИНИНГ САМАРАДОРЛИГИНИ БАҲОЛАШ………………………………………………………………………..66 17. Ражабов Отабек Асрорович МЕТАЛЛОКЕРАМИК ПРОТЕЗИ БЎЛГАН БЕМОРЛАР ГУРУҲИДА ДАВОЛАШДАН ОЛДИНГИ ВА КЕЙИНГИ ИММУНОЛОГИК ТАДҚИҚОТЛАР ТАҲЛИЛИЙ НАТИЖАЛАРИ………………………………………….69 18. Taylakova Dildora Ibragimovna PREVENTION AND TREATMENT OF DENTAL FLUOROSIS IN CHILDREN…………………………………………………72 19. Ташева Гулчехра Сулямановна ОДОНТОГЕН ЯЛЛИҒЛАНИШ БИЛАН КАСАЛЛАНГАН БОЛАЛАРНИ КОМПЛЕКС ДАВОЛАШ УСУЛЛАРИ………..76 20. Фозилов Уктам Абдураззоқович ЮҚОРИГИ ЖАҒНИНГ ТОРАЙИШИ ҲИСОБИГА ЮЗАГА КЕЛГАН ПАТОЛОГИК ОКЛЮЗИЯНИ ДАВОЛАШ УСУЛЛАРИНИ ТАКОМИЛЛАШТИРИШ…………………………………………………………………………80 daWXcYS`[X¬ɹ   10. Ахмедов Хуршид Камалович 13. Атоева Максад Амановна 14. Нурова Шоҳсанам Норпўлотовна Ё 16. Астанов Отабек Миржонович Ў 17. Ражабов Отабек Асрорович 18. Taylakova Dildora Ibragimovna 5  ɮ ɮ  ɖɹ ɮ ɖɹɮ ɮɹɮ ɮɹ¬Yfc`S^dea_Sea^aV[[[]cS`[agSi[S^o`nh[dd^XWaUS`[\ №2 | 2023 АННОТАЦИЯ Ц Сегодня пациенты с одонтогенными воспалительными заболеваниями составляют 10-20% стоматологических заболеваний. А 30-70% одонтогенных воспалительных заболеваний - это абсцессы и флегмоны, совершенствование диагностики этих заболеваний и эффективное лечение считается одной из актуальных проблем. Важно разработать специальные меры лечения детей с этим заболеванием. Ключевые слова: одонтогенные воспалительные заболевания, абсцессы и флегмоны. Ташева Гулчехра Сулямановна Бухоро давлат тиббиёт институти Ташева Гулчехра Сулямановна Ташева Гулчехра Сулямановна Бухарский государственный медицинский институт Бухарский государственный медицинский институт http://dx.doi.org/10.5281/zenodo.8033098 http://dx.doi.org/10.5281/zenodo.8033098 АННОТАЦИЯ Ц Бугунги кунда одонтоген яллиғланиш касалликлари билан оғриган беморлар стоматологик касалликларни 10 – 20 % ташкил этади. Одонтоген яллиғланиш касалликларини 30 - 70 % ни эса абцесс ва флегмоналар ташкил этиб, ушбу касалликларга ташхис қўйишни мукаммаллаштириш ва самарали даволаш долзарб муаммолардан бири бўлиб ҳисобланади. Ушбу касаллик билан касалланган болаларда махсус даволаш чораларини ишлаб чиқиш муҳим аҳамиятга эга. у р р у Калит сўзлар: одонтоген яллиғланиш касалликлари, абцесс ва флегмоналар. 1-жадвал 1 жадва Одонтоген яллиғланиш асоратлари билан касалланган болаларда ёши ва жинсига қараб, гурухлараро тақсимланиши. Гурухлар Ўғил болалар сони Қиз болалар сони 0-6 ёшлилар сони 6-12 ёшлилар сони 12-18 ёшлилар сони Жами сони I 28 21 20 19 10 49 II 29 24 16 27 10 53 III 12 8 2 9 9 20 д иш асоратлари билан касалланган болаларда ёши ва жинсига қараб, гурухлараро тақсимланиши. Одонтоген яллиғланиш асоратлари билан касалланган болаларда ёши ва жинсига қараб, гурухл фаоллиги ва самарадорлигини одонтоген яллиғланиш касалликлар асоратларини олдини олишни таъминлайди. Қўллаш усуллари, бактериофаг воситаси қон ва лимфа ичига кириб, яллиғланиш медиаторларини бужмайтирувчи таъсир кўрсатади. Бактериофагнинг асосий қисми буйраклар орқали чиқарилади, сийдик йўлларида дезинфекциялаш таъсирига эга, қолганлари эса ошқозон - ичак тракти орқали чиқарилиши аниқланган. Бактериофаг одонтоген яллиғланиш касалликларига антисепти ва антибактериал таъсир хусусиятига эга. Одонтоген яллиғланиш асоратлари билан касалланган болаларда I гурухда ўғил болалар 28 нафарни, қиз болалар эса 21 наафрни ташкил этди. Бу гурухда ўғил болаларда қиз болаларга нисбатан касалланиш тенденцияси юқорилигида дарак беради. II ва III гурухларда ҳам ўғил болаларда касалланиш юқориги аниқланди. Бактириофаг воситасини одонтоген яллиғланиш касалликларига таъсир этиш механизмлари: ANNOTATION С. Счаде, Э. С. 19819, Э. Булл. ). Тадқиқот материали ва усуллари: Одонтоген яллиғланиш билан касалланган болаларни комплекс даволашда 122 нафар одонтоген яллиғланиш билан мурожаат қилган бемор болалар олинди. Илмий изланишимизда беморларни 3 гурухга бўлиб ўрганилди. I гурух одонтоген яллиғланиш касаллиги асорати тиш – жағ тизими абцесс ва флегмонаси билан касалланган 49 нафар бемор болаларда текширув олиб борилди ва комплекс даволанди. I гурух одонтоген яллиғланиш касаллиги асорати тиш – жағ тизими абцесс ва флегмонаси билан касалланган 49 нафар бемор болаларда текширув олиб борилди ва комплекс даволанди. II гурух одонтоген яллиғланиш касаллиги асорати тиш – жағ тизими периостити билан касалланган 53 нафар бемор болаларда текширув олиб борилди ва комплекс даволанди. II гурух одонтоген яллиғланиш касаллиги асорати тиш – жағ тизими периостити билан касалланган 53 нафар бемор болаларда текширув олиб борилди ва комплекс даволанди. Бизга маълумки, одонтоген яллиғланиш патологияларининг тарқалиши пасайишга мойил эмас, даволаш жараёни машаққатли ва узоқ давом этади ва асоратларнинг частотаси доимий равишда юқорилиги аниқланган. Одонтоген яллиғланиш жараёнларининг ўчоғи организмнинг гетеро- ва аутосенсибилизациясининг манбаи ҳисобланади ва маҳаллий ҳамда умумий патологиянинг ривожланишдаги этакчи омилларидан биридир. III гурух одонтоген яллиғланиш касаллиги асорати тиш – жағ тизими одонтоген остеомилети билан касалланган 20 нафар бемор болаларда текширув олиб борилди ва комплекс даволанди. Тадқиқот натижалари ва муҳокамалар Текширилган бемор болаларда одонтоген яллиғланиш касалликлар асоратларини ананавий даволаш усулидан тубдан фарқ қиладиган, Бактериофаг дори воситаси орқали комплекс даволашда касалликнинг тузалиш даврини камайтиришга эришилди. Болалар орасида одонтоген яллиғланиш касалликларининг тарқалиши ўткир йирингли - яллиғланиш касалликларининг оғир шакллари ва грипп, тонзиллит, юқумли гепатит ва бошқалар каби умумий юқумли касалликларининг асорати натижасида ривожланади. Одонтоген яллиғланиш касалликлари асоратлари билан касалланган бемор болаларни ёши, жинс бўйича гурухлараро тақсимланиши 1-жадвалда келтирилган. Одонтоген йирингли-яллиғланиш жараёнларининг боришини эрта ташҳислаш стоматологияда кенг тарқалганлиги ва оғир асоратларни ривожланишга олиб келади. ANNOTATION Today, patients with odontogenic inflammatory diseases account for 10-20% of dental diseases. And 30-70% of odontogenic inflammatory diseases are abscesses and phlegmons, improving the diagnosis of these diseases and effective treatment is considered one of the urgent problems. It is important to develop special treatment measures for children with this disease. Key words: odontogenic inflammatory diseases, abscesses and phlegmons. В.В., Коринская Н.Н., 1996; Ушаков Р. Б. 1995; Каваи Т. ва бошқалар, 1998 ва бошқалар). Кириш. Стоматологиянинг энг долзарб муаммоларидан бири ўткир одонтоген яллиғланиш жараёнларидир (Губин М.А., 2013; Балин В.Н., 2000; Карнаухов А.Т., 1999). Одонтоген яллишланиш касалликларига қарши курашнинг янги усулларини ишлаб чиқишга қарамасдан, яллиғланиш касалликлари билан касалланган болалар сони, айниқса тез-тез касал бўлган болаларда кўпайиш тенденциясига эга. (Робустова Т.Г. ва бошқалар, 2011; Шаргородский А.Г., 2010 ва бошқалар). Айни пайтда, маълумки, болаларда тиш – жағ тизими ўткир яллиғланиш жараёнлари кўпинча тананинг иммунологик реактивлигининг пасайиши билан ривожланади ва касалликнинг кечиши ва асоратлар эҳтимоли асосан иммунитетнинг дастлабки параметрлари билан белгиланади. Болаларда одонтоген яллиғланишларнинг кечиши боланинг тиш – жағ тизими ва тўқималарининг нисбий этуклиги, иммунитетнинг номукаммаллиги, лимфа тўқималарининг кўплиги, тишлар тузилишининг анатомик ва физиологик хусусиятларининг мавжудлиги сабабли бир қатор хусусиятларга эга ҳамда жағлар, шикастланишнинг осонлиги ва табиий ҳимоя тўсиқларининг микробларга ўтказувчанлигини ошириш ҳам муҳим аҳамиятга эгадир. Маълумки, тиш – жағ тизимининг яллиғланиш касалликларини ташхислаш ва даволаш болалар стоматологиясининг асосий муаммоларидан биридир. Бир қатор муаллифларнинг маълумотларига кўра, тиш –жағ тизимининг яллиғланиш касалликлари болалик давридаги барча жарроҳлик касалликларининг 21 % тиш касалликларининг 52 % ташкил қилади. Кўплаб эпидемиологик тадқиқотлар натижалари шуни кўрсатадики, ҳар бир бола йилига ўртача 3 дан 5 гача ўткир респиратор вирусли инфекциялар (ЎРВИ) эпизодларидан азият чекади. Ўткир респираторли инфекцияларнинг энг юқори Тиш – жағ тизими йирингли-яллиғланиш жараёнларининг этиологияси, патогенези, олдини олиш ва даволашнинг кўплаб масалалари ҳали ҳам ҳал этилмаган бўлиб, бу тадқиқотчиларнинг уларга доимий қизиқиши ва эътиборини тушунтиради (Рогинский 76  ɮ ɮ  ɖɹ ɮ ɖɹɮ ɮɹɮ ɮɹ¬Yfc`S^dea_Sea^aV[[[]cS`[agSi[S^o`nh[dd^XWaUS`[\ №2 | 2023 даражаси ёш болалар, мактабгача ёшдаги болалар ва кичик мактаб ўқувчиларида кузатилади. Ҳаётнинг биринчи 3 ёшидаги болалар 10 ёш ва ундан катта ёшдаги болаларга қараганда йил давомида ЎРВИ билан 2-2,5 марта тез-тез касаланиш кузатилади. Такрорий респиратор инфекциялар тананинг функционал ҳолатининг бузилишига олиб келади, мослашув этишмовчилигини келтириб чиқариши ва сурункали патологиянинг ривожланишига олиб келиши мумкин [Ключников С.О. ва бошқ., 2017]. Болаларда одонтоген яллиғланиш касалликларининг кечиши ва тарқалиши қуйидаги омилларга боғлиқ. 1. тиш кариесининг бирламчи ва иккиламчи профилактикаси, шунингдек, тиш кариесининг мураккаб шаклларини даволашнинг этарли даражада эмаслиги. 2. ANNOTATION бирламчи стоматологик касалликларни ўз вақтида даволанмаслиги ҳамда оғиз бўшлиғи шахсий гигиена қоидаларига риоя қилмаслик. 3. одонтоген микрофлоранинг антибиотикларга сезувчанлигининг пасайиши. 3. одонтоген микрофлоранинг антибиотикларга сезувчанлигининг пасайиши. Бу тиш – жағ тизими одонтоген йирингли яллиғланиш жарроҳликдаги муаммонинг долзарблигини кўрсатади. Қайта респиратор инфекциялари бўлган болаларда жарроҳлик касалликлари курси мураккаб иммунитет механизмлари ва уларнинг ошқозон ости бези, гипофиз, қалқонсимон без ва буйрак усти гормонлари билан ўзаро таъсири билан белгиланади. Маълумки, кўпинча одонтоген яллиғланиш касалликлари оғиз бўшлиғининг нормал микрофлорасининг патологик микрофлорага ўтишидан келиб чиқади. Одонтоген йирингли яллиғланишлар ривожланиши аслида иммун тизимининг компенсацион имкониятларининг пасайиши туфайли юзага келади. Иммунитет тизимининг бостирилиши, метаболик касалликлар, оғриқ сезувчанлигининг пасайиши кўплаб жарроҳлик касалликларининг клиник кўринишини ва курсини ўзгартиради, бу жиддий диагностика хатоларига олиб келиши ва жарроҳлик патологиясининг натижасига салбий таъсир кўрсатиши мумкин. (Галимов О.В. ва бошқалар, 2018; Заитсева Э.Л., 2018; Пиаггесиетал, 2018). Иммунитет тизимининг бостирилиши, метаболик касалликлар, оғриқ сезувчанлигининг пасайиши кўплаб жарроҳлик касалликларининг клиник кўринишини ва курсини ўзгартиради, бу жиддий диагностика хатоларига олиб келиши ва жарроҳлик патологиясининг натижасига салбий таъсир кўрсатиши мумкин. (Галимов О.В. ва бошқалар, 2018; Заитсева Э.Л., 2018; Пиаггесиетал, 2018). Триггер механизми юқумли касалликлар, масалан, грипп, тонзиллит ва бошқалар туфайли келиб чиққан вақтинчалик иммуносупрессия бўлиши ҳам одонтоген яллиғланиш касалликларини ривожланишини келтириб чиқаради. Тадқиқотнинг мақсади одонтоген яллиғланиш билан касалланган болаларни комплекс даволаш усулларини такомиллаштиришдан иборат. Тез-тез касал бўлган болаларда одонтоген яллиғланиш касалликлари ва уларнинг асоратлари аниқ микросиркуляция бузилиши, микротромблар, дистрофик ва некротик жараёнларнинг мавжудлиги, яллиғланиш компонентининг репаративдан устунлиги, ҳужайралар кўпайишини ҳосил қилиш билан тавсифланган маълум хусусиятларга эга. Лейкоцитларнинг фагоцитлар фаоллиги, тўлиқ бўлмаган фагоцитоз, яра тўқималарининг микробиал ифлосланишининг юқори даражаси, умумий ва маҳаллий иммунологик реактивликнинг пасайиши (Н. И. Камзалакова, 2000, А. Ю. Токмакова, 2003, Д. С. Счаде, Э. С. 19819, Э. Булл. ). Тез-тез касал бўлган болаларда одонтоген яллиғланиш касалликлари ва уларнинг асоратлари аниқ микросиркуляция бузилиши, микротромблар, дистрофик ва некротик жараёнларнинг мавжудлиги, яллиғланиш компонентининг репаративдан устунлиги, ҳужайралар кўпайишини ҳосил қилиш билан тавсифланган маълум хусусиятларга эга. Лейкоцитларнинг фагоцитлар фаоллиги, тўлиқ бўлмаган фагоцитоз, яра тўқималарининг микробиал ифлосланишининг юқори даражаси, умумий ва маҳаллий иммунологик реактивликнинг пасайиши (Н. И. Камзалакова, 2000, А. Ю. Токмакова, 2003, Д. С. Счаде, Э. С. 19819, Э. Булл. ). Тез-тез касал бўлган болаларда одонтоген яллиғланиш касалликлари ва уларнинг асоратлари аниқ микросиркуляция бузилиши, микротромблар, дистрофик ва некротик жараёнларнинг мавжудлиги, яллиғланиш компонентининг репаративдан устунлиги, ҳужайралар кўпайишини ҳосил қилиш билан тавсифланган маълум хусусиятларга эга. Лейкоцитларнинг фагоцитлар фаоллиги, тўлиқ бўлмаган фагоцитоз, яра тўқималарининг микробиал ифлосланишининг юқори даражаси, умумий ва маҳаллий иммунологик реактивликнинг пасайиши (Н. И. Камзалакова, 2000, А. Ю. Токмакова, 2003, Д. 2 – жадвал д Одонтоген яллиғланиш касалликлар асоратларини анавий даволаш ва Бактериофаг дори воситаси орқали комплекс даволашни гурухлараро таққослашнинг таҳлилий натижалари Гурухлар Ананавий даволаш Соғайиш тенденсияси Бактериофаг орқали комплекс даволаш Соғайиш тенденсияси I Антибактериал воситалар 7 кун Бактериофаг 5 кун II Антибактериал воситалар 5 кун Бактериофаг 3 кун III Антибактериал воситалар 14 кун Бактериофаг 10 кун Одонтоген яллиғланиш касалликлар асоратларини анавий даволаш ва Бактериофаг дори воситаси даволашни гурухлараро таққослашнинг таҳлилий натижалари ланиш касалликлар асоратларини анавий даволаш ва Бактериофаг дори воситаси орқали комплекс даволашни гурухлараро таққослашнинг таҳлилий натижалари Бактириофаг воситасини одонтоген яллиғланиш касалликларига таъсир этиш механизмлари: Бактериофаг-бу гомологик бактериянинг ҳужайра мембранасида адсорбсияланган, ҳужайра ичига кириб, уни лизисга учратади. Бактериянинг ўзига хос вирусига таъсир кўрсатиб, таркибига бактерияларнинг селектив, фақат вирусли фаглари - Staphylococcus aureusга тасир қилади. Бактериофаг юқори Жароҳат майдонига бактериофаг воситасини қўллаш зарарланган тўқима ҳажмига қараб индивидуал равишда белгиланади. Жарохат майдонидан йирингни олиб ташлаганидан кейин оғиз бўшлиғига пуркаш тавсия қилинади. Беморларда 77  ɮ ɮ  ɖɹ ɮ ɖɹɮ ɮɹɮ ɮɹ¬Yfc`S^dea_Sea^aV[[[]cS`[agSi[S^o`nh[dd^XWaUS`[\ №2 | 2023 кейинги қатнов кунларда бактериофаг дренаж ёрдамида инфекцияланган бўшлиққа қўйилади. кейинги қатнов кунларда бактериофаг дренаж ёрдамида инфекцияланган бўшлиққа қўйилади. операциядан кейинги 40 мл миқдоридаги юқумли асоратларни олдини олишда ишлатилади. Одонтоген яллиғланиш касалликларини даволашда бактериофаг дозаси беморларнинг аҳволига қараб белгиланади. Агар стафилококк бактериофаг препаратини қўллашдан олдин яраларни даволаш учун кимёвий антисептиклар ишлатилган бўлса, ярани стерил 0,9% натрий хлорид эритмаси билан яхшилаб ювиш тавсия этилди. Илмий тадқиқотимиз давомида ушбу усулни қўллаш тан нархи арзон қўллаш қулай, даволаш самарадорлиги юқори ҳисобланади. Ушбу жараён кунига 1 марта, 3-5 кун давомида амалга оширилади. Оғиз бўшлиғи дренажланган бўлса, бактериофаг кунига 2 марта 5-10 мл юборилади. Бактериофаг намланган турундни ювиш, томизиш, юбориш учун ишлатилади. Оғиз бўшлиғи йирингли яллиғланиш ҳолатларида даволаш учун восита чайиш учун ишлатилади ва бир вақтнинг ўзида буюрилади. Стоматит ва сурункали умумий периодонтитни даволашда восита кунига 3-4 марта 10-20 мл дозада оғиз ювиш воситаси шаклида қўлланилади, шунингдек, пиобактериофаг билан сингдирилган турунданинг периодонтал чўнтакларига 5-10 дақиқа давомида қўлланилади. Профилактика мақсадида бактериофаг операциядан кейинги ва одонтоген яллиғланиш даволашда, шунингдек, тиш – жағ тизими ва оғиз бўшлиғи касалликларини олдини олишда, Одонтоген яллиғланиш касалликлар асоратларини ананавий даволаш усулидан тубдан фарқ қиладиган, Бактериофаг дори воситаси орқали комплекс даволашда касалликнинг соғайиш тенденсиясининг самарали натижага эришилганлиги 2 – жадвалда келтирилган. Хулосалар Ху Одонтоген йирингли яллиғланиш касалликлари асоратлари билан оғриган учала гурухдаги беморларни Бактериофаг дори воситаси билан комплекс даволашда беморларни тузалиш кунини биринчи гурухда 7 кундан 5 кунга, иккинчи гурухда 5 кундан 3 р кунга, учунчи гурухда эса 14 кундан 10 кунга камайтиришга эришилди. Беморларни комплекс даволаш курсидан сўнг чайнов самарадорлик ҳолатини тўлиқ тиклашга эришилди. Фойдаланилган адабиётлар у 3. Азимов М. Диагностика и патогенетические аспекты терапии воспалительных заболеваний челюстно-лицевой области: Автореф. дис. ... д-ра мед. наук: 14.00.21. - Ташкент, 1991.-316 с. р ф р у 4. Алешина О.Е. Дифференциальная диагностика гнойных и негнойных вос-палительных процессов челюстно-лицевой области у детей: Автореф. дис. ... канд. мед. наук; 14.00.21. - М., 2002. - 28 с. 4. Алешина О.Е. Дифференциальная диагностика гнойных и негнойных вос-палительных процессов челюстно-лицевой области у детей: Автореф. дис. ... канд. мед. наук; 14.00.21. - М., 2002. - 28 с. имизация комплексного лечения гнойно-воспалительных заболеваний челюстно-лицевой области: Дис.... канд / ИГМУ. - Иркутск, 1997.- 131 с. 5. Алешкин И.Г. Оптимизация комплексного лечения гнойно-воспалительных заболеваний челюстно- мед. наук: 14.00.21 / ИГМУ. - Иркутск, 1997.- 131 с. 5. Алешкин И.Г. Оптимизация комплексного лечения гнойно-воспалительных заболеваний челюстно-лицевой области: Дис.... канд. мед. наук: 14.00.21 / ИГМУ. - Иркутск, 1997.- 131 с. 6 Бажанов Н Н Итоги обсуждения классификации острых одонтогенных воспалительных процессов / Н Н Бажанов М М 6. Бажанов Н.Н. Итоги обсуждения классификации острых одонтогенных воспалительных проц Соловьев, В.В. Рогинский // Стоматология. - 1990. -№ 3. - С. 87-89. 7. Бажанов Н.Н. Фагоцитоз при острых воспалительных заболеваниях челюстно лицевой области / Н.Н. Бажанов, М.В. Ларская, М.Т. Александров // Специализированная медицинская помощь и современные проблемы ее интеграции: Тез. докл. Всесоюзн. конф. - М., 1986 - С. 143-146. 7. Бажанов Н.Н. Фагоцитоз при острых воспалительных заболеваниях челюстно лицевой области / Н.Н. Бажанов, М.В. Ларская, М.Т. Александров // Специализированная медицинская помощь и современные проблемы ее интеграции: Тез. докл. Всесоюзн. конф. - М., 1986 - С. 143-146. , 8. Баиров Г.А. Гнойная хирургия детей / Г.А. Баиров, Л.М. Рошаль. - Л.: Медицина, 1991. - С. 72-74,237. 9 Б ф й б й б / Н Н Б Е П 8. Баиров Г.А. Гнойная хирургия детей / Г.А. Баиров, Л.М. Рошаль. - Л.: Медицина, 1991. - С. 72-74,237. 9. Бактериальная микрофлора при одонтогенных острых гнойных заболева¬ниях челюстно-лицевой области / Н.Н. Бажанов, Е.П. Пашков, М.С. Култа- ев va boshq. // Стоматология. - 1985. - № 1. - С. 31-32. Баиров Г.А. Гнойная хирургия детей / Г.А. Баиров, Л.М. Рошаль. - Л.: Медицина, 1991. - С. 72-74,237. Бактериальная микрофлора при одонтогенных острых гнойных заболева¬ниях челюстно-лицевой области / Пашков, М.С. Култа- ев va boshq. // Стоматология. - 1985. - № 1. - С. 31-32. 10. Баркаган З.С. Геморрагические заболевания и синдромы / З.С. Баркаган. - М., 1988.-60 с. 10. Баркаган З.С. Геморрагические заболевания и синдромы / З.С. Баркаган. 17. Rizaev Jasur Alimjanovich, Ahrorova Malika Shavkatovna, Kubaev Aziz Saydalimovich, Hazratov Alisher Isamiddinovich, Morphological Changes in the Oral Mucous Membrane in Patients with COVID-19, American Journal of Medicine and Medical Sciences, Vol. 12 No. 5, 2022, pp. 466-470. doi: 10.5923/j.ajmms.20221205.04. Фойдаланилган адабиётлар - М., 1988.-60 с. Д у у р у Д 12. Кубаев А. и др. Comparative analysis of methods for treating depressed frontal sinus fractures //Журнал стоматологии и краниофациальных исследований. – 2020. – Т. 1. – №. 1. – С. 25-28 13. Ризаев Ж., Кубаев А., Бузрукзода Ж. Современный подход к комплексной реабилитации пациентов с приобретенными дефектами верхней челюсти (обзор литературы) //Журнал стоматологии и краниофациальных исследований. – 2021. – Т. 2. – №. 3. – С. 77- 83. 14. Rizaev Elyor Alimdjanovich, & Buzrukzoda Javokhirkhon Davron. (2023). HEALING WITH THE USE OF TITANIUM THREADS OF CONTROLLED BONE RESORPTION. American Journal of Interdisciplinary Research and Development, 16, 9–14. Retrieved from http://ajird.journalspark.org/index.php/ajird/article/view/649 p j j p g p p j 15. Rizaev, E. A., & Buzrukzoda, J. D. (2022). OPTIMIZATION OF GUIDED BONE REGENERATION IN CONDITIONS OF JAW BONE ATROPHY. Applied Information Aspects of Medicine (Prikladnye informacionnye aspekty mediciny), 25(4), 4-8. 16. Rizaev J.A., Akhrorova M.Sh., Kubaev A.S., Khazratov A.I. Clinical and immunological aspects of the relationship of the oral cavity and covid-19, TJE - Thematic journal of Education, 7, 3-15, 2022 p j j p g p p j 15. Rizaev, E. A., & Buzrukzoda, J. D. (2022). OPTIMIZATION OF GUIDED BONE REGENERATION IN CONDITIONS OF JAW BONE ATROPHY. Applied Information Aspects of Medicine (Prikladnye informacionnye aspekty mediciny), 25(4), 4-8. ATROPHY. Applied Information Aspects of Medicine (Prikladnye informacionnye aspekty mediciny), 25(4), 4-8. 16. Rizaev J.A., Akhrorova M.Sh., Kubaev A.S., Khazratov A.I. Clinical and immunological aspects of the relationship of the oral cavity and covid-19, TJE - Thematic journal of Education, 7, 3-15, 2022 16. Rizaev J.A., Akhrorova M.Sh., Kubaev A.S., Khazratov A.I. Clinical and immunological aspects of the relationship of the oral cavity and covid-19, TJE - Thematic journal of Education, 7, 3-15, 2022 78  ɮ ɮ  ɖɹ ɮ ɖɹɮ ɮɹɮ ɮɹ¬Yfc`S^dea_Sea^aV[[[]cS`[agSi[S^o`nh[dd^XWaUS`[\№2 | 2023 17. Rizaev Jasur Alimjanovich, Ahrorova Malika Shavkatovna, Kubaev Aziz Saydalimovich, Hazratov Alisher Isamiddinovich, Morphological Changes in the Oral Mucous Membrane in Patients with COVID-19, American Journal of Medicine and Medical Sciences, Vol. 12 No. 5, 2022, pp. 466-470. doi: 10.5923/j.ajmms.20221205.04.  ɮ ɮ  ɖɹ ɮ ɖɹɮ ɮɹɮ ɮɹ¬Yfc`S^dea_Sea^aV[[[]cS`[agSi[S^o`nh[dd^XWaUS`[\ №2 | 2023 №2 | 2023 17. Rizaev Jasur Alimjanovich, Ahrorova Malika Shavkatovna, Kubaev Aziz Saydalimovich, Hazratov Alisher Isamiddinovich, Morphological Changes in the Oral Mucous Membrane in Patients with COVID-19, American Journal of Medicine and Medical Sciences, Vol. 12 No. 5, 2022, pp. 466-470. doi: 10.5923/j.ajmms.20221205.04. 17. Rizaev Jasur Alimjanovich, Ahrorova Malika Shavkatovna, Kubaev Aziz Saydalimovich, Hazratov Alisher Isamiddinovich, Morphological Changes in the Oral Mucous Membrane in Patients with COVID-19, American Journal of Medicine and Medical Sciences, Vol. 12 No. 5, 2022, pp. 466-470. doi: 10.5923/j.ajmms.20221205.04. 79 90 ISSN 2181-0966 Doi Journal 10.26739/2181-0966 Yfc`S^dea_Sea^aV[[[ ]cS`[agSi[S^o`nh[dd^XWaUS`[\ ea_•`a_Xc   ɮ ɮ  ɖɹ ɮ ɖɹɮ ɮɹɮ ɮɹ   •  Контакт редакций журналов. www.tadqiqot.uz ООО Tadqiqot город Ташкент, улица Амира Темура пр.1, дом-2. Web: http://www.tadqiqot.uz/; Email: info@tadqiqot.uz Тел: (+998-94) 404-0000 Editorial staff of the journals of www.tadqiqot.uz Tadqiqot LLC The city of Tashkent, Amir Temur Street pr.1, House 2. Web: http://www.tadqiqot.uz/; Email: info@tadqiqot.uz Phone: (+998-94) 404-0000 90 Контакт редакций журналов. www.tadqiqot.uz ООО Tadqiqot город Ташкент, улица Амира Темура пр.1, дом-2. Web: http://www.tadqiqot.uz/; Email: info@tadqiqot.uz Тел: (+998-94) 404-0000 Editorial staff of the journals of www.tadqiqot.uz Tadqiqot LLC The city of Tashkent, Amir Temur Street pr.1, House 2. Web: http://www.tadqiqot.uz/; Email: info@tadqiqot.uz Phone: (+998-94) 404-0000 Контакт редакций журналов. www.tadqiqot.uz ООО Tadqiqot город Ташкент, улица Амира Темура пр.1, дом-2. Web: http://www.tadqiqot.uz/; Email: info@tadqiqot.uz Тел: (+998-94) 404-0000
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O arrependimento após a esterilização cirúrgica e o uso das tecnologias reprodutivas
Revista brasileira de ginecologia e obstetrícia
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O arrependimento após a esterilização cirúrgica e o uso das tecnologias reprodutivas Repentance after surgical sterilization and the use of reproductive technologies Repentance after surgical sterilization and the use of reproductive technologies Editorial As informações mais recentes que temos sobre o uso de métodos anticoncepcionais para o território nacional são os dados da Pesquisa Nacional sobre Demografia e Saúde1 conduzida em 1996, que apontavam para alta prevalência de esterilização feminina (40,1%) entre as mulheres de 15 a 49 anos em união sexual (casada ou em coabitação). O segun­ do método mais usado, em proporção bastante inferior, 21,4%, era o anticoncepcional hormonal oral. Em estudo comparativo entre o Estado de São Paulo e o Brasil, de 1986 e 19962, observam-se algumas tendências: até os 30 anos de idade, o anticoncepcional hormonal oral é prevalente e, a partir dessa idade, a esterilização feminina cresce. A esterilização feminina aumenta com o número de filhos e diminui com a escolaridade. A vasectomia era, naquela época, cinco vezes menos utilizada que a laqueadura, embora já se percebesse uma tendência de aumento de uso de métodos masculinos. As altas taxas de esterilização feminina no país chamaram a atenção, na década de 80 e 90, de estudiosos de várias áreas: demografia, saúde pública, ginecologia/obstetrícia e sociologia. Até mesmo uma Comissão Parlamentar de Inquérito (CPI)3 foi criada para entender como ocorria essa desenfreada oferta. Vários estudos conduzidos no país4-9 ten­ taram entender como e por que a esterilização feminina fazia (e ainda faz) tanto sucesso, apesar de ser, na época, um procedimento que não estava legalmente regulamentado. Por isso, os Conselhos de Medicina afirmavam não ser ético realizar a esterilização, visto que o médico poderia sofrer as conseqüências previstas no parágrafo 3º do Artigo 129 do Código Penal, que trata de lesões corporais, perda de órgãos ou funções10. Apesar das dúvidas sobre a sua legalidade, a oferta da esterilização, tanto feminina quanto masculina, tornou-se uma prática no setor privado11. As mulheres obtinham a esterili­ zação clandestinamente pelo pagamento “por fora”, na hora do parto, freqüentemente por meio de uma cirurgia cesariana12. Em Ribeirão Preto, cerca de 60% das mulheres esterilizadas durante uma cesariana, em 1998, pagaram pelo procedimento13. Outros estudos tentaram entender como a esterilização feminina foi usada como moeda de troca no país, favores políticos e votos14. Desnecessário dizer que durante esse processo ocorre acentuada queda na taxa de fecundidade no país. Elisabeth Meloni Vieira Elisabeth Meloni Vieira Professora Associada do Departamento de Medicina Social da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo – USP – Ribeirão Preto (SP), Brasil. Correspondência: Elisabeth Meloni Vieira Rua Bernardino de Campos, 50, apto.1101 – Centro CEP 14015-130 – Ribeirão Preto/SP E-mail: bmeloni@fmrp.usp.br Recebido 28/05/2007 Aceito com modificações 01/06/2007 O arrependimento após a esterilização cirúrgica e o uso das tecnologias reprodutivas A noção de direito reprodutivo como direito humano desenvolveu-se ao longo do século XX, sendo um fenômeno recente, relacionado às mudanças no papel das mulheres e seu valor na sociedade, às mudanças de consciência das mulheres sobre seu papel e valor e ao desenvolvimento das tecnologias médicas. Embora a Declaração Universal dos Direitos Humanos não mencionasse os Direitos Reprodutivos, a partir de 1968 a reprodução humana tornou-se objeto de preocupação. Os direitos reprodutivos foram definitivamente incorporados na Conferência Internacional sobre População e Desenvolvimento do Cairo, em 1994: “Os direitos reprodutivos baseiam-se no reconhecimento do direito básico de todos os casais e indivíduos decidirem livre e responsavelmente sobre o número, espaçamento e quando devem ter os filhos e de terem acesso a informação sobre a forma como fazê-lo, bem como o direito de se beneficiarem de saúde sexual e reprodutiva do mais alto nível. Também incluem o direito de todos tomarem decisões sobre a reprodução sem discriminação, coerção nem violência.” (p.58)19. Desde 1988, no Brasil, o planejamento familiar é um direito do cidadão, e é definido como um conjunto de ações de regulação da fertilidade, que objetiva garantir direitos iguais de constituição (limitação ou au­ mento de prole) pela mulher, pelo homem ou pelo casal. Deve ser parte integrante do atendimento à saúde, com ações preventivas e educativas no acesso igualitário às informações e meios de anticoncepção cientifi­ camente aceitos. É dever do Estado assegurar o livre exercício do planejamento familiar. Mas foi somente a partir de 1997 que a oferta da esterilização cirúrgica voluntária passou a ser regulamentada no Brasil, com a aprovação da Lei 9.263, regulamentando o parágrafo 7o do Artigo 226 da Constituição Federal. A lei 9.263 foi aprovada em janeiro de 1996, mas o Artigo 10o, sobre a esterilização cirúrgica, permaneceu vetado pelo Presidente até agosto de 1997. Composta de 25 Artigos e organizada em três Capítulos, a Lei aborda vários aspectos do planejamento familiar, estabelecendo que nenhum programa deva visar a metas demográficas. Fornece diretrizes claras para questões institucionais, alocando no Sistema Único de Saúde (SUS) as ações de planejamento familiar, como parte integrante da atenção à saúde reprodutiva, incluindo as atividades básicas de assistência à concepção e contracepção, o atendimento pré-natal, a assistência ao parto, ao puerpério e ao neonato, o controle das doenças sexualmente transmissíveis e o controle e prevenção do câncer cérvico-uterino, do câncer de mama e de pênis. O arrependimento após a esterilização cirúrgica e o uso das tecnologias reprodutivas i A insuficiência do Estado em implantar e fornecer um planejamento reprodutivo adequado, que já havia sido desenhado no Programa de Atenção Integral à Saúde da Mulher (Paism) em 1984, entrou em primeiro lugar na lista de explicações para a altís­ sima prevalência de esterilização feminina. Também se chamou atenção para o caráter de Vieira EM dissociação entre sexo e reprodução no uso da esterilização5, com a ação legitimadora do médico que confirma para a mulher que seu desejo de contracepção é válido, sério e deve ser considerado15, explicando, então, a preferência cultural da esterilização. Discutiu-se muito seu uso em uma situação quase obrigatória após duas cesáreas (outra preferência nacional)16. Berquó (1993) alertou para uma cultura da esterilização16. Nesse contexto, destaca-se o papel da medicalização, que tomou lugar no vácuo de uma política pública que não foi implementada17. Podemos olhar melhor para a alta prevalência da esterilização feminina no contexto maior da oferta de métodos contraceptivos pelo serviço público. O relatório técnico de uma missão do Fundo de População das Nações Unidas (UNFPA)18 ao Brasil, em 1994, revelou vários problemas relativos ao planejamento familiar no país: a imensa lacuna entre a demanda por contraceptivos e o acesso a eles, a falta de insumos nos serviços públicos de saúde, a ausência de gerenciamento logístico, a falta de capacidade técnica das equipes. Uma análise detalhada sobre as características dos serviços prestados na área de saúde reprodutiva apontava para várias deficiências: predominância de apenas dois métodos anticoncepcionais, leque de escolhas reduzido, os métodos oferecidos demonstram medi­ calização excessiva, predominância do setor privado (compra de pílulas e ofertas de cirurgias), acesso tardio (quase 50% das mulheres começavam a fazer contracepção depois do primeiro filho), uso inadequado de contraceptivos, alta proporção de gravidez indesejada, falta de informação e desigualdade no acesso17. Acrescenta-se a isso uma questão de gênero importante, pois são as mulheres que mais contribuem com a contracepção, já que a participação masculina, embora crescente, ainda não atinge os níveis de outros países, onde parece haver maior equilíbrio de gênero, principalmente no que se refere à esterilização. A oferta de métodos contraceptivos em programas de saúde baseia-se nos direitos reprodutivos. 226 Rev Bras Ginecol Obstet. 2007; 29(5):225-9 226 Rev Bras Ginecol Obstet. 2007; 29(5):225-9 O arrependimento após a esterilização cirúrgica e o uso das tecnologias reprodutivas O arrependimento após a esterilização é uma séria questão médica, porque, na maioria dos casos, a escolha da laqueadura é eletiva, não se pode garantir o retorno à fertilidade e existem alternativas contraceptivas mais reversíveis do que a esterilização que podem ser utilizadas com grande eficácia. O arrependimento já foi estimado entre 11 e 15% das mulheres brasileiras esterilizadas24. Reconhecendo a dificuldade de medir com precisão o arrependimento, Marcil-Gratton25 usou um gradiente de sentimentos de arrependimento para classificar as mulheres em alguns grupos: 1. mulheres que nunca sentiram arrependimento; i 2. mulheres que tentariam ter outro filho se não estivessem esterilizadas; i 3. mulheres que conversaram com o médico sobre a reversão do procedimento; 4. mulheres que solicitaram a reversão da laqueadura para seus médicos. Alguns fatores como idade jovem, falta de estabilidade no relacionamento conjugal e morte de filho são apon­ tados em todo o mundo como predisponentes ao arrependimento23. No Brasil, além de ter motivos diferentes para satisfação com o número de filhos, destaca-se a idade jovem ao ter o primeiro filho como fator para o arrependi­ mento, visto que predispõe à laqueadura jovem. Observou-se, também, que o pagamento atua como obstáculo ao arrependimento, de maneira que este pode aumentar após a oferta de esterilização gratuita pelo SUS6. Dessa forma, é importante garantir que a escolha da esterilização seja feita de forma consciente e infor­ mada pelos indivíduos e que estes recebam aconselhamento e orientação sobre todos os métodos anticon­ cepcionais, inclusive os reversíveis, tal como prevê a Lei 9.263. Tem sido o caso do município de Ribeirão Preto, que, desde 1999, oferece a esterilização masculina e feminina pelo SUS com adequado suporte de aconselhamento realizado por profissionais de saúde capacitados. Mesmo neste caso não existem garantias de que não haja arrependimento entre os esterilizados, pois ele pode ocorrer mesmo após a escolha informada, como já foi explorado por vasta literatura em estudos realizados em diversos países23. A oferta conjunta da esterilização, tanto feminina quanto masculina, pelo sistema público representa uma novidade em termos de serviços de contracepção no Brasil, pois concentra, em um mesmo serviço, a oferta das duas cirurgias que anteriormente eram oferecidas por especialistas de duas áreas distintas e, aparentemente, sem conexões: ginecologistas e urologistas. O arrependimento após a esterilização cirúrgica e o uso das tecnologias reprodutivas Estabelece, ainda, que é dever do Estado assegurar o livre exercício do plane­ jamento familiar e que a esterilização voluntária é somente permitida em duas situações: • em homens e mulheres com capacidade civil plena e maiores de 25 anos ou, pelo menos, dois filhos vivos, desde que observado o prazo mínimo de 60 dias entre a manifestação da vontade e o ato cirúrgico, período pelo qual será propiciado à pessoa interessada acesso a serviço de regulação da fecundidade, incluindo aconselhamento por equipe multidisciplinar, visando desencorajar a esterilização precoce; • em homens e mulheres com capacidade civil plena e maiores de 25 anos ou, pelo menos, dois filhos vivos, desde que observado o prazo mínimo de 60 dias entre a manifestação da vontade e o ato cirúrgico, período pelo qual será propiciado à pessoa interessada acesso a serviço de regulação da fecundidade, incluindo aconselhamento por equipe multidisciplinar, visando desencorajar a esterilização precoce; • risco à vida ou à saúde da mulher ou do futuro concepto, testemunhado em relatório escrito e assinado por dois médicos. Para desvincular a esterilização feminina das cesarianas, a lei veda a esterilização cirúrgica em mulher nos períodos de parto ou aborto, exceto nos casos de comprovada necessidade, por cesarianas anteriores. Prevê ainda fiscalização e controle pelo SUS das instituições que realizam planejamento familiar. Várias portarias e resoluções no nível federal e estadual, estabelecendo formas de controle, foram publicadas20,21. Em seu capítulo II, a Lei 9.263 prevê penalidades, definindo como crime as esterilizações em desacordo com a lei. Embora a regulamentação legal dos direitos assegurados na Constituição Brasileira seja fundamental para garantir os direitos reprodutivos e melhorar a qualidade de vida das pessoas, apenas a regulamentação não seria suficiente para criar situações para o exercício dos direitos reprodutivos, como afirmam alguns autores22. Quando os mecanismos legais não são suficientes, então outros aspectos sociais, materiais e institucionais da sociedade precisam ser desenvolvidos para assegurá-los6. É o caso da oferta do planejamento familiar de boa qualidade que necessita de investimento na capacitação de profissionais, na compra de insumos e de bom desenvolvimento técnico e tecnológico para se efetivar. Em sua ausência, há situações como testemunhamos neste estudo relevante realizado por pesquisadores da Uni­ versidade de Brasília apresentado nesta edição de RBGO23. Este nos informa sobre um contingente de 98 mulheres arrependidas que procuraram um serviço de reprodução assistida para conceber após terem sido esterilizadas. O arrependimento após a esterilização cirúrgica e o uso das tecnologias reprodutivas É importante notar, em concordância com alguns estudos6,23, que apenas uma pequena parcela de mulheres arrependidas estaria disposta a se submeter a essas técnicas para conceber e uma parcela menor ainda atinge seus objetivos. Em estudo realizado no Brasil, entre 79 mulheres arrependidas, apenas 19 (24%) procuraram por infor­ mação sobre reversão26. No caso do estudo em questão, das 98 mulheres, apenas 8% tiveram o sucesso almejado. Das 20 mulheres submetidas à reversão da laqueadura, seis (30%) tiveram o bebê a termo, e, entre oito mulheres submetidas às técnicas de fertilização in vitro com transferência de embrião, duas (25%) obtiveram sucesso. Chama Vieira EM Vieira EM atenção que para 22 mulheres estava indicada fertilização in vitro com transferência de embrião, mas infere-se que elas fazem parte das que desistiram do procedimento. O uso das tecnologias reprodutivas, cirurgia de recanalização tubária ou transferência de embriões não é inó­ cuo, e pode causar dor e sofrimento. Existem riscos de gravidez tubária, abortamento, óbito neonatal e gravidez múltipla. O mais estarrecedor é que entre as 98 mulheres estudadas, 83,6% afirmaram não terem tido informação suficiente a respeito do caráter definitivo da laqueadura tubária. Ou seja, uma decisão definitiva como a esterilização cirúrgica foi tomada sem a informação necessária. Essa distorção fere os direitos reprodutivos. A sociedade deve aperfeiçoar suas regulamentações e normas, de forma a garantir o bem-estar dos indivíduos e os limites de respon­ sabilidade profissional. O currículo médico na área deve incluir discussões sobre bioética e direitos reprodutivos, para evitar situações como essas. É necessário também ao médico ter consciência do processo de medicalização, pois a sociedade pode colocar em suas mãos decisões e responsabilidades enormes. li p p p Refletindo recentemente sobre um texto de Bateman27, que afirma que a atuação médica na manipulação das tecnologias reprodutivas difere de outros procedimentos médicos já atuantes sobre o campo da não-doença, separando definitivamente o sexo da reprodução e colocando para a medicina questões éticas, morais e políticas jamais pensadas pela humanidade, apontamos para a medicalização como processo de tomada do objeto do co­ nhecimento do campo do não-médico para o médico. Para Conrad28, a medicalização contém, em si, uma questão conceitual, ou seja: o problema é definido em termos médicos, usa-se uma linguagem médica para descrevê-lo, adota-se um referencial teórico médico para entendê-lo ou usa-se a intervenção médica para tratá-lo. Sociedade Civil Bem-Estar Familiar no Brasil (Bemfam). Pesquisa nacional sobre demografia e saúde – Pnds 1996. Rio de Janeiro: Bemfam/Macro; 1997. 1. O arrependimento após a esterilização cirúrgica e o uso das tecnologias reprodutivas É um processo sociocultural que pode ou não envolver a profissão, conduz a um controle médico social ou tratamento médico, ou é resultado de uma expansão intencional da profissão médica. No caso de mulheres cirurgicamente esterilizadas e tratadas com tecnologias reprodutivas, pode-se colocar o problema desta medicalização, provavelmente, como a de expansão não intencional da prática médica, mas que coloca em jogo um dos princípios éticos essenciais a essa prática revelado pelo aforisma primum non nocere (primeiro, não ferir). Na análise dessa situação, outras questões poderiam ser incluídas: em que condições se dão essas escolhas repro­ dutivas? Houve aconselhamento? Houve consciência dos sujeitos envolvidos nessa decisão? Deve aconselhar o proce­ dimento de esterilização o indivíduo que receberá pagamento por ele? Devem ter acesso às tecnologias reprodutivas todos os indivíduos que se arrependeram de um procedimento tão definitivo quanto a esterilização cirúrgica?l Há necessidade de maior debate sobre a bioética e suas condições de desenvolvimento, promovendo a reflexão dos profissionais sobre sua prática que, provavelmente, se realizou fundada em boas intenções. É necessário que o entendimento e o desenvolvimento de tecnologias educacionais de aconselhamento, orientação, conversa e amplo acesso a métodos sejam esgotados antes de uma decisão definitiva como essa. Para tal, o profissional médico ne­ cessita de outros profissionais para compartilhar seu trabalho. A ausência de uma estrutura de múltiplos olhares profissionais que apóie os envolvidos nos processos de decisão os abandona à própria sorte em tomadas de decisões complexas e difíceis. A ausência de regulamentações sobre as tecnologias deixa médicos e requerentes à margem de um processo justo e igualitário, em termos de direitos e escolhas. Referências Osis MJD. Laqueadura e representações acerca da sexualidade e do papel reprodutivo [tese]. São Paulo: Universidade de São Paulo; 2001. 7. Vieira EM, Badiani R, Fabbro ALD, Rodrigues Junior AL. Características do uso de métodos anticoncepcionais no Estado de São Paulo. Rev Saúde Pública. 2001;36(3):263-70. 2. Serruya S. Mulheres esterilizadas: submissão e desejo. Belém: Naea/Afpa/Uepa; 1996. 8. Minella LS. A produção científica sobre esterilização feminina no Brasil nos anos 80 e início dos anos 90: um debate em aberto. Rev Bras Estud Popul. ���������������� 1998;15(1):3-22. 9. Barroso C. Esterilização no Brasil. Brasília: Comissão de Estudos de Direitos da Reprodução/MS; 1988. 3. Berquó E. A esterilização feminina hoje. Ciênc Tecnol. 1989;88(5):598-610. 4. Berquó E. A esterilização feminina hoje. Ciênc Tecnol. 1989;88(5):598-610. 4. Conselho Regional de Medicina do Estado de São Paulo (Cremesp). Aspectos éticos que envolvem a esterilização. Ética Méd. 1988;1(1):67. 10. Barroso C. Esterilização feminina: liberdade e opressão. Rev Saúde Pública. 1984;18(2):170-80. 5. Berquó E, Cavenaghi SM. Direitos reprodutivos de mulheres e homens face à nova legislação brasileira sobre esterilização voluntária. Cad Saúde Pública. 2003;19 Supl 2:441-53. 11. Vieira EM. Regret after female sterilization among low-income women in São Paulo, Brazil [thesis]. Exeter: University of Exeter������� ; 1994. 6. 228 Rev Bras Ginecol Obstet. 2007; 29(5):225-9 Vieira EM, Ford NJ. The provision of female sterilization in São Paulo, Brazil: a study among low income women. Soc Sci Med. 1996;42(10):1427-32. 12. Resolução SS-5, de 11 de janeiro de 2000. Dispõe sobre Estabelece critérios para a efetivação dos procedimentos de esterilização no âmbito do Estado de São Paulo. Diário Oficial do Estado de São Paulo, Poder Executivo, São Paulo jan. 2000; Seç.I (110): p.13. 21. Vieira EM, Ford NJ. Provision of female sterilization in Ribeirão Preto, São Paulo, Brazil. Cad Saúde Pública. 2004;20(5):1201-10. 13. Freedman LP, Isaacs SL. Human rights and reproductive choice. Stud Fam Plann. ����������������� 1993;24(1):18-30. 22. Caetano AJ. Sterilization for votes in the Brazilian Northeast: the case of Pernambuco [thesis]. Austin: University of Texas; 2000. 14. Cunha ACR, Wanderley MS, Garrafa V. Fatores associados ao futuro reprodutivo de mulheres desejosas de gestação após ligadura tubária. Rev Bras Ginecol Obstet. 29(5):230-4. 23. Vieira EM. A medicalização do corpo feminino. Rio de Janeiro: Fiocruz; 2002. 15. Berquó E. Brasil, um caso exemplar: anticoncepção e partos cirúrgicos à espera de uma ação exemplar. Estud Fem. 1993;1(2):366-81. 16. Vieira EM. O arrependimento após a esterilização feminina: é possível prevenir? ������������������������������� Reprod Clim. 1999;14(4):178-84. Referências 24. Vieira EM. Políticas públicas e contracepção no Brasil. In: Berquó E, organizador. Sexo e vida: panorama de saúde reprodutiva no Brasil. Campinas: Unicamp; 2003. p. 151-96. 17. Marcil-Gratton N. Sterilization regret among women in metropolitan Montreal. ������������������������������������� Fam Plann Perspect. 1988;20(5):222-7. 25. United Nations Population. Contraceptive requirements and logistics management needs in Brazil. New York: FNUAP; 1995. (Technical Report, 21). 18. 26. Vieira EM. Arrependimento após a esterilização feminina. Cad Saúde Pública. 1998;14 Supl 1:59-68. 26. Bateman S. When reproductive freedom encounters medical responsibility: changing conceptions of reproductive choice. In: Vayena E, Rowe PJ, Griffin PD, editors. Current practices and controversies in assisted reproduction. Report of a WHO meeting. Geneva: World Health Organization; 2002. p. 320-32. 27. Nações Unidas. Relatório da Conferência Internacional sobre População e Desenvolvimento; 5-13 set 1994; Cairo. Brasília: Comissão Nacional de População e Desenvolvimento/ FNUAP; 1994. 19. Ministério da Saúde. Secretaria de Assistência à Saúde. Portaria n.o 144 de novembro de 1997. Dispõe sobre..... Diário Oficial da União, Brasília (DF) 20 nov. 1997;(227). 20. Conrad P. Medicalization and social control. Annu Rev Sociol. 1992;18:209-32. 28.
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Mining Region Economy Servization (on example of Kemerovo Region-Kuzbass)
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Mining Region Economy Servization (on example of Kemerovo Region-Kuzbass) Olga Esina1, Natalia Tereshchenko1,*, and Tatiana Pakshina2 1 Siberian Federal University, 660075 Lida Prushinskaya St. 2, Krasnoyarsk, Russia 2 Vitebsk State Technological University, 210038, 72 Moskovsky av., Vitebsk, Belarus Olga Esina1, Natalia Tereshchenko1,*, and Tatiana Pakshina2 1 Siberian Federal University, 660075 Lida Prushinskaya St. 2, Krasnoyarsk, Russia 2 Vitebsk State Technological University, 210038, 72 Moskovsky av., Vitebsk, Belarus Abstract. The social-and-economic development of the regions is largely determined by the state and specific features of the service industries’ development. The high-quality level of transport, housebuilding, communications, education and science, health care and other sectors of this industry creates the preconditions for sustainable growth of all spheres of the national economy. The article defines the development dynamics in the service sector in Kuzbass, its significance in the formation of the gross regional product, analyzes the average per capita volumes and the structure of service sector revenue in the Kemerovo region in comparison with the indicators in the Russian Federation and Siberian Federal District. Based on the research carried out, the specific features and trends in the development of the service sector in the region have been identified. 1 Introduction The formation of the regional economy structure occurs under the influence of a number of objective factors, such as historical, climatic, infrastructural, technological conditions of development. However, regardless of the cumulative impact of many constituent indicators, one of the most dynamically developing sectors of economy is the service sector. In modern conditions of economic development and globalization processes, the service sector significantly affects both the volume of economy and the structure of the gross domestic product. According to the information resource CIAWordFactbook [1], the share of the service sector in the formation of the gross product in 19 countries with the largest GDP, accounts for from 45.4% in India to 80.2% in the UK. Russia ranks the 6th by this indicator and over the past decade the share of the service sector in the country's GDP ranges from 70.66% in 2014 to 62% in 2017 [2]. At the same time, all other values for this indicator arranged by the years are within the specified interval. In the context of the federal districts of the Russian Federation, the share of the service sector in the gross regional product ranges from 42% in Urals Federal District to 75% in Central Federal District (Fig. 1). E3S Web of Conferences 278, 03025 (2021) SDEMR-2021 E3S Web of Conferences 278, 03025 (2021) SDEMR-2021 https://doi.org/10.1051/e3sconf/202127803025 © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). * Corresponding author: tereshchenko.65@mail.ru 2 Materials and Methods The issues of research and modeling of the service sector development are described in the works of both foreign and domestic researchers. The issues of research and modeling of the service sector development are described in the works of both foreign and domestic researchers. A number of scientists (Kitova O.V., Savinova V.M., Dyakonova L.P., Bruskin S.N., Beshmelnitskiy A.A., Danko T.P., Sekerin V.D.) [3] offer methodological approaches to the study of the service sector through the creation of situational centers. They focus on assessing social-and-economic indicators at different levels, planning and factor analysis in real time and forecasting the development of the service sector using a variety of qualitative and quantitative methods. Many foreign and domestic scientists (Razumova, E., Ilienko, O., [4] Dalevska N., Khobta V., Kwilinski A., Kravchenko S. [5], Dmitrieva M., Strekaleva T., Suslov D., Tereshchenko N. [6], Demchenko, S.K., Zlotnikov M.S., Melnikova T.A., Demchenko O.S. [7,8]) propose a methodological tool in the field of analysis and economic modeling of the service sector as a whole and for individual constituent industries. Generalization and critical analysis of the works of domestic and foreign authors allows determining a number of basic characteristics of the service sector as a central sector of the economy at all levels. Firstly, the service sector is an indicator of the social-and-economic development of the region. The civilizational approach to the study of the society development (theories of D. Bell, E. Tafler, A. Lesch) puts forward the state of the service sector as the main characteristics of industrial and post-industrial society. As this sector of economy determines the transformed resources in the form of knowledge, information, storage systems and data processing and key intelligent technologies. Thus, a high or constantly increasing level of development of the service sector testifies to the successful social and economic development of the regions. g Secondly, the service sector acts as the main factor in the development of production. By improving the occupancy and content of service industries, the formation and interaction of the main classical factors of production takes place, since it is with the use of intelligent technologies that individual and collective skills, production and exchange technologies are developed, the significance of the results achieved increases. Thirdly, the service sector can be viewed as an indicator of ensuring the quality of life of the population. * Corresponding author: tereshchenko.65@mail.ru E3S Web of Conferences 278, 03025 (2021) E3S Web of Conferences 278, 03025 (2021) SDEMR-2021 https://doi.org/10.1051/e3sconf/202127803025 Fig. 1. The share of the service sector by Federal Districts of the Russian Federation in 2019, %. Fig. 1. The share of the service sector by Federal Districts of the Russian Federation in 2019, %. Thus, we can say that the structural composition of the regional economy has a significant impact on its social-and-economic development, while the service sector, along with the extractive industries and the production sector, plays an important role in this process. 2 Materials and Methods A number of basic characteristics identified by UNESCO as part of assessing the life quality, such as safety, health, education, rational nutrition, are formed with the direct participation of certain service industries. Therefore, the higher the level of development of 2 https://doi.org/10.1051/e3sconf/202127803025 E3S Web of Conferences 278, 03025 (2021) SDEMR-2021 a given sector of economy, the more qualitative characteristics are determined by the standard of living of the population. a given sector of economy, the more qualitative characteristics are determined by the standard of living of the population. As already noted, the development of the service sector is influenced by many factors that determine its state in various territories. The object of our research is Kuzbass. The Kemerovo region is one of the technologically advanced mining regions of the Russian Federation, in connection with which the service sector in Kuzbass has a certain specificity, which is advisable to consider through a number of indicators: - the share of the service sector in the formation of the gross regional product; - the dynamics of the revenues’ volume in service industries for a number of years; the average per capita revenue in service industries per 1000 inhabitants; - the average per capita revenue in service industries per 1000 inhabitants; - the structural changes in main types of services provided to the population. - the structural changes in main types of services provided to the population. The Russian practice of statistical research reflects the results of the activities of economic sectors in accordance with the Russian National Classifier of Economic Activities Types, which introduced a number of changes that do not imply a clear separation of three main sectors of economy: extractive industries and agriculture, manufacturing and services. In this regard, the structuring of statistical data provided by the Federal State Statistics Service [2] makes it possible to determine the share of participation of the above groups of industries in the formation of the gross domestic product and gross regional products of the objects of research. 3 Results and Discussion A comparative characteristic of the service sector importance in the economy of the Russian Federation, Siberian Federal District and the Kemerovo region is shown in Figure 2. Fig. 2. Dynamics of the share of the service sector in the GDP of the Russian Federation and the GRP of Siberian Federal District and the Kemerovo region in 2008 – 2019, %. The data analysis shows that in the Russian Federation in the period from 2008 to 2019 the share of service industries accounted for from 71.01% in 2013 to 62% in 2017. In recent years, there has been a slowdown in the rate of increase in the volume of services, and, Fig. 2. Dynamics of the share of the service sector in the GDP of the Russian Federation and the G of Siberian Federal District and the Kemerovo region in 2008 – 2019, %. Fig. 2. Dynamics of the share of the service sector in the GDP of the Russian Federation and the GRP of Siberian Federal District and the Kemerovo region in 2008 – 2019, %. The data analysis shows that in the Russian Federation in the period from 2008 to 2019 the share of service industries accounted for from 71.01% in 2013 to 62% in 2017. In recent years, there has been a slowdown in the rate of increase in the volume of services, and, accordingly, its share in the gross domestic product. A similar situation is observed in the 3 E3S Web of Conferences 278, 03025 (2021) SDEMR-2021 https://doi.org/10.1051/e3sconf/202127803025 economy of Siberian Federal District. Here the interval of participation of the service sector in the formation of the gross regional product ranges from 58.4% in 2013 to 53.1% in 2017. The share of the service sector in the formation of the gross regional product in the Kemerovo region, ranges from 50.7% in 2014 to 41.2% in 2017 and is slightly lower than in the Russian Federation and Siberian Federal District. economy of Siberian Federal District. Here the interval of participation of the service sector in the formation of the gross regional product ranges from 58.4% in 2013 to 53.1% in 2017. The share of the service sector in the formation of the gross regional product in the Kemerovo region, ranges from 50.7% in 2014 to 41.2% in 2017 and is slightly lower than in the Russian Federation and Siberian Federal District. 3 Results and Discussion Thus, it can be noted that the importance of the service sector in the economy of Kuzbass is not so significant, although it accounts for more than a third of the enterprises revenues in the region, which is explained by the focus of the region on the mining and manufacturing sectors. Comparative characteristics of the dynamics of the change chain rates in the revenue of the subjects in the service sector, presented in Figure 3, shows that in all three analyzed regions there is a positive change in this indicator, with the exception of 2017, when there was a decrease in Siberian Federal District (86.75%) and in the Kemerovo region (99.44%). Fig. 3. Dynamics of revenue in service industries in the Russian Federation, Siberian Federal District and the Kemerovo region in 2010-2019, %. Fig. 3. Dynamics of revenue in service industries in the Russian Federation, Siberian Federal District and the Kemerovo region in 2010-2019, %. At the same time, it should be noted that the largest increase in the revenue of service sector entities was observed in 2010 for all research objects. The rate of increase in the volume of service industries in the Kemerovo Region is comparable to the average Russian and the indicators of Siberian Federal District, but we can note some lag in the last 4 years. Thus, we can note the positive dynamics of the development of the service industries in Kuzbass, which, however, is taking place at a slower pace than in the Russian Federation and Siberian Federal District. One of the characteristics of the service sector development is the average per capita revenue of industries per 1000 residents, which makes it possible to form an estimate of the consumption of services both in dynamics and in comparison by regions. Table 1. GDP of the Russian Federation and GRP of Siberian Federal District and the Kemerovo region from the service industries per 1000 residents of the region, million rubles. (Compiled according to [2]). 2009 2011 2013 2015 2017 2019 The Russian Federation 142.9 254.56 316.85 351.29 323.9 326.6 Table 1. GDP of the Russian Federation and GRP of Siberian Federal District and the Kemerovo region from the service industries per 1000 residents of the region, million rubles. (Compiled according to [2]). 3 Results and Discussion 4 E3S Web of Conferences 278, 03025 (2021) SDEMR-2021 https://doi.org/10.1051/e3sconf/202127803025 Siberian Federal District 100.5 134.5 167.8 191.8 213.1 218.3 The Kemerovo region 90.4 120.3 128.9 146.1 160.9 166.4 Analysis of the data summarized in Table 1 makes it possible to reveal the positive dynamics of the development of this indicator for almost all objects of study and in all periods, however, its cost characteristic is ambiguous. It should be noted that in the Kemerovo region in 2019 the volume of services per 1000 residents amounted to 0.51 points from the national average and 0.76 points from the indicator of Siberian Federal District. Thus, every resident of Kuzbass was provided services for about 13 thousand rubles per month (including utilities, medical, educational, communication services, transport, etc.), despite the fact that the national average of this figure was about 27 thousand rubles, and in Siberian Federal District it was about 18 thousand rubles. Thus, we can conclude that the development of the service sector in the region lags behind the average Russian and subject indicators and creates an opportunity for setting up and developing of business in the relevant sectors of economy. As it was noted earlier, there is no special form of accounting for services, including by type of activity in the State Statistics. Certain information is presented in the information and analytical system "Statistics" [9] and statistical collections of the Higher School of Economics [10]. Generalization of the data from the above sources allows making a judgment about the consumption structure of services by the population of the Kemerovo region in Table 2. Table 2. Dynamics of the structure of revenue from the provision of services in 2000-2019 in the Kemerovo region (compiled according to [9, 10]). Services 2000 2010 2019 Deviations 2010 from 2000 2019 from 2010 2019 from 2000 Domestic services 19.36 8.60 11.34 -10.76 2.74 -8.02 Veterinary services 0.17 0.13 0.14 -0.04 0.01 -0.03 Housing services 6.92 8.24 9.46 1.32 1.22 2.54 Utilities services 14.90 23.38 23.58 8.48 0.20 8.68 Medical services 4.38 6.51 7.12 2.13 0.61 2.74 Legal services 1.09 1.84 1.09 0.75 -0.75 - Sanitary and wellness 2.88 1.90 1.40 -0.98 -0.05 -1.48 Educational services 5.62 6.78 7.12 1.16 0.34 1.50 Transport services 28.10 13.20 12.85 -14.90 -0.35 -15.25 Tourist and excursion 0.38 3.93 3.97 3.55 0.04 3.59 Table 2. 3 Results and Discussion Dynamics of the structure of revenue from the provision of services in 2000-2019 in th Kemerovo region (compiled according to [9, 10]). 5 5 https://doi.org/10.1051/e3sconf/202127803025 E3S Web of Conferences 278, 03025 (2021) SDEMR-2021 Hotels, motels - 1.41 1.10 1.41 -0.31 1.10 Communications 8.78 20.30 16.02 11.52 -4.25 7.24 Cultural institutions 1.24 1.43 1.57 0.19 0.14 0.33 Physical culture and sports 0.26 0.76 1.10 0.50 0.34 0.84 Other services 5.92 1.58 2.15 -4.34 0.57 -3.77 Total 100.0 100.0 100.0 - - - Analysis of the data in the table allows determining that over the past twenty years, the consumption structure of services by the population of Kuzbass has changed significantly. If in 2000 the main share was accounted for transport (28.1%), domestic (19.36%) and utilities (14.9%) services, then in 2019, the share of communication (16.02%) and utilities (23.56%) services is significantly increasing, at the same time, the share of transport services remains significant (12.85%) too. Veterinary services (0.14%), legal services (1.09%), services of hotels and motels, and services of physical culture and sports (1.10%) have insignificant shares in the structure of revenues from the provision of services. Thus, a thesis about the possibility of entrepreneurship development in these service sectors arises. 4 Conclusion The service sector is one of the most important sectors of economy, determining many aspects of the national economy development. The studies carried out made it possible to determine a number of specific features of the service sector development on the territory of the Kemerovo region – Kuzbass, which may be summarized in the following provisions: g y g - There is lower importance of the service sector in the formation of the gross regional product than in the Russian Federation and Siberian Federal District. - There is lower importance of the service sector in the formation of the gross regional product than in the Russian Federation and Siberian Federal District. - There are slower rates of development of this sector of economy in comparison with the average Russian and subject indicators. - There are slower rates of development of this sector of economy in comparison with the average Russian and subject indicators. - There is a significant lag in the volume of services per 1000 residents from the average in the country and Siberian Federal District. - There is poor development of certain branches of the service sector related to the development of health, legal support, culture, recreation. - There is poor development of certain branches of the service sector related to the development of health, legal support, culture, recreation. All of the above allows making the conclusion that business development in this territory has great opportunities in the service sector. All of the above allows making the conclusion that business development in this territory has great opportunities in the service sector. It should be noted that the “Strategy for the social-and-economic development of the Kemerovo region – Kuzbass for the period up to 2035” [11] pays considerable attention to the development of the service industries. The main principles are high quality and reliable provision of a new standard of living and meeting the needs of residents. In this regard, four main development priorities have been formed, ensuring professional excellence, high quality of life, and a green economy for residents. The presented provisions will undoubtedly contribute to the development of the service sector in Kuzbass. 6 6 https://doi.org/10.1051/e3sconf/202127803025 E3S Web of Conferences 278, 03025 (2021) SDEMR-2021 References 1. С.I.A. theWorldFactbook. URL: https://www.cia.gov/library/publications/the-world- factbook/. 2. Rosstat, Official Website. URL: www.gks.ru 3. O. Kitova, V. Savinova, L. Dyakonova, S. Bruskin, European Research Studies Journal, 20(4), 275-283 (2017) 4. E. Razumova, O. Ilienko, V. Ihnatiuk, Baltic journal of economic studies, 4(2), 191-197 (2018) 5. N. Dalevska, V. Khobta, A. Kwilinski, S. Kravchenko, Entrepreneurship and Sustainability Issues, 6(4), 1839-1860 (2019) 6. M. Dmitrieva, T. Strekaleva, D. Suslov, N. Tereshchenko, Proceedings The 32nd IBIMA conference (Seville, Spain, 2018) 7. S.K. Demchenko, M.S. Zlotnikov, T.A. Melnikova, O.S. Demchenko, International Journal of Civil Engineering and Technology, 10(2), 1877-1884 (2019) 8. S. Zhironkin, S. Demchenko, G. Kayachev, E. Taran, O. Zhironkina, E3S Web Conf. 105, 03008 (2019) 9. Information and analytical system "Statistics". URL: www.ias-stat.ru/ 10. HSE, Statistical collections. URL: https://www.hse.ru/primarydata/ 11. Strategy of socio-economic development of the Kemerovo region-Kuzbass for the period up to 2035 (AKO, Kemerovo, 2019) 7
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FTL004, an anti-CD38 mAb with negligible RBC binding and enhanced pro-apoptotic activity, is a novel candidate for treatments of multiple myeloma and non-Hodgkin lymphoma
Journal of hematology & oncology
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Zhang et al. Journal of Hematology & Oncology (2022) 15:177 https://doi.org/10.1186/s13045-022-01395-0 Zhang et al. Journal of Hematology & Oncology (2022) 15:177 https://doi.org/10.1186/s13045-022-01395-0 Zhang et al. FTL004, an anti‑CD38 mAb with negligible RBC binding and enhanced pro‑apoptotic activity, is a novel candidate for treatments of multiple myeloma and non‑Hodgkin lymphoma Guangbing Zhang1†, Cuiyu Guo1†, Yan Wang1†, Xianda Zhang2†, Shuang Liu1,2, Wen Qu3, Chunxia Chen4, Lingli Yan4, Zhouning Yang2, Zhixiong Zhang1, Xiaohua Jiang1, Xiaofeng Chen1, Hong Liu1, Qinhuai Lai1, Xian Wei1, Ying Lu1, Shengyan Zhao1, Han Deng1, Yuxi Wang1,5, Lin Yu3, Hongbin Yu6, Yu Wu6, Zhaoming Su1, Pengyu Chen2, Ziqing Ren2, Meng Yu2, Feng Qu2, Yong Luo7, Lantu Gou1, Qing Li2,8, Ying Huang2†, Fanxin Ma1,2† and Jinliang Yang1,9*† Abstract Anti-CD38 monoclonal antibodies (mAbs), daratumumab, and isatuximab have represented a breakthrough in the treatment of multiple myeloma (MM). Recently, CD38-based mAbs were expected to achieve increasing poten- tial beyond MM, which encouraged us to develop new anti-CD38 mAbs to meet clinical needs. In this study, we developed a novel humanized anti-CD38 antibody, FTL004, which exhibited enhanced pro-apoptotic ability and negligible binding to red blood cells (RBCs). FTL004 presented a better ability to induce direct apoptosis independ- ent of Fc-mediated cross-linking against lymphoma and MM cell lines as well as primary myeloma cells derived from MM patients. For instance, FTL004 induced RPMI 8226 cells with 55% early apoptosis cells compared with 20% in the isatuximab-treated group. Of interest, FTL004 showed ignorable binding to CD38 on human RBCs in contrast to tumor cells, even at concentrations up to 30 μg/mL. Furthermore, with an engineered Fc domain, FTL004 displayed stronger antibody-dependent cellular cytotoxicity (ADCC) against CD38+ malignant cells. In vivo MM and non-Hodg- kin lymphoma tumor xenograft models showed that FTL004 possessed an effective anti-tumor effect. Cryo-electron microscopy structure resolved two epitope centers of FTL004 on CD38: one of which was unique while the other © The Author(s) 2022. Open Access This arti permits use, sharing, adaptation, distribution original author(s) and the source, provide a li other third party material in this article are in to the material. If material is not included in t regulation or exceeds the permitted use, you licence, visit http://​creat​iveco​mmons.​org/​lice mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies †Guangbing Zhang, Cuiyu Guo, Yan Wang, and Xianda Zhang equally contributors †Ying Huang, Fanxin Ma, and Jinliang Yang contributed equally to the article as Co-senior authors *Correspondence: jinliangyang@scu.edu.cn 1 State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, 3‑17 People Road, Chengdu, Sichuan 610041, People’s Republic of China Full list of author information is available at the end of the article †Guangbing Zhang, Cuiyu Guo, Yan Wang, and Xianda Zhang equally contributors †Ying Huang, Fanxin Ma, and Jinliang Yang contributed equally to the article as Co-senior authors *Correspondence: jinliangyang@scu.edu.cn 1 State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, 3‑17 People Road, Chengdu, Sichuan 610041, People’s Republic of China Full list of author information is available at the end of the article CORRESPONDENCE Open Access To the editor CD38 is a type II transmembrane glycoprotein with mul- tiple functions and expressed lower on normal blood cells and higher on hematologic tumor cells [1, 2]. Anti-CD38 mAbs were effective in some hematological malignan- cies, especially multiple myeloma (MM) [3]. Although both daratumumab and isatuximab have significantly improved the outcome of patients with MM, incomplete responses and on-target/off-tumor effects emerge dur- ing the treatment [4, 5]. Herein, we described FTL004, a novel humanized IgG1κ anti-CD38 mAb possessing novel properties. Then, the CD38/FTL004 complex structure was deter- mined using Cryo-electron microscopy  at 3.86  Å reso- lution (Fig. 1h, i). Of note, there seem to be two epitope centers of FTL004, one of them includes from Pro52 to His56 and the other one is 91TQTV94. Superimposition of the CD38/daratumumab (PDB code 7DHA) complex and CD38/isatuximab (PDB code 4CMH) complex onto the CD38/FTL004 complex revealed that the epitopes of FTL004 share no common residue of CD38 with dara- tumumab, whereas one of the epitopes (91TQTV94) of FTL004 partly overlaps with that of isatuximab (Fig. 1j). FTL004 exhibited similar affinities to CD38 (KD of 2.55, 3.84, and 1.2 nM, respectively) in surface plasmon resonance (Fig. 1a) and to CD38-positive cell lines in flow cytometry (Fig. 1b), compared with daratumumab or isatuximab. However, these antibodies bound differently to blood cells. Daratumumab or isatuximab bound to RBCs to different extents from healthy donors, while the binding of FTL004 to RBCs was virtually undetectable (Fig. 1c). This property should result in a more favorable pharmacokinetic of FTL004 by circumventing binding to CD38 on circulating RBCs [6] and a better safety profile by minimizing side effects associated with RBC binding. Moreover, FTL004 did not agglutinate RBCs in indirect antiglobulin tests (Fig. 1d), which could avoid the inter- ferences with blood transfusion that are observed in other anti-CD38 mAbs [7, 8]. Next, to evaluate the tumor-killing capacity of FTL004, pro-apoptotic activity was first analyzed. We found that FTL004 had a superior ability to induce direct apoptosis independent of cross-linking rea- gents against CD38-positive cell lines (Fig. 2a) and primary MM cells (Fig. 2b) than isatuximab. Particu- larly, the highest apoptosis rate of FTL004 on pri- mary MM cells was detected to be up to 60%. On the other hand, the Fc domain of FTL004 was engineered (S240D/I333E mutation) to elevate the affinity to human Fcγ receptors, leading to the enhancement of antibody-dependent cellular cytotoxicity (ADCC) [10]. (See figure on next page.) Fig. 1  Distinct binding profile of FTL004 to CD38+ tumor cells, RBCs, and immune cells. a Affinity analysis of FTL004 to human CD38 as measured by Biacore 8 K (KD = 2.55 × ­10–9 mol/L). b EC50 values for anti-CD38 mAbs binding to diverse cells. DARA, daratumumab; ISA, isatuximab. c Binding of FTL004, daratumumab or isatuximab to freshly isolated RBCs from healthy donors. d Indirect antiglobulin tests for CD38 mAbs to the same three healthy donors RBCs above. e The percentage of binding of CD38 mAbs to normal PBMCs (n = 4), including to CD45+ CD3+ T cells, CD3+ CD4+ T cells, CD3+ CD8+ T cells, CD45+ CD19+ B cells and CD3-CD56+ NK cells. Data show means ± SD and unpaired Student’s t tests were used to determine the statistical significance between daratumumab versus FTL004. *P < 0.05, **P < 0.01 and ***P < 0.001. f Flow cytometry dot plots showing the binding of anti-CD38 mAbs to NK cells and CD8+ T cells. g PBMCs from normal donors (n = 6) were pretreated with 1 μg/mL FTL004 or isatuximab for 3 days. Flow cytometry was used to determine the percentage of CD3+ T cells, CD4+ T cells, CD8+ T cells, B cells, and NK cells in lymphocytes. Data were then normalized to isotype controls and fold changes (means ± SD) are shown. Isotype cells were used as controls in settings. h The overall structure of the CD38/FTL004 Fab complex. CD38 and the heavy and light chains of FTL004 are colored gray, purple, and green, respectively. i Stereoscopic view of the direct hydrogen bonds, salt bridge, in the interface between CD38 and FTL004. j Comparison of the binding of FTL004, daratumumab, and isatuximab. Surface representation of the epitopes of FTL004 (green), daratumumab (blue), isatuximab (red) and superposition part of FTL004 and isatuximab (yellow) on CD38 (gray) © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Zhang et al. Journal of Hematology & Oncology (2022) 15:177 Page 2 of 6 partly overlapped with that of isatuximab. Taken together, FTL004 distinguishes it from other CD38 targeting mAbs and represents a potential candidate for the treatment of MM and non-Hodgkin lymphoma. Keywords:  Multiple myeloma, Monoclonal antibodies, CD38, Red blood cells, Direct apoptosis partly overlapped with that of isatuximab. Taken together, FTL004 distinguishes it from other CD38 targeting mAbs and represents a potential candidate for the treatment of MM and non-Hodgkin lymphoma. Keywords:  Multiple myeloma, Monoclonal antibodies, CD38, Red blood cells, Direct apoptosis partly overlapped with that of isatuximab. Taken together, FTL004 distinguishes it from other CD38 targeting mAbs and represents a potential candidate for the treatment of MM and non-Hodgkin lymphoma. Keywords:  Multiple myeloma, Monoclonal antibodies, CD38, Red blood cells, Direct apoptosis reflected that the epitope of FTL004 binding on CD38 may be distinct. Although anti-CD38 mAbs require a high threshold for CD38 expression to induce cell death, we must be wary of its possible off-target effects. PBMCs coculture assays showed that there was no depletion in T and B cells, indicating the limited off-tumor toxicity of FTL004 (Fig. 1g). But as expected, NK cells, with higher expression of CD38, were reduced to an acceptable level [9].h To the editor In ADCC bioassays with Jurkat-Lucia-CD16a-NFAT cells [11], FTL004 showed a distinctly higher intensity of ADCC, with EC50 values of 2–8 ng/mL, compared For other immune cells, FTL004 bound to most sub- population cells of peripheral blood mononuclear cells (PBMCs) in a comparable intensity with daratumumab or isatuximab (Fig. 1e). Unexpectedly, FTL004 exhib- ited a slightly higher binding to CD8+ T cells and a much higher binding to NK cells (Fig. 1f). These events Zhang et al. Journal of Hematology & Oncology (2022) 15:177 Page 3 of 6 Page 3 of 6 Zhang et al. Journal of Hematology & Oncology (2022) 15:177 Fig. 1 (See legend on previous page.) Fig. 1  (See legend on previous page.) Fig. 1  (See legend on previous page.) Fig. 1  (See legend on previous page.) Zhang et al. Journal of Hematology & Oncology (2022) 15:177 Page 4 of 6 Fig. 2  Anti-tumor efficacy of FTL004. a Induction of apoptosis by FTL004, daratumumab, or isatuximab in various cell lines, cells were treated with anti-CD38 mAbs 1.5 μg/mL for 24 h. Data represent means of triplicates and SD. Statistical significance between FTL004 and hIgG groups was determined by unpaired Student’s t tests. *P < 0.05; **P < 0.01, ***P < 0.001. b Apoptosis induction by FTL004 in primary MM cells. c, d ADCC reporter bioassays to various CD38+ tumor cell lines (c) and MM primary cells (d) of FTL004, daratumumab, or isatuximab. Data represent means of triplicates and SD. e–g Representative dose–response curve examples for ADCC activity (e), ADCP activity (f), and CDC activity (g) of FTL004 or daratumumab to CHO-CD38+ cells. Data show mean ± SD of three experiments. h Anti-tumor efficacy of FTL004 in lymphoma cell Ramos, MM cell H929, MM1S xenograft models. NOD-SCID mice bearing xenografts were treated with administration of PBS, daratumumab, or FTL004. Tumor burdens were monitored. Each point on the graph represents the average tumor volume Fig. 2  Anti-tumor efficacy of FTL004. a Induction of apoptosis by FTL004, daratumumab, or isatuximab in various cell lines, cells were treated with anti-CD38 mAbs 1.5 μg/mL for 24 h. Data represent means of triplicates and SD. Statistical significance between FTL004 and hIgG groups was determined by unpaired Student’s t tests. *P < 0.05; **P < 0.01, ***P < 0.001. b Apoptosis induction by FTL004 in primary MM cells. Consent for publication Not applicable. Consent for publication Not applicable. References 1. Chini CCS, Peclat TR, Warner GM, Kashyap S, Espindola-Netto JM, de Oliveira GC, et al. CD38 ecto-enzyme in immune cells is induced during aging and regulates NAD(+) and NMN levels. Nat Metab. 2020;2(11):1284–304. Competing interests Q Qing Li, Ying Huang, Fanxin Ma, Xianda Zhang, Shuang Liu, Zhouning Yang, Pengyu Chen, Ziqing Ren, Meng Yu, and Feng Qu are employees at Sound Biopharmaceuticals Co., Ltd. No potential conflicts of interest were disclosed by the other authors. Acknowledgements The authors thank all of the patients and healthy donors who consented to this study. They also thank Yiran Tao (West China-California Research Center for Predictive Intervention Medicine, West China Hospital, Sichuan University) for FACS data acquisition and analysis. To the editor c, d ADCC reporter bioassays to various CD38+ tumor cell lines (c) and MM primary cells (d) of FTL004, daratumumab, or isatuximab. Data represent means of triplicates and SD. e–g Representative dose–response curve examples for ADCC activity (e), ADCP activity (f), and CDC activity (g) of FTL004 or daratumumab to CHO-CD38+ cells. Data show mean ± SD of three experiments. h Anti-tumor efficacy of FTL004 in lymphoma cell Ramos, MM cell H929, MM1S xenograft models. NOD-SCID mice bearing xenografts were treated with administration of PBS, daratumumab, or FTL004. Tumor burdens were monitored. Each point on the graph represents the average tumor volume Fig. 2  Anti-tumor efficacy of FTL004. a Induction of apoptosis by FTL004, daratumumab, or isatuximab in various cell lines, cells were treated with anti-CD38 mAbs 1.5 μg/mL for 24 h. Data represent means of triplicates and SD. Statistical significance between FTL004 and hIgG groups was determined by unpaired Student’s t tests. *P < 0.05; **P < 0.01, ***P < 0.001. b Apoptosis induction by FTL004 in primary MM cells. c, d ADCC reporter bioassays to various CD38+ tumor cell lines (c) and MM primary cells (d) of FTL004, daratumumab, or isatuximab. Data represent means of triplicates and SD. e–g Representative dose–response curve examples for ADCC activity (e), ADCP activity (f), and CDC activity (g) of FTL004 or daratumumab to CHO-CD38+ cells. Data show mean ± SD of three experiments. h Anti-tumor efficacy of FTL004 in lymphoma cell Ramos, MM cell H929, MM1S xenograft models. NOD-SCID mice bearing xenografts were treated with administration of PBS, daratumumab, or FTL004. Tumor burdens were monitored. Each point on the graph represents the average tumor volume Zhang et al. Journal of Hematology & Oncology (2022) 15:177 Page 5 of 6 with daratumumab and isatuximab (Fig. 2c). Consistent results were obtained in primary MM cells (Fig. 2d). Abbreviations Ab M l mAb: Monoclonal antibody; MM: Multiple myeloma; RBCs: Red blood cells; PBMCs: Peripheral blood mononuclear cells; ADCC: Antibody-dependent cellular cytotoxicity. Received: 24 October 2022 Accepted: 20 December 2022 Received: 24 October 2022 Accepted: 20 December 2022 Funding h k This work was supported by the National Natural Science Foundation of China (81,872,907, 32,070,049, 32,222,040), Science and Technology Plan Project of Sichuan Province (Provincial Academy and Provincial University Cooperation Project) (2020YFSY0025), Sichuan Science and Technology Program (2021YJ0454, 2019YFS0003), Ministry of Science and Technology of China (MoST) (2021YFA1301900), Sichuan University start-up funding (20822041D4057), the CAMS Innovation Fund for Medical Sciences (CIFMS) (2021-I2M-5-075), and the National Major Scientific and Technological Special Project for Significant New Drugs Development (2018ZX09201018-021, 2017ZX09302010). 5. Ofran Y. Daratumumab: new indications revolving around “off-targets.” Haematologica. 2021;106(12):3032–3. 6. Fedyk ER, Idamakanti N, Chen J, Estevam J, Palumbo A. The binding of CD38 therapeutics to red blood cells and platelets subverts depletion of target cells. Blood. 2019;134(Supplement_1):3136. 7. Werle E, Ziebart J, Wasmund E, Eske-Pogodda K. Daratumumab interfer- ence in pretransfusion testing is overcome by addition of daratumumab Fab fragments to patients’ plasma. Transfus Med Hemother. 2019. https://​ doi.​org/​10.​1159/​00049​5773. 7. Werle E, Ziebart J, Wasmund E, Eske-Pogodda K. Daratumumab interfer- ence in pretransfusion testing is overcome by addition of daratumumab Fab fragments to patients’ plasma. Transfus Med Hemother. 2019. https://​ doi.​org/​10.​1159/​00049​5773. 8. Lancman G, Arinsburg S, Jhang J, Cho HJ, Jagannath S, Madduri D, et al. Blood transfusion management for patients treated with anti-CD38 monoclonal antibodies. Front Immunol. 2018;9:2616. 8. Lancman G, Arinsburg S, Jhang J, Cho HJ, Jagannath S, Madduri D, et al. Blood transfusion management for patients treated with anti-CD38 monoclonal antibodies. Front Immunol. 2018;9:2616. Author details 1 State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innova- tion Center for Biotherapy, West China Hospital, Sichuan University, 3‑17 People Road, Chengdu, Sichuan 610041, People’s Republic of China. 2 Sound Biopharmaceuticals Co., Ltd., Tianfu International Bio‑Town, Huigu Dong 2nd Road 8, Chengdu, Sichuan 610200, People’s Republic of China. 3 Department of Clinical Laboratory, Mianyang Central Hospital, Mianyang, People’s Republic of China. 4 Department of Transfusion, West China Hospital, Sichuan University, Chengdu, People’s Republic of China. 5 Department of Clinical Research Man- agement, West China Hospital, Sichuan University, Chengdu, People’s Republic of China. 6 Department of Hematology, West China Hospital, Sichuan Univer- sity, Chengdu, People’s Republic of China. 7 Department of Head and Neck Oncology, West China Hospital, Sichuan University, Chengdu, People’s Repub- lic of China. 8 West China School of Public Health, Sichuan University, Chengdu, People’s Republic of China. 9 Research Unit of Gene and Immunotherapy, Chinese Academy of Medical Sciences, Chengdu, People’s Republic of China. In summary, FTL004 is a novel CD38 mAb that owns unique properties. Although the mechanism by which FTL004 binds differently to tumor cells versus RBCs remains unclear, it is expected that FTL004 owns lim- ited on-target/off-tumor effects. The results above sug- gest that FTL004 is a therapeutic Ab with high potential for the treatment of MM and non-Hodgkin lymphoma. Author contributions 2. Mehta K, Shahid U, Malavasi F. Human CD38, a cell-surface pro- tein with multiple functions. FASEB J Off Publ Fed Am Soc Exp Biol. 1996;10(12):1408–17. ZGB, GCY, ZXD, MFX, HY, LQ, GLT, and YJL were involved in study conception and design; ZGB, WY, LS, YZN, ZZX, JXH, and WYX contributed to the develop- ment of methodology; QW, LQH, YLL, CCX, YL, YHB, WY, and SZM participated in acquisition of data; LH, CPY, RZQ, YM, and QF were involved in analysis and interpretation of data; ZGB, WX, LY, ZSY, CXF, DH, and LY wrote and reviewed the manuscript. All authors read and approved the final manuscript. 3. van de Donk NW, Janmaat ML, Mutis T, van Lammerts Bueren JJ, Ahmadi T, Sasser AK, et al. Monoclonal antibodies targeting CD38 in hematologi- cal malignancies and beyond. Immunol Rev. 2016;270(1):95–112. 3. van de Donk NW, Janmaat ML, Mutis T, van Lammerts Bueren JJ, Ahmadi T, Sasser AK, et al. Monoclonal antibodies targeting CD38 in hematologi- cal malignancies and beyond. Immunol Rev. 2016;270(1):95–112. 4. Bhatnagar V, Gormley NJ, Lola L, et al. FDA approval summary: dara- tumumab for treatment of multiple myeloma after one prior therapy. Oncologist. 2017. https://​doi.​org/​10.​1634/​theon​colog​ist.​2017-​0229. Ethics approval and consent to participate To  investigate further, we examined the ADCC of FTL004 with healthy PBMCs. PBMCs exhibited enhanced ADCC activity against CHO-CD38+ cells in the presence of FTL004, with threefold higher than daratumumab (Fig. 2e). Meanwhile, FTL004 induced stronger ADCP with 74% phagocytosed CHO- CD38+ cells in an EC50 value of 40  ng/mL (Fig. 2f). However, FTL004 induced a lower CDC than daratu- mumab of CHO-CD38+ cells (Fig. 2g). In vivo studies were further carried out, and FTL004 was examined in Ramos, H929, and MM1S-bearing NOD-SCID mice (Fig. 2h). FTL004 treatment resulted in significant inhi- bition of tumor growth compared to PBS treatment. The human subject studies were approved by the Ethnic Committee of West China Hospital of Sichuan University. All patients provided written informed consent. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. 9. Naeimi Kararoudi M, Nagai Y, Elmas E, de Souza Fernandes Pereira M, Ali SA, Imus PH, et al. CD38 deletion of human primary NK cells eliminates daratumumab-induced fratricide and boosts their effector activity. Blood. 2020;136(21):2416–27. Page 6 of 6 Zhang et al. Journal of Hematology & Oncology (2022) 15:177 Zhang et al. Journal of Hematology & Oncology (2022) 15:177 Zhang et al. Journal of Hematology & Oncology (2022) 15:177 10. Jebamani P, Sriramulu DK, Jung ST, Lee S-G. Structural study on the impact of S239D/I332E mutations in the binding of Fc and FcγRIIIa. Biotechnol Bioprocess Eng. 2021;26(6):985–92. 11. Hong Y, Guo H, Wei M, Zhang Y, Fang M, Cheng T, et al. Cell-based reporter assays for measurements of antibody-mediated cellular cytotox- icity and phagocytosis against SARS-CoV-2 spike protein. J Virol Methods. 2022;307: 114564. 11. Hong Y, Guo H, Wei M, Zhang Y, Fang M, Cheng T, et al. Cell-based reporter assays for measurements of antibody-mediated cellular cytotox- icity and phagocytosis against SARS-CoV-2 spike protein. J Virol Methods. 2022;307: 114564. 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http://conservancy.umn.edu/bitstream/11299/174058/1/JTLU_vol8_no2_pp171-189.pdf
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Has Mexico City’s shift to commercially produced housing increased car ownership and car use?
Journal of transport and land use
2,015
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       http://jtlu.org vol. 8 no. 2 [2015] pp. 171–189        http://jtlu.org vol. 8 no. 2 [2015] pp. 171–189 Article history: Abstract: Mexico City’s principal form of housing production has shifted over the past two decades. More households now purchase houses in large commercially built housing developments than move into informal settlements. Looking at 1500 households in two subur- ban municipalities from a 2007 metropolitan travel survey, this study is the first to quantify differences in car ownership and car use across households in informal settlements and commercial housing develop- ments. Accounting for income, proxies for wealth, household com- position, and geography, households living in commercial housing developments are likely to own more cars and drive more than similar households in neighboring informal settlements. A test for residen- tial self-selection finds no unobserved correlations across households that own cars and live in commercial housing developments, suggest- ing that the included controls do a good job of capturing the effects of residential self-selection or that the effects are limited. Something about the local land use and design of new commercial housing devel- opments appears conducive to car ownership and use. Differences be- tween the two settlement types, including more parking, wider streets, less-connected street-grids, and less accessible transit stops in commer- cial settlements, likely play a role. Article history: Received: December 28, 2013 Received in revised form: August 25, 2014 Accepted: February 16, 2015 Available online: May 29, 2015 Introduction 1 At 35,000 feet—a typical cruising altitude for a long-distance commercial flight—Los Héroes Tecámac looks a bit like an urban airport. Two long, thick, near-perpendicular, gray lines intersect like massive runways providing access from northwest to southeast and northeast to southwest. To the north are the unmistakable tan and green rectangles of agricultural land. To the east and west are the equally unmis- takable curving black, gray, and tan specks and lines of informal urbanization that is so characteristic of Mexico City’s peripheral neighborhoods and many like them around the world (Figure 1). Located al- most 20 miles to the northeast of the historic center, Los Héroes, despite its somewhat unusual appear- ance from the air, is typical of contemporary housing developments in Mexico City on the ground. Over the past two decades, Mexico City’s principal form of new housing production has shifted from an informal process, where households built their own properties generally in informal settlements on Copyright 2015 Erick Guerra http://dx.doi.org/10.5198/jtlu.2015.714 ISSN: 1938-7849 | Licensed under the Creative Commons Attribution – Noncommercial License 3.0 Copyright 2015 Erick Guerra http://dx.doi.org/10.5198/jtlu.2015.714 ISSN: 1938-7849 | Licensed under the Creative Commons Attribution – Noncommercial License 3.0 Has Mexico City’s shift to commercially produced housing increased car ownership and car use? Erick Guerra University of Pennsylvania erickg@design.upenn.edu Copyright 2015 Erick Guerra http://dx.doi.org/10.5198/jtlu.2015.714 ISSN: 1938-7849 | Licensed under the Creative Commons Attribution – Noncommercial License 3.0 py g http://dx.doi.org/10.5198/jtlu.2015.714 ISSN: 1938-7849 | Licensed under the Creative Commons Attribution – Noncommercial License 3.0 The Journal of Transport and Land Use is the official journal of the World Society for Transport and Land Use (WSTLUR) and is published and sponsored by the University of Minnesota Center for Transportation Studies. This paper is also published with sponsorship from WSTLUR and the Institutes of Transportation Studies at the University of California, Davis, and the University of California, Berkeley. 172 JOURNAL OF TRANSPORT AND LAND USE 8.2 the periphery, to a formal process, where private developers acquire large peripheral lots, build new hous- ing speculatively, and sell completed homes in massive developments to households that qualify for publicly subsidized mortgages (Monkkonen 2011a, 2011b, Pardo and Velasco Sánchez 2006). What look like thick gray lines from the air are rows of identical one-, two-, and three-story attached houses and condominiums along intersecting rectangular grids of arterial roads and linear culs-de-sac. Small parking spaces separate the building facades from the sidewalk and street. Figure 1: Aerial view of Los Héroes and surroundings in Ecatepec and Tecamac, State of Mexico (Google Earth 02-08-2013) Figure 1: Aerial view of Los Héroes and surroundings in Ecatepec and Tecamac, State of Mexico (Google Earth 02-08-2013) Changes in public policy, rather than demand, primarily drove this shift in housing production. Since Mexico’s 1994 economic crisis and currency devaluation, the central government has pursued financial liberalization reforms including the reform of Infonavit, a national housing provident fund that requires private-sector employers to pay 5 percent of salaried workers’ wages into the fund, which subsidizes loans for qualified low- to moderate- income workers to purchase newly built, privately produced homes (Monkkonen 2011a, Pardo and Velasco Sánchez 2006). Between 1995 and 2005, public agencies pro- vided 75 percent of all housing loans by value—and even more by volume—in Mexico. Infonavit accounted for 81 percent of these publicly financed loans (Monkkonen 2011b). Smaller but similar housing funds for low-income workers with informal employment, government workers, and the state- owned oil company (Pemex) also contributed to the increase in commercial housing production. The legalization of sales of communally owned farmland, a simplification of housing construction regula- tions, and active political engagement with private developers also facilitated the rapid shift in housing production (Monkkonen 2011a). Copyright 2015 Erick Guerra http://dx.doi.org/10.5198/jtlu.2015.714 ISSN: 1938-7849 | Licensed under the Creative Commons Attribution – Noncommercial License 3.0 In 1980, commercial developers produced just 15 percent of housing in the Mexico City me- tropolis (Dowall and Wilk 1989). By 2010, they produced the majority, particularly in fast-growing Has Mexico City’s shift to commercially produced housing increased car ownership and car use? 173 suburban municipalities like Tecamac. By the early 21st century, Mexico’s largest commercial housing builders—Geo, Homex, Javer, Sadasi, and Urbi—were among the world’s largest housing producers by volume. In 2010, Geo produced and sold more homes per year than the combination of D.R. Horton and the Pulte Group, the two largest housing producers by volume in the United States. All five of the above-mentioned Mexican companies would have made recent top-10 lists of the largest US housing producers (Hanley Wood LLC 2013, Nava 2011). Over the same period as the shift in housing production occurred, car ownership and use have increased steadily. Between 2000 and 2010, the number of registered cars increased an average of 14 percent annually (INEGI 2013). Between 1994 and 2007, total vehicle kilometers traveled (VKT) on an average weekday increased from 24 to 32 million (Guerra 2014, INEGI 2007, 1994). Most of this growth has occurred in suburban municipalities outside of the Federal District of Mexico City where the shift in housing production has been so pronounced. In general, the new commercial housing develop- ments are denser and closer to the city center than recently constructed housing in informal settlements (Monkkonen 2011c) but have less connected local street networks, and each housing unit comes with a single, exterior parking space. Although many residents convert parking into commercial retail spaces or residential additions over time, the default state has moved from one in which households expend land, energy, and resources to build a parking space to one in which they must expend time and energy to convert the parking to something else. This paper provides the first empirical analysis of whether the shift in Mexico’s housing policy is contributing to the rapid growth in car ownership and VKT in Mexico City. Are households that live in new commercial housing developments more likely to own and drive cars than similar households in nearby informal settlements? To what extent do differences in car ownership and use relate to local land use and design, or unobserved differences between households, such as a preference for driving and thus a preference to locate in new, more car-friendly neighborhoods? Copyright 2015 Erick Guerra http://dx.doi.org/10.5198/jtlu.2015.714 ISSN: 1938-7849 | Licensed under the Creative Commons Attribution – Noncommercial License 3.0 Given the level of public involvement in housing production and the public desire to reduce emis- sions and congestion, the answers to these questions matter. If the increased driving of households in commercial housing developments is related to local land use or design, then the public sector has the opportunity to reduce car use by redesigning commercial housing developments and requiring new construction to follow design guidelines. If, by contrast, differences are primarily related to regional accessibility or varying household preferences, local design and land-use changes will have little to no influence on car use. The answers also matter outside of Mexico City. There is a long and lively debate over whether and to what extent local land use and design influence travel behavior (Boarnet and Crane 2001, Brown- stone 2008, Crane 2000, 1996, Ewing and Cervero 2010, Handy, Cao, and Mokhtarian 2005, Pickrell 1999). One point of particular contention is the extent to which residential self-selection influences the observed relationships with urban design and land use (for a discussion, see Mokhtarian and Cao 2008). In Mexico City, however, the recent and dramatic change in housing type may provide something akin to a natural experiment. The new housing product is uniform, giving households little chance to select amenities based on preferences. Furthermore, due to the housing subsidy, households are most likely choosing between living in a peripheral informal settlement or a peripheral commercial housing devel- opment based on loan eligibility rather than travel preferences. Since the new developments have the same levels of regional transit- and car-accessibility as the informal neighborhoods around them, differ- ences in travel behavior that are related to the built environment stem from neighborhood rather than regional differences. If local land use and design influence travel in peripheral Mexico City, they also likely influence travel in the fast-growing peripheral neighborhoods of many other cities where popula- tion, suburbanization, and car ownership are increasing rapidly. JOURNAL OF TRANSPORT AND LAND USE 8.2 JOURNAL OF TRANSPORT AND LAND USE 8.2 174 2 Research framework and methods 2.1 Study area and data 2 Study area and data 2.1 This study focuses on two suburban municipalities: Ixtapaluca and Tecamac. Both grew rapidly between 1990 and 2010—a period that coincides with the rapid shift from informal to formal housing produc- tion. Ixtapaluca, located about 30 kilometers east of Mexico City’s historic center, grew at an average annual rate of 12 percent, adding 330,000 new residents between 1990 and 2010. At an average an- nual growth rate of 10 percent, Tecamac’s population tripled from 120,000 to 360,000 residents over the same period. Figure 2 maps metropolitan population growth by municipality between the last two decennial censuses and shows the location of Ixtapaluca and Tecamac. Large-scale, high-density private commercial housing developments accounted for a substantial portion of the growth in both munici- palities. Figure 2: Mexico City’s average annual population growth by municipality Figure 2: Mexico City’s average annual population growth by municipality Based on 2007 and 2008 satellite imagery from Google Earth, and first-hand knowledge of the new development sites, I drew geographic shapefiles around commercial housing developments and matched them with existing Census Tract shapefiles from the National Institute of Geography and Statistics (INEGI). Figure 3 shows the location of commercial housing developments in urbanized areas of Ix- tapaluca and Tecamac. Each Census Tract then received a score ranging from 0 to 1 indicating the pro- portion of the tract that fell within a commercial housing development around the time of the 2007 metropolitan household travel survey (INEGI 2007). Figure 5 shows the distribution of the proportion 175 Has Mexico City’s shift to commercially produced housing increased car ownership and car use? of each Census Tract that overlapped the drawn shapefiles of commercial housing developments. Two- thirds of Census Tracts contain either no commercial housing developments or only commercial hous- ing developments. I then matched these data to household location, trip origin, and trip destination data from the 2007 metropolitan household travel survey. Figure 3: Location of large-scale commercial developments in Ixtapaluca and Tecamac Figure 3: Location of large-scale commercial developments in Ixtapaluca and Tecamac JOURNAL OF TRANSPORT AND LAND USE 8.2 car ownership and use. For example, commercial housing residents are less likely to live in poverty since the new housing finance program has minimum income requirements. This is likely associated with higher car ownership and use. Computer ownership and home ownership are included as proxies for wealth, since income captures earnings, rather than total assets. Owning a computer may also correlate with lifestyle preferences that favor car ownership and use. p Table 1: Proposed control variables and expected correlation with whether a household lives in a commercial housing development, car ownership, and car use p Table 1: Proposed control variables and expected correlation with whether a household lives in a commercial housing development, car ownership, and car use a household lives in a commercial housing development, car ownership, and car use Variable Commercial Housing (Treatment Group) Car Ownership and Use Car ownership (+) NA Average daily VKT (+) NA Household income (+) (+) Owns home (+) (+) Owns a computer (+) (+) Average age of household (-) (?) Presence of children (?) (+) Distance to downtown (?) (-) Distance to highway (?) (+) Distance to Metro (?) (-) Local population density (+) (-) Source: 2007 Metropolitan Mexico City Household Travel Survey (INEGI 2007) and author’s calculations 2.2 Research hypotheses This study tests two overarching hypotheses about the relationship between living in a commercial hous- ing development and owning and using cars. H1: Households living in commercial housing developments are more likely to own and use cars than similar households in nearby informal settlements. Commercial housing developments tend to have more parking, wider streets, and a more discon- nected street grid than nearby informal housing developments. As a result, residents are more likely to own and use cars than those in informal settlements. Limiting the sample to two municipalities with similar levels of regional accessibility and including municipal-level fixed effects ensures that any differ- ences found across households relates to local rather than regional differences. gf Table 1 presents a list of control variables and their likely association with housing type and car ownership and use. Even within Ixtapaluca and Tecamac, there are likely some systematic differences between households in formal developments and those in informal ones. Many are also correlated with 176 JOURNAL OF TRANSPORT AND LAND USE 8.2 Written formally, the null hypothesis is H0: E( Yi | Di , Xi ) = E( Yi | Xi ) Written formally, the null hypothesis is H0: E( Yi | Di , Xi ) = E( Yi | Xi ) Where E is an expectation function; Where E is an expectation function; Y is a variable indicating whether a household owns one or more cars and the number of VKT generated on the weekday of the household travel survey; D is a dummy variable that indicates whether a household lives in a commercial housing development; X is the vector of demographic and geographic control variables; and i denotes a household. H2: The relationship between commercial housing and car ownership and VKT is independent of un- identified differences between households in the two types of housing Households tend to choose to live in neighborhoods that meet their transportation preferences. (For a discussion, see Cervero 2007, Chatman 2009, or Mokhtarian and Cao 2008.) As a result, households that favor transit often move into locations with good transit access, while households that wish to own a car tend to move to locations with available parking. To the extent that these preferences vary within the population and are independent of observed socioeconomic controls, they may bias estimates of the Has Mexico City’s shift to commercially produced housing increased car ownership and car use? 177 relationship between the built environment and travel behavior, particularly in cross-sectional studies. What may appear to be a causal relationship between living in dense neighborhoods and walking, for example, could be partially or even entirely caused by people with a strong preference for walking (and who would have walked in any environment) selecting to the densest, most walkable neighborhoods. In a review of 38 empirical studies, Cao, Mohktarian, and Handy (2009) found that this residential self-selection bias tends to lead to overestimates of the relationship between the built environment and travel, but that it only accounts for a portion of the correlation. To the extent that the built environ- ment allows people to exercise preferences (if, for example, people who prefer to walk would not do so as much unless they could select into denser neighborhoods), the relationship is causal and not biased by residential self-selection. Given an undersupply or oversupply of specific neighborhood types, self- selection may even lead to underestimates of the influence of the built environment on travel (Chatman 2009, Levine 2006). Written formally, the null hypothesis is H0: E( Yi | Di , Xi ) = E( Yi | Xi ) In Mexico City, the recent and dramatic change in housing type may provide something akin to a natural experiment in regard to residential self-selection. Instead of households choosing to live in a housing unit based on neighborhood preferences, the primary determinant of whether a household chooses to move into a new housing development is whether its members qualify for a subsidized home and are purchasing a new house. Since the commercial housing developments are all similar, there is little opportunity to select based on urban design or land-use preferences. Since the subsidy makes the housing cheaper relative to market alternatives, there is little incentive to purchase market-rate housing. By limiting the sample to households in two peripheral municipalities, the study design also limits po- tential selection bias to local rather than regional residential preferences, such as preference to live near downtown or near a Metro station. I therefore hypothesize that the effects of residential self-selection will be weak or unobservable, when conditioned on income and other socioeconomic attributes that correlate with living in a commercial housing development and owning and driving cars. g g g g Mohtktarian and Cao (2008) and Cao, Mohtkarian, and Handy (2009) identify nine principal methods to identify and control for residential self-selection bias. A subset is applicable to this analysis, which relies on cross-sectional data and does not include a variable indicating residential preference. I use two such methods. The first is an instrument variable approach. For examples of applications to address residential self-selection bias, see Boarnet and Sarmiento (1998) or Vance and Hedel (2007). In theory, a good instrument is one that exerts influence on the outcome of interest (car ownership and use), but only through its influence on the treatment of interest (residence type) (Wooldridge 2010). In practice, finding good instruments is difficult (Angrist and Pischke 2008, Bound, Jaeger, and Baker 1995), and in LaLonde’s (1986) landmark study of econometric methods, using them failed to replicate experimental results of the influence of worker training programs on employment—presumably a much more straightforward relationship than that between residential selection, the built environment, and travel behavior. For instruments, I used whether households had members who worked in public-sector, adminis- trative, or professional jobs. Written formally, the null hypothesis is H0: E( Yi | Di , Xi ) = E( Yi | Xi ) In theory, these job types make households more likely to qualify for Info- navit loans but no less likely to own a car or drive outside of their effect on wealth and income, which are included as controls. In practice, the instrument variable approach produced inconsistent parameter estimates depending on which job types were included as instruments and what other controls were included. I chose not to report the results. The second approach in this study models car ownership and residential location simultaneously with a nested logit model, an approach used by Cervero (2007) and Salon (2009). Each household faces six different discrete choices: whether to live in a commercial housing development and whether to own zero, one, or two or more cars. If residential self-selection or other unobserved household attributes JOURNAL OF TRANSPORT AND LAND USE 8.2 178 influence the correlation between living in a commercial development and owning a car, then nesting these choices and estimating a parameter for the nest should produce an estimate that ranges from zero to one, but is statistically different from one. The closer the estimate is to zero, the stronger the unob- served correlation between living in a commercial development and owning a car. For example, a nest value of 0.4 implies an unobserved correlation of 0.6, and a value of 0.2, a correlation of 0.8. Cervero (2007) found a statistically significant unobserved correlation across households that live near rail transit and use it of almost 0.75. If the nest value is either greater than or not statistically different from one, as in Salon’s (2009) study of car ownership and residential location within New York, it indicates that the nesting parameter should not be included in the estimation. 2.3 This section provides sources, means, and standard deviations for each of the independent and depen- dent variables modeled. It also describes the choice of expectation function used for car ownership and car use. As discussed in Section 2.1, the borders of commercial housing developments and Census Tracts do not match precisely (Figure 4). Most Census Tracts, however, have no commercial housing develop- ments or only have commercial housing developments (Figure 5). Furthermore, since Census Tracts contain non-residential land uses, including roads, vacant land, and open space, the Census Tracts with a high percentage of commercial housing developments often have no other residential uses. Although somewhat of a simplification, I treat households residing in Census Tracts with 50 percent or more land comprised of commercial housing developments as residents of commercial housing developments. This may weaken the statistical relationships tested, but otherwise is unlikely to bias parameter estimates. Figure 4: Matching and overlap of Census Tracts and commercial housing developments Figure 4: Matching and overlap of Census Tracts and commercial housing developments Has Mexico City’s shift to commercially produced housing increased car ownership and car use? 179 Figure 5: Histogram of households’ residential location by the proportion of the Census Tract that is comprised of commercial housing developments Figure 5: Histogram of households’ residential location by the proportion of the Census Tract that is comprised of commercial housing developments Table 2 presents the dependent and independent variables, based on whether households are assumed to live in traditional informal settlements or commercial housing developments. As expected, residents of commercial housing developments tend to own more cars and drive more frequently than households in informal settlements. On average, households in commercial housing developments in Tecamac and Ixtapaluca own 56 percent more cars and generate 160 percent more weekday VKT than residents in traditional, informal housing developments. They also tend to have higher incomes, though only by 17 percent, and higher rates of home and computer ownership. The distribution of residents’ income is also much tighter, presumably because households must meet mini- mum and maximum income requirements to qualify for a loan. In terms of geographic location, those in commercial housing developments tend to live in more densely populated neighborhoods that are marginally closer to Mexico City’s historic center, the Metro system, and regional highways than those in traditional informal developments. JOURNAL OF TRANSPORT AND LAND USE 8.2 JOURNAL OF TRANSPORT AND LAND USE 8.2 180 Table 2: Sample means and standard deviations of independent and dependent variables by whether house- holds reside in a Census Tract that is primarily comprised of commercial housing developments Traditional Development (Control Group) Commercial Housing* (Treatment Group) Mean SD Mean SD Average number of cars1 0.39 0.60 0.61 0.64 Has one or more cars1 34% 48% 54% 50% Has two or more cars1 4% 19% 7% 26% Average daily VKT2 6.0 20.1 15.6 33.2 Monthly income (in pesos)1 $7431 $11,909 $8678 $6426 People in household1 4.5 2.0 4.0 1.4 Household has children1 56% 50% 62% 49% Owns house1 81% 39% 90% 30% Average age of adults1 36.1 7.8 35.7 5.9 Has a computer1 23% 42% 43% 50% A member is college-educated1 45% 77% 70% 80% A member works in public sector1 1% 12% 2% 14% A member works in administration1 13% 40% 28% 52% A member works in professional sector1 19% 47% 37% 61% People per hectare in Census Tract3 107 74 204 100 Kilometers to Zocalo4 33.4 6.0 31.1 2.9 Accessibility score2 61,026 32,767 69,541 31,312 Kilometers to highway4 1.2 0.9 0.9 0.6 Kilometers to Metro4 16.8 7.3 13.8 2.9 In Ixtapaluca1 54% 50% 68% 47% Total Households (N) 1070 - 456 - *indicates that a household lives in a Census Tract that is 50 percent or more comprised of commercial housing *indicates that a household lives in a Census Tract that is 50 percent or more comprised of commercial housing developments. Sources: 1) 2007 Metropolitan Mexico City Household Travel Survey (INEGI, 2007); 2) Travel survey and author calculations; 3) 2005 Census; and 4) Network-distance based calculation using household travel survey and road network shapefiles. To estimate household car-ownership levels, I fit binomial logit, multinomial logit, and nested logit models. Only 5 percent of all households owned two or more cars. Sixty percent owned none. In the multinomial models, households choose between no cars, one car, or two-or-more cars. The nested logit model accounts for unobserved correlations between households that own a single car and those that own two or more. The nest, however, is not statistically different from one, and I opt to report the unor- dered multinomial logit model. To estimate VKT, I fit a left-censored Tobit model, which is commonly applied to joint discrete and continuous data including VKT. 2.3 This conforms to Monkkonen’s (2011c) findings about com- mercial housing developments in 128 of Mexico’s urban areas. A higher proportion of households in commercial developments were in Ixtapaluca than Tecamac, where several massive commercial housing developments have opened since 2007. 3 New commercial housing development, car ownership, and car use 3 After controlling for household income, age, composition, and location, the odds that the average household owns a single car are 61 percent higher in a commercial housing development than in a tra- ditional neighborhood. The odds of owning two or more cars are 87 percent higher. Wealthier house- holds that own their homes and a computer and have college-educated members are also significantly more likely to own cars. Neither the average age of adults in the household nor the number of people in the household is statistically correlated with a higher probability of car ownership. By contrast, house- holds with one or more children are more likely to own a car. With the exception of the fixed effect for whether a household lives in Ixtapaluca, none of the geographic controls is statistically significantly as- sociated with higher or lower car ownership. I opted to drop distance from the Metro, distance from Mexico City’s historic center, and the accessibility score from the model since they were not significant and they are highly correlated with one another and the municipal fixed effect. This does not indicate that regional accessibility does not matter, but that there is insufficient variation within each municipal- ity to estimate the correlation with car ownership. Table 3: Multinomial logit model of car ownership in Tecamac and Ixtapaluca Estimate Std. Error Odds-ratio Commercial housing development: 1 cars 0.48 0.15 1.61 ** Commercial housing development: 2+ cars 0.62 0.32 1.87 . JOURNAL OF TRANSPORT AND LAND USE 8.2 Three quarters of households did not have any members who drove on the survey day and thus produced no VKT. For a complete description of the model forms and estimation procedures, see Wooldridge (2010). Due to a long-tailed distribution, I log-transform the positive VKT data. This produces a better model fit and more homoscedastic residual plots. Has Mexico City’s shift to commercially produced housing increased car ownership and car use? 181 Due to non-linearity, logit model coefficients and odds ratios only provide behavioral information about a theoretical average household. Table 4 shows the car ownership levels that the model predicts assuming that either no households or all households live in a commercial housing development. If no households live in commercial housing developments, the model predicts that 38 percent of households would own JOURNAL OF TRANSPORT AND LAND USE 8.2 more cars, compared to 48 percent if all households lived in commercial housing developments. one or more cars, compared to 48 percent if all households lived in commercial housing developments. Table 4: Predicted car ownership rates based on no, some, or all households living in commercial housing developments No cars One car Two-plus cars No commercial housing 61.9% 33.6% 4.4% Existing conditions (31% commercial housing) 58.6% 36.3% 5.0% All commercial housing 51.7% 42.2% 6.0% Table 4: Predicted car ownership rates based on no, some, or all households living in commercial housing developments g p No cars One car Two-plus cars No commercial housing 61.9% 33.6% 4.4% Existing conditions (31% commercial housing) 58.6% 36.3% 5.0% All commercial housing 51.7% 42.2% 6.0% Living in a commercial housing development also correlates with producing statistically significantly more VKT per weekday than living in a traditional, informal neighborhood (Table 5). On average, a household in a commercial development generates four times more latent VKT (the exponent of 1.408 equals 4.088) than a similar household in an informal settlement. The Tobit regression, however, pri- marily predicts negative values since two-thirds of households produced no VKT on the survey day. This coefficient must therefore be interpreted with caution. At the mean predicted latent VKT of -2.479, the model does not expect a change in actual VKT from a household moving from an informal settlement to a commercial housing development, since latent VKT would still be negative. An ordinary least squares regression on the subset of households with VKT above zero produces a weaker, though still statistically significant result: Households in commercial developments generate 80 percent more VKT (with a T-value of 4.2) than households in traditional neighborhoods. The Tobit model also finds strong positive correlations between car use and households’ income, educational attainment, and computer ownership. Higher population densities and residence in Ixtapaluca are associated with fewer weekday VKT Table 5: Type I Tobit model of log of households’ weekday VKT in Tecamac and Ixtapaluca Table 5: Type I Tobit model of log of households’ weekday VKT in Tecamac and Ixtapal Estimate Std. 3 New commercial housing development, car ownership, and car use Log of income (in pesos): 1 car 0.58 0.10 1.79 *** Log of income (in pesos): 2+ cars 1.29 0.21 3.63 *** Owns home: 1 car 0.39 0.18 1.48 * Owns home: 2+ cars 1.54 0.74 4.65 * Has a computer: 1 car 0.80 0.14 2.22 *** Has a computer: : 2+ cars 1.39 0.29 4.01 *** College education: 1 car 0.24 0.08 1.27 ** College education: 2+ cars 0.66 0.15 1.93 *** Number of household members: 1 car -0.03 0.04 0.97 Number of household members: 2+ cars 0.04 0.09 1.05 Average age of adults: 1 car 0.02 0.06 1.02 Average age of adults: 2+ cars 0.07 0.18 1.07 Average age of adults squared: 1 car 0.00 0.00 1.00 Average age of adults squared: 2+ cars 0.00 0.00 1.00 In Ixtapaluca: 1 car -0.55 0.13 0.58 *** In Ixtapaluca: 2+ car - 0.71 0.28 0.49 * Population per hectare: 1 car 0.00 0.00 1.00 Population per hectare: 2+ cars 0.00 0.00 1.00 Constant: 1 car -6.61 1.37 *** Constant: 2+ cars -18.42 3.74 *** Significance codes: ***0.001, **0.01 , *0.05, .0.1, McFadden R2: 0.128, N = 1478 Table 3: Multinomial logit model of car ownership in Tecamac and Ixtapaluca Due to non-linearity, logit model coefficients and odds ratios only provide behavioral information about a theoretical average household. Table 4 shows the car ownership levels that the model predicts assuming that either no households or all households live in a commercial housing development. If no households live in commercial housing developments, the model predicts that 38 percent of households would own 182 JOURNAL OF TRANSPORT AND LAND USE 8.2 JOURNAL OF TRANSPORT AND LAND USE 8.2 Error Commercial housing development 1.408 0.340 *** Log of monthly income (in pesos) 1.310 0.215 *** Owns home 0.047 0.402 Has a computer 1.165 0.310 *** College education 0.898 0.174 *** People in household 0.023 0.089 Household has children 0.769 0.315 ** Average age of adults -0.187 0.144 Average age squared 0.002 0.002 In Ixtapaluca -1.088 0.288 *** People per hectare in Census Tract -0.003 0.002 * Constant -11.435 3.075 *** Significance codes: ***0.001, **0.01 , *0.05, .0.1, McFadden R2: 0.063 To demonstrate the stability of the estimated average effect of living in a commercial housing develop- ment on car ownership and VKT and the relationship to the control variables, Table 6 provides coeffi- cient estimates from four additional multinomial logit and Tobit models with fewer controls, as well as the coefficients with full controls as presented in Tables 4 and 5. Without any controls, households in commercial housing developments have more than twice the odds of owning one or more cars than those in traditional informal neighborhoods. Introducing controls for income, wealth, household com- Has Mexico City’s shift to commercially produced housing increased car ownership and car use? 183 position, and geographic features, the odds drop to 50 percent to 60 percent higher. Similarly, predicted VKT drops when controlling for household income, wealth, and demographics. The correlation, how- ever, strengthens when geographic variables are introduced. Commercial housing developments tend to have higher population densities, which are associated with fewer weekday VKT per household. The association between living in a commercial housing development and owning two or more cars is not always statistically different from zero with controls included, which is likely due to the small sample of households with two-or-more cars. In none of the models is it statistically different from the relationship to owning one car. Table 6: Relationship between living in commercial housing development and household car ownership and weekday VKT Multinomial logit model Tobit model Controls One car Two-plus cars Log of VKT Income and wealth Household composition Geography 0.73*** 0.89*** 1.67*** (No) (No) (No) 0.44** 0.40 0.96*** (Yes) (No) (No) 0.40** 0.37 0.96*** (Yes) (Yes) (No) 0.48** 0.62. 1.41*** (Yes) (Yes) (Yes) Significance codes: ***0.001, **0.01 , *0.05, .0.1 p between living in commercial housing development and household car ownership and weekday VKT Significance codes: ***0.001, **0.01 , *0.05, .0.1 3.1 The nested logit model of residential location and car ownership produces generally similar estimates of the relationship between socioeconomic control variables and car ownership (Table 7). It also indicates that a number of the control variables are indeed associated with whether households live in commercial housing developments. For example, smaller, wealthier households with computers are more likely to live in commercial housing developments. As expected, households with members who work in profes- sional or administrative jobs are more likely to live in commercial housing developments. Government employment is not statistically correlated, likely because government employees qualify for a separate fund, the Housing Fund of the Institute of Social Security of State Workers. The model includes two nest parameters, one for car ownership and one for the combination of car ownership and residence in a commercial housing development. The car-ownership nest of 0.83 indicates an unobserved 17 per- cent correlation across households that own cars. However, the value is not statistically different from one. The nest for car ownership and commercial residence is greater than one, which indicates that the choices are not correlated and should not be nested. Again, however, the value is not statistically dif- ferent from one. Residential self-selection does not appear to play a significant role in influencing the car-ownership parameter. This supports the hypothesis that the rapid change in housing production has limited the effects of residential self-selection. The rich set of socioeconomic control variables may also do a good job of capturing the effects of residential self-selection as found in studies by Brownstone and Golob (2009) and Bhat and Guo (2007). 184 JOURNAL OF TRANSPORT AND LAND USE 8.2 Table 7: Nested logit model of joint decision of how many cars to own and whether to live in a com- mercial housing development Estimate Std. Error Odds-ratio Car ownership Log of monthly income: 1 car 0.587 0.093 1.799 *** Log of monthly income: 2+ cars 1.223 0.235 3.397 *** Owns home: 1 car 0.438 0.177 1.550 * Owns home: 2+ cars 1.622 0.856 5.064 . 3.1 Has a computer: 1 car 0.794 0.140 2.213 *** Has a computer: 2+ cars 1.353 0.331 3.870 *** College education: 1 car 0.234 0.083 1.264 ** College education: 2+ cars 0.633 0.172 1.884 *** People in household: 1 car -0.030 0.038 0.970 People in household: 2+ cars 0.019 0.099 1.019 Average age of adults: 1 car 0.018 0.066 1.018 Average age of adults: 2+ cars 0.057 0.174 1.058 Average age squared: 1 car 0.000 0.001 1.000 Average age squared: 2+ cars 0.000 0.002 1.000 In Ixtapaluca: 1 car -0.605 0.130 0.546 *** In Ixtapaluca: 2+ cars -0.779 0.298 0.459 ** Population per hectare: 1 car 0.001 0.001 1.001 Population per hectare: 2+ cars -0.001 0.002 0.999 Lives in commercial housing development Log of monthly income 0.178 0.103 1.195 . Has a computer 0.421 0.136 1.524 ** College education 0.007 0.083 1.007 People in household -0.322 0.051 0.725 *** Average age of adults 0.340 0.075 1.405 *** Average age squared -0.005 0.001 0.995 *** In Ixtapaluca 0.566 0.136 1.761 *** Professional job 0.562 0.164 1.755 *** Administrative job 0.770 0.164 2.161 *** Public sector job 0.050 0.424 1.051 Nests Car ownership and commercial development 1.246 0.662 Car ownership 0.831 0.240 Significance codes: ***0.001, **0.01 , *0.05, .0.1, McFadden R2: 0.118, N = 1478, Constants not reported to conserve space Table 7: Nested logit model of joint decision of how many cars to own and whether to live in a com- mercial housing development 4 Discussion 4 There is a clear and statistically significant positive correlation between living in commercial housing developments and owning and using cars in Tecamac and Ixtapaluca, two suburban municipalities of Mexico City. Given the rapid shift in housing production from an informal process to a formal one, whether, how, and to what extent this relationship is causal is of great policy concern. Congestion, in Has Mexico City’s shift to commercially produced housing increased car ownership and car use? 185 particular, has large social and economic costs; the average one-way commute lasted 72 minutes for residents of the two municipalities in 2007 (INEGI 2007). Furthermore, due to the federal role in fi- nancing mortgages for commercial housing developments, the central government has unusually strong control over the design, location, and regulation of future commercial housing developments like those in Ixtapaluca and Tecamac. While this study cannot definitively quantify a causal relationship, it does suggest that one exists. This section discusses the evidence for the positive relationship being causal and then considers why commercial housing might produce higher rates of car ownership and use. There are two principal reasons that living in commercial housing developments is likely to corre- late with higher car ownership and use. Either the developments attract households with a stronger pref- erence for driving and car ownership or there is something about the design and location of the develop- ments that is conducive to driving and car ownership. The descriptive statistics and regression results strongly indicate that commercial housing developments attract wealthier households with observed preferences for driving. Including income, home ownership, and computer ownership in the statistical models reduces the average correlation between residing in a commercial housing development and car ownership and weekday driving by around 40 percent. There may also be unobserved variables that cor- relate with car ownership and driving as well as living in a commercial housing development, and bias estimates of the relationship between commercial housing development and car ownership and driving. Possibilities include personal preference, job location, and social status above and beyond what might be captured by income, home ownership, educational attainment, and computer ownership. However, the results from the nested logit model suggest that this is not the case. 4 Discussion Furthermore, the strength of the observed relationship and the rapid and exogenous shift in housing production—since selection into a commercial development is largely determined by whether a household qualifies for a loan, rather than residential preference—both suggest that there is something about the commercial housing develop- ments that encourages higher rates of car ownership and use. Since the analysis draws data from two similar suburban municipalities and includes a fixed effect for one of them, any of the observed relationship between the physical form of commercial housing developments and car ownership and use is related to local features, like design or local transit access, rather than regional ones, like accessibility to job centers. There are also likely to be differences related to regional accessibility, as found in a study of car use in metropolitan Mexico City (Guerra 2014), but this study would not capture them. Unfortunately, the analysis cannot provide insight into which aspects of commercial housing devel- opments correlate most strongly with higher car ownership or use. There is simply not enough variation in the design and land use of commercial housing developments. Furthermore, this shortcoming is com- mon in studies of the relationship between the built environment and travel, either because a variable like neighborhood population density serves as a proxy (intentional or not) for other measures (see for example Brownstone and Golob 2009), or the measure of the built environment is a qualitative dummy variable for a neighborhood type (see for example Handy, Cao, and Mokhtarian 2005). Given the evidence that there may be a causal link between the local features of commercial hous- ing developments and car ownership and car use, it is worth considering how peripheral commercial housing developments differ from nearby informal neighborhoods. Both tend to contain small, fairly uniform, cement housing units on small lots. Both are primarily constructed on former communal farmland. In Tecamac and Ixtapula, commercial developments tend to be more densely populated. Per- haps the most apparent difference is in how these neighborhoods develop over time. Informal neighbor- hoods grow organically over time, with residents and entrepreneurs building housing units and shops to suit their needs. By contrast, residents move into fully constructed single-use residential neighborhoods in commercial housing developments, although residents often convert units or parking spaces into JOURNAL OF TRANSPORT AND LAND USE 8.2 186 shops over time. 4 Discussion With limited access points, furthermore, informal transit, which serves around half of all metropolitan trips (INEGI 2007), is generally less well-integrated into commercial housing develop- ments than informal neighborhoods. Geo built the 11,000-unit Villas de Santa Barbara in Ixtapaluca with just three access points (Peralta and Hofer 2006). Rather than providing stations at the center of a 10,000-unit development, informal transit operators will generally pick up and drop off passengers at the edges. Ample parking likely plays a particularly important role. Roadways are wider than in informal settlements allowing for more on-street parking and each unit has an off-street parking space. Many households would not voluntarily choose to expend time, land, and money to create a parking space from scratch—indeed many take the time and effort to convert parking into some other use—but the default suburban housing product in Mexico City now comes with parking, when previously it did not. If indeed, “[m]inimum parking requirements are truly a great planning disaster—perhaps the greatest of all time [in the United States] (Shoup 2005, p. 218),” the potential costs in rapidly motorizing met- ropolitan areas like Mexico City are alarming. More than three in four children born today will live in a city in a developing country (United Nations Population Division 2007). Most will live in newly form- ing neighborhoods in rapidly motorizing suburban locations like Tecamac and Ixtapaluca. The parking policies adopted by these metropolitan areas will have tremendous economic, social, and environmental costs, both locally and globally. 5 Conclusion While it is Has Mexico City’s shift to commercially produced housing increased car ownership and car use? 187 unclear precisely how Infonavit or housing production will respond to the growing and often conflicting interests and priorities of private homebuilders, workers, and the public sector, it is clear that they are changing. This study finds that, from the perspective of limiting the growth in car ownership and use, the local design and land use of commercial housing developments matter. In addition to promoting more centralized, vertical development, policymakers should pay more attention to designing and retro- fitting peripheral commercial housing developments in ways that do less to encourage driving. Theory, previous findings, and differences with informal settlements suggest that fewer parking spaces, more connected street grids, and better local transit access would help. 5 Conclusion 5 This paper examined the correlation between living in a commercial housing development and owning and driving a car across 1500 households in two suburban municipalities in Mexico City. Accounting for income, proxies for wealth, household composition, and geography, residents of an average house- hold living in commercial housing developments are 61 percent more likely to own a car and 87 percent more likely to own two or more cars than those of a similar household in a neighboring informal settle- ment. They also generate significantly more private vehicle travel on an average weekday. While some of the differences in car ownership and use may relate to unobserved household preferences, the rapid shift in housing production and the results of a nested logit model of car ownership and residential lo- cation suggest that something about the new commercial housing developments is more conducive to car ownership and use. Since the sample only includes households from two similar suburban munici- palities and the models include regional controls, the observed statistical correlations likely stem from differences in local land use and design. The shift in housing policy has almost certainly increased car ownership and use. In the wake of the Great Recession, this policy is starting to change. Hundreds of thousands of new homes sit vacant throughout Mexico. Not only have new homes not been purchased, each year Infona- vit and other lenders foreclose on tens of thousands of homes (Juarez 2013). Homes in the most periph- eral new developments have been particularly vulnerable. Housing fund administrators have responded by cutting lending and reorienting the fund’s portfolio to the renovation and resale of existing proper- ties. Meanwhile, the national homebuilders have hit rock bottom. Geo and Urbi, both publicly traded, failed to report second quarter earnings in 2013 and were suspended from trading on the Mexican stock exchange (Purnell 2013). Days earlier, Homex, which reported earnings, experienced the steepest drop in value of any highly traded company on the Bloomberg World Index (Levin 2013). Workers’ unions are demanding better, larger housing in more central locations, greater leniency for unemployed work- ers with mortgages, and expanded access to credit for poorer households. 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Relationship of attitudes toward uncertainty and preventive health behaviors with breast cancer screening participation
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Relationship of attitudes toward uncertainty and preventive health behaviors with breast cancer screening participation Miho Satoh  (  miho.sth@mail.com ) Yokohama Shiritsu Daigaku https://orcid.org/0000-0001-8939-5595 Fukushima Kenritsu Ika Daigaku Abstract Background: Early detection of breast cancer is effective for prolonging survival, but the participation rate in breast cancer screening among target Japanese women remains low. This study examined the relationships between tendencies in decision-making under conditions of uncertainty, health behaviors, demographics, and breast cancer screening participation in Japanese women. Methods: The study population consisted of the 2,945 households that completed the 2017 Keio Household Panel Survey (KHPS). Data were obtained from the KHPS for women aged 40 years or older. Breast cancer screening participation in the past year, risk aversion, time preference, health behaviors (e.g., smoking, alcohol consumption, and medical treatment received in the past year), and demographic variables were analyzed. Results: Data from 708 women were analyzed. Among the respondents, 28.8% had attended breast cancer screening in the past year. Factors found to significantly contribute to breast cancer screening participation included higher risk aversion (odds ratio [OR], 2.34; 95% confidence interval [CI] = 1.03-5.32; p = 0.043), medical treatment received in the past year (OR, 1.56; 95% CI = 1.06-2.30; p = 0.026), higher self-rated health (OR, 1.47; 95% CI = 1.18-1.83; p = 0.001), living above the poverty line (OR, 2.31; 95% CI = 1.13-4.72; p = 0.022), and having children (OR, 1.57; 95% CI = 1.02-2.42; p = 0.042). Factors not significantly associated with screening participation were smoking (OR, 0.20; 95% CI = 0.10-0.42; p < 0.000), alcohol consumption (OR, 0.56; 95% CI = 0.37-0.86; p = 0.007), being self-employed (OR, 0.22; 95% CI = 0.10-0.46; p < 0.000), and being unemployed (OR, 0.48; 95% CI = 0.26-0.90; p = 0.022). No significant relationship was observed between time preference and screening participation. Conclusions: The results indicate that women who recognize the actual risk of developing breast cancer or have high awareness of breast cancer prevention tend to participate in breast cancer screening. Barriers to screening participation are not working for an organization that encourages screening and low income. Conclusions: The results indicate that women who recognize the actual risk of developing breast cancer or have high awareness of breast cancer prevention tend to participate in breast cancer screening. Barriers to screening participation are not working for an organization that encourages screening and low income. Research article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at BMC Women's Health on April 21st, 2021. See the published version at https://doi.org/10.1186/s12905-021-01317-1. Page 1/20 Page 1/20 Page 1/20 Background Breast cancer is the most common cancer among women worldwide [1]. In Japan, it is the leading cause of cancer mortality among women aged 30 to 64 years [2]. Both the number of women who are diagnosed with breast cancer and the number who die of the disease is increasing; 14,653 per 100,000 population died of breast cancer in 2018 in Japan [2]. Early detection and early treatment of breast cancer can lead to favorable prognosis [3, 4]. The 5-year net survival rate for breast cancer in the United Kingdom is reported to be 97.9% for stage 1 disease and 89.6% for stage 2, but this decreases to 72.0% for stage 3 disease and 26.2% for stage 4 [5]. In the United States, the 5-year survival rate is 98.8% for localized female breast cancer but 27.4% for female breast cancer with distant metastases [6]. It is therefore critical to detect breast cancer early. Page 2/20 Page 2/20 Mammography is the most effective breast cancer screening tool and can help to decrease breast cancer mortality by allowing earlier treatment [7-9]. Since 2004, the Japan Ministry of Health, Labour and Welfare has recommended mammography screening for women aged 40 years or over every 2 years [10]. In the United States in 2016, 72.5% of women aged 40 years or over had undergone mammography screening within the past 2 years [11]; in Western industrialized countries, the Organization for Economic Co- operation and Development reported that 70-80% of women underwent mammography within the past 5 years [12]. However, the participation rate among Japanese women targeted for breast cancer screening continues to be low. In 2016, only 44.9% of the target women had undergone mammography screening within the past 2 years [13], falling short of the targeted participation rate of 50% set in the Basic Plan to Promote Cancer Control Programs [14]. Effective strategies are therefore urgently needed to increase the number of target women who undergo breast cancer screening in Japan. Previous studies have revealed that breast cancer screening participation is associated with health- related lifestyle and behavioral factors, such as knowledge or health literacy about breast cancer [15-17], smoking [18, 19], alcohol consumption [19, 20], physical activity level [21], and self-rated health (SRH) [20, 22]. Economic factors are also barriers to breast cancer screening participation [23, 24]. Background According to Japan’s Census on Cancer Control [25], one of the reasons for women not attending cancer screening was “economic burden” (12.6%). Several other demographic factors are also associated with breast cancer screening participation, including higher level of education completed, lower occupational class [26, 27], regular visits to doctors [28, 29], living with a partner [30], having children [31], and older age [24, 28, 29]. Determinants of preventive health behaviors are receiving increased attention in the field of behavioral economics [32, 33]. Preventive health behaviors and the use of preventive medical care to treat fear of disease and death are likely affected by tendencies or preferences in behavior or decision-making under conditions of uncertainty [34, 35]. People often decide to use disease prevention services in consideration of intertemporal tradeoff—whether they prefer receiving an immediate small reward or a delayed larger reward—as explained by time preference theory [34, 36]. For example, the decision to participate in cancer screening is influenced by psychological value, that is, time preference as to whether to place emphasis on the present or the future. Another tendency in behavior or decision-making under conditions of uncertainty is risk aversion. This concerns the attitude toward risk that people have when deciding to use disease prevention services to decrease the probability of disease or death [34, 35]. Even a highly satisfactory behavior may be viewed as harmful to health and thus avoided. Therefore, participation in cancer screening may be affected by an individual’s attitude toward confronting or avoiding health problems caused by cancer [34, 35]. Those who have a higher time preference tend to place value on the current situation without considering the future and therefore prefer not to engage in preventive health behaviors. Those who are likely to avoid risk tend to actively engage in preventive health behaviors [34, 35, 37]. Although decision-making tendencies Page 3/20 Page 3/20 under conditions of uncertainty are a crucial factor that could predict preventive health behavior, there are few empirical research studies addressing this topic. under conditions of uncertainty are a crucial factor that could predict preventive health behavior, there are few empirical research studies addressing this topic. Communities in Japan are struggling with improving breast cancer screening rates. Given that women who attend breast cancer screening tend to have healthy habits, strategies have usually been proposed for people with poorer health behaviors or who are not interested in health behaviors. Background However, such strategies would not be fully effective considering the low rate of breast cancer screening participation in Japan. More effective tactics are needed both to encourage women to participate in breast cancer screening in order to decrease their risk of breast cancer-related health problems in the future and to improve screening participation rates. Against this background, in this study we examined the relationship between decision-making tendencies under conditions of uncertainty and breast cancer screening participation among Japanese women. We also analyzed the relationship of health behaviors and demographics with breast cancer screening to explore more effective strategies for increasing the breast cancer participation rate in Japanese women. Variables The primary outcome was breast cancer screening participation in the past year (participated or did not participate). Explanatory variables were variables related to risk aversion, time preference, health behaviors, and demography. Risk aversion was assessed using a question about deciding whether to take an umbrella out depending on the percentage likelihood of rain in a weather forecast. Time preference was measured using a question about the relative value placed on smaller immediate rewards or larger later rewards [40]: “How satisfied are you with receiving JPY 10,000 after 13 months instead of receiving it after 1 month?” (1 “interest rate = -5%” to 8 “interest rate = 40%”). The KHPS asked about the following health- related lifestyle variables: alcohol consumption (yes or no), smoking status (smoker or non-smoker), weekly physical exercise (yes or no), sleep duration (hours per night), and medical treatment received in the past year (yes or no). SRH (1 “poor” to 5 “excellent”) was also evaluated. The survey also asked about the following employment characteristics: working hours per week, relative poverty (yearly household disposable income < JPY 122,000 or not), and type of employment (regular, non-regular, self-employed, or unemployed). Demographic attributes obtained included gender (male or female), year of birth, marital status (married or single), children (none or ≥ 1), and level of education completed (junior or senior high school, junior college or vocational school, or university or graduate school). Residential area was also recorded. Ratios of time preference and risk aversion were obtained as percentages, with a lower time preference ratio indicating lower time preference and a higher risk aversion ratio indicating higher risk aversion. To account for relative poverty, yearly household disposable income was calculated by dividing household income by the square root of household size and then dividing participants into two groups: “living below the poverty line” (yearly household disposable income less than JPY 122,000; this is the median yearly household disposable income in Japan and is defined as the poverty line) and “living above the poverty line” (yearly household disposable income equal to or more than JPY 122,000). Age was calculated from year of birth. Methods Data from the Keio Household Panel Survey (KHPS) were used in this preliminary cohort study. The KHPS survey, conducted by Keio University, provides representative data from panel surveys of Japanese households [39]. Briefly, the KHPS was approved by the Ministry of Education, Culture, Sports, Science and Technology in Japan and has been conducted annually since 2004, surveying a total of 4,005 households nationwide. A stratified two-stage sampling method is used for the survey. KHPS respondents in 2004 were men and women born between 1935 and 1984. The demographic characteristics of the respondents are representative of Japanese households nationwide. The KHPS questionnaire includes items on place of residence, basic demographic data (e.g., year of birth, level of education completed, and gender), employment status, health status, health-related behaviors, and household economic status. All participants in the KHPS were informed about the purpose of the research, potential use of their data, the survey methodology, data anonymity, strict protection of individual data, and secondary use of data. The KHPS data are collected by mail, Internet survey, and visit survey by researchers. Applications for secondary use of these data must be submitted to and approved by Keio University, with assurance provided that ethical considerations will be met. Before we started this study, Keio University approved our use of the KHPS data in accordance with our application and assurance that all ethical considerations will be met. Accordingly, Keio University then provided us with the KHPS data sets. The empirical analysis in this study primarily used the 2017 wave of the KHPS and partially relies on the 2004 wave for basic demographic data. The sample analyzed included women aged 40 years or older, which corresponds with the recommended age for breast cancer screening in Japan. Page 4/20 Page 4/20 The aim of the study was to explore the relationships between decision-making tendencies under conditions of uncertainty, health behaviors, demographics, and likelihood of breast cancer screening participation in Japanese women. Data analysis Frequency (percentage) and descriptive statistics (mean and standard deviation [SD]) were first confirmed for all variables. Chi-squared tests were then performed and adjusted standardized residuals (ASRs) were calculated in a preliminary analysis to compare those who participated in breast cancer screening with those who did not according to age, child status, marital status, level of education completed, type of employment, relative poverty, medical treatment received in the past year, smoking status, alcohol consumption, and weekly physical exercise. Furthermore, t-tests were performed as a Page 5/20 Page 5/20 preliminary analysis to compare differences in risk aversion, time preference, SRH, and sleep duration between the groups. Subsequently, logistic regression analysis was performed and odds ratios were calculated to identify the factors associated with breast cancer screening participation. Variables that were significant in the univariate analysis were entered into the model as exploratory variables. preliminary analysis to compare differences in risk aversion, time preference, SRH, and sleep duration between the groups. Subsequently, logistic regression analysis was performed and odds ratios were calculated to identify the factors associated with breast cancer screening participation. Variables that were significant in the univariate analysis were entered into the model as exploratory variables. All statistical analyses were conducted using SPSS Statistics 25.0 for Mac (IBM Corp., Armonk, NY, USA). Statistical significance was set at p < 0.05 (two-tailed). Results Demographic characteristics Demographic characteristics Conversely, breast cancer screening participation was less likely among those who were self-employed (ASR = 5.5) and those who were living below the poverty line (ASR = 3.1). Those who had participated in breast cancer screening during the past year had a significantly shorter sleep duration (p = 0.005), higher SRH (p = 0.025), and greater risk aversion (p = 0.022) than those who did not. No significant difference was observed in time preference between those who did and did not attend for breast cancer screening. Demographic characteristics In 2017, the KHPS questionnaire was distributed to 2,945 households, of which 2,729 responded (response rate: 92.7%). A total of 708 questionnaires had complete responses and met the inclusion criterion (answered by women aged ≥ 40 years). These responses were analyzed in the course of the present study (valid response rate: 24.0%). Table 1 shows the descriptive statistics of the sample. Page 6/20 Of the respondents, almost a third underwent breast cancer screening in the past year. Those in the age ranges of 60-69 years and 50-59 years comprised just over half of all respondents, and those aged 40-49 years comprised almost a quarter. Most lived with families; approximately three-quarters were married and over half had children. Most had not completed university or graduate school. Relatively few were regular employees whereas more than a third were unemployed. Analysis of health behavior variables revealed that most were non-smokers, more than two-thirds did not consume alcohol, almost a quarter Page 7/20 Page 7/20 performed weekly exercise, and almost half had received medical treatment in the past year. Mean time preference was 0.17 ± 0.15 and mean risk aversion was 0.60 ± 023. performed weekly exercise, and almost half had received medical treatment in the past year. Mean time preference was 0.17 ± 0.15 and mean risk aversion was 0.60 ± 023. Univariate analysis of explanatory variables and breast cancer screening participation Univariate analysis of explanatory variables and breast cancer screening participation Chi-squared tests and t-tests were performed to examine the differences in explanatory variables according to breast cancer screening participation (Tables 2 and 3). Chi-squared tests and t-tests were performed to examine the differences in explanatory variables according to breast cancer screening participation (Tables 2 and 3). Chi-squared tests showed significant differences in breast cancer screening participation according to family type (χ2 = 3.91, p < 0.048), child status (χ2 = 13.37, p < 0.000), level of education completed (χ2 = 11.18, p < 0.004), type of employment (χ2 = 48.75, p < 0.000), relative poverty (χ2 = 9.61, p < 0.002), smoking status (χ2 = 32.06, p < 0.000), and alcohol consumption (χ2 = 16.77, p < 0.000). Demographic characteristics Breast cancer screening participation during the past year was more likely among those who lived with family (ASR = 2.0), had children (ASR = 3.7), had completed junior college or vocational school (ASR = 3.2), had precarious employment (ASR = 5.4), had received medical treatment in the past year (ASR = 2.3), were a non-smoker (ASR = 5.7), and did not consume alcohol (ASR = 4.1). Conversely, breast cancer screening participation was less likely among those who were self-employed (ASR = 5.5) and those who were living below the poverty line (ASR = 3.1). Chi-squared tests showed significant differences in breast cancer screening participation according to family type (χ2 = 3.91, p < 0.048), child status (χ2 = 13.37, p < 0.000), level of education completed (χ2 = 11.18, p < 0.004), type of employment (χ2 = 48.75, p < 0.000), relative poverty (χ2 = 9.61, p < 0.002), smoking status (χ2 = 32.06, p < 0.000), and alcohol consumption (χ2 = 16.77, p < 0.000). Breast cancer screening participation during the past year was more likely among those who lived with family (ASR = 2.0), had children (ASR = 3.7), had completed junior college or vocational school (ASR = 3.2), had precarious employment (ASR = 5.4), had received medical treatment in the past year (ASR = 2.3), were a non-smoker (ASR = 5.7), and did not consume alcohol (ASR = 4.1). Conversely, breast cancer screening participation was less likely among those who were self-employed (ASR = 5.5) and those who were living below the poverty line (ASR = 3.1). Chi-squared tests showed significant differences in breast cancer screening participation according to family type (χ2 = 3.91, p < 0.048), child status (χ2 = 13.37, p < 0.000), level of education completed (χ2 = 11.18, p < 0.004), type of employment (χ2 = 48.75, p < 0.000), relative poverty (χ2 = 9.61, p < 0.002), smoking status (χ2 = 32.06, p < 0.000), and alcohol consumption (χ2 = 16.77, p < 0.000). Breast cancer screening participation during the past year was more likely among those who lived with family (ASR = 2.0), had children (ASR = 3.7), had completed junior college or vocational school (ASR = 3.2), had precarious employment (ASR = 5.4), had received medical treatment in the past year (ASR = 2.3), were a non-smoker (ASR = 5.7), and did not consume alcohol (ASR = 4.1). Discussion This study investigated the relationships between decision-making tendencies under conditions of uncertainty, health behaviors, demographics, and breast cancer screening attendance to explore strategies that would be more effective than those currently implemented for increasing the participation rate of Japanese women in breast cancer screening. The results showed that those with higher risk aversion tended to participate in screening. However, time preference was not observed to significantly affect participation. Women who underwent breast cancer screening were found to have healthy preventive behaviors, such as not smoking, not drinking alcohol, and having received medical treatment in the past year. Higher SRH was also associated with breast cancer screening participation. Analysis of demographic characteristics revealed that the following factors were barriers to breast cancer screening: being self-employed, being unemployed, and living in relative poverty. Furthermore, having children was positively associated with participation. This study investigated the relationships between decision uncertainty, health behaviors, demographics, and breast c strategies that would be more effective than those current rate of Japanese women in breast cancer screening. The r aversion tended to participate in screening. However, time affect participation. Women who underwent breast cancer preventive behaviors, such as not smoking, not drinking al in the past year. Higher SRH was also associated with bre demographic characteristics revealed that the following fa being self-employed, being unemployed, and living in relat positively associated with participation. Association of explanatory variables with breast cancer screening participation The variables that were significantly associated with breast cancer screening participation in the preliminary analysis (chi-squared tests and t-tests) were subjected to logistic regression analysis, with age and region of residence used as control variables (Table 4). Page 9/20 Page 10/20 Factors found to significantly contribute to breast cancer screening participation in the past year included being more risk averse (odds ratio [OR), 2.34; 95% confidence interval [CI] = 1.03-5.32; p = 0.043), receiving medical treatment in the past year (OR, 1.56; 95% CI = 1.06-2.30; p = 0.026), having a higher SRH (OR, 1.47; 95% CI = 1.18-1.83; p = 0.001), living above the poverty line (OR, 2.31; 95% CI = 1.13-4.72; p = 0.022), and having children (OR, 1.57; 95% CI = 1.02-2.42; p = 0.042). Conversely, factors found not to contribute significantly to breast cancer screening participation were being a smoker (OR, 0.20; 95% CI = 0.10-0.42; p < 0.000), alcohol consumption (OR, 0.56; 95% CI = 0.37-0.86; p = 0.007), being self-employed Page 10/20 Page 10/20 (OR, 0.22; 95% CI = 0.10-0.46; p < 0.000), and being unemployed (OR, 0.48; 95% CI = 0.26-0.90; p = 0.022). Time preference was not found to be significantly associated with breast cancer screening participation. (OR, 0.22; 95% CI = 0.10-0.46; p < 0.000), and being unemployed (OR, 0.48; 95% CI = 0.26-0.90; p = 0.022). Time preference was not found to be significantly associated with breast cancer screening participation. Characteristics of the sample in this study The rate of participation in breast cancer screening in the past year was low (28.8%) in our sample compared with the rate of participation within the past 2 years obtained in the Comprehensive Survey of Living Conditions in 2016 (44.9%) [13], even though there was just a 1-year difference in the survey periods. Analysis of descriptive statistics indicated that most participants in the present study were non- smokers and did not drink alcohol. Also, their average sleep duration was the same as that reported in the National Health and Nutrition Survey 2016 [41]. Thus, we can view these women as likely to have healthier behavior. On the other hand, the women in this study were less likely to have healthy behavior in relation to weekly physical exercise compared with data from a census on physical fitness [42] showing that 37.8-71.5% of women engaged in physical exercise on more than 1 day a week. Relationships between decision-making tendencies under conditions of uncertainty and breast cancer screening participation Page 11/20 Our results indicate that women with higher risk aversion might actively participate in breast cancer screening to decrease the risk of delayed cancer detection. There is increasing awareness that early detection of breast cancer leads to more effective treatment and thus to a better prognosis. Therefore, women with an accurate perception of breast cancer might undergo breast cancer screening to mitigate their breast cancer risk. A meta-analytic review demonstrated that perceived risk is a predictor of the likelihood of attending breast cancer screening [43]. Women who recognize the actual risk of developing breast cancer or are anxious about developing breast cancer tend to participate in cancer screening [43, 44]. Therefore, the breast cancer screening participation rate could be increased by messages that emphasize the benefits of attending screening, the necessity of screening for improving quality of life even for women with breast cancer, and the risk of breast cancer being overlooked. Also, to increase participation, it might be effective to provide appropriate health education information about the accuracy of breast cancer screening, advances in breast cancer treatment, and improvement of prognosis after treatment. However, the association between risk aversion and breast cancer screening participation has not yet been fully examined and results are not consistent across studies. Characteristics of the sample in this study Some studies have reported that women with higher risk aversion do not participate in breast cancer screening to avoid the risk of a diagnosis of breast cancer, which provokes anxiety or psychological stress [34, 45]. Moreover, Sasaki and Ohtake suggest that individuals perceive the consequences of decision-making related to breast cancer screening differently depending on whether they take a gain-framing (safety concerns) or loss-framing (risk concerns) perspective [35]. For example, on the one hand, individuals who are risk averse are unlikely to participate in breast cancer screening when considering the risky condition of “having breast cancer but its treatment might not be successful”. On the other hand, individuals who are risk-seeking are unlikely to participate in breast cancer screening because they are more tolerant of the uncertain condition of “not having breast cancer, but it might be detected.” Their survey measured risk aversion from only the perspective of loss-framing, but future studies should measure it from both the loss-framing and gain-framing perspectives to examine more effective strategies for improving the breast cancer screening participation rate. The results of the present study also showed that time preference did not significantly affect the likelihood of breast cancer screening participation, unlike the findings of past studies [34, 35, 46]. However, a meta-analysis suggested that time preference is associated with addictive health behavior (e.g., smoking, alcohol consumption, or drug use) and not with preventive health behaviors such as attending cancer screenings or medical checkups [36]. Some studies have shown that the relationship of time preference with breast cancer screening participation is weaker than that with other factors [45, 47]. Time preference may be reflected in impulsive, addictive, or emotional behaviors rather than considered behaviors such as vaccination, medication compliance, or screening [36]. The effect of time preference might depend on mood or thoughtfulness in decision-making or decision-taking behaviors [35, 36] but remains incompletely understood. Further research is needed to clarify this effect. Relationship between health behaviors and breast cancer screening participation Smoking and alcohol consumption are risk factors for various cancers, including breast cancer [48]. Past research has shown a positive association between high breast cancer knowledge and breast cancer screening participation, and suggested individuals might be more likely to undergo breast cancer screening if they are knowledgeable about breast cancer, including its risk factors, the usefulness of mammography, and causes [17, 49, 50]. In addition, individuals who are interested in preventing health problems or who actively engage in preventive health behaviors would be likely to engage more in healthy behaviors that can reduce the risk of disease or death. In contrast, individuals who engage in unhealthy behaviors such as smoking, alcohol consumption, or not attending medical checkups are less likely to engage in preventive activities [51, 52]. In particular, women who usually engage in preventive health behaviors would likely participate in breast cancer screening as a part of their preventive health behaviors [53, 54]. The results of the present study might reflect this behavioral characteristic. The survey that Page 12/20 Page 12/20 provided the data analyzed in this study did not ask about knowledge of breast cancer; further research is needed to clarify the associations of attending breast cancer screenings with health-related behaviors like smoking and alcohol consumption and breast cancer knowledge. provided the data analyzed in this study did not ask about knowledge of breast cancer; further research is needed to clarify the associations of attending breast cancer screenings with health-related behaviors like smoking and alcohol consumption and breast cancer knowledge. Several studies have demonstrated that frequent visits to medical doctors could increase the opportunities to recommend breast cancer screening and promote awareness of cancer control [20, 28, 29], whereby women who received medical treatment tended to undergo screening. The association between higher SRH and participation in breast cancer screening supports the findings of previous research [55, 56]. Individuals perceived as being in poorer physical health are less likely to attend health checkups or screenings to avoid knowing the cause of their physical health status [57]. In the present study, participants with lower SRH had high risk aversion, which might have led to their non-participation in breast cancer screening. Relationships between demographic factors and breast cancer screening participation Regular employees have been shown to have the highest breast cancer screening participation rate [26, 58, 59], whereas unemployed and self-employed individuals are less likely to participate [58, 60, 61]. Employees of organizations are more likely to be exposed to recommendations for breast cancer screening or attend organized breast cancer screenings, further promoting their participatory behavior. Self-employed or unemployed women likely have limited cancer control service benefits, so they miss opportunities for screening. Self-employed women might also find it difficult to take time off to attend screenings [62, 63]. This might explain the differences in breast cancer screening participation according to employment status. Exposure to breast cancer screening opportunities or frequent recommendations for breast cancer screening is likely dependent on living environment and socioeconomic status; inequalities in either or both could lead to health disparities, and breast cancer is no exception. As such, carefully designed recommendation strategies for breast cancer screening should be jointly implemented by the government, medical facilities, and organizations. Social marketing communications could also be effective, such as combining mass media, social network services, and individual mailings. Our analysis suggests that relative poverty should not be overlooked as a barrier to breast cancer screening. Income and medical insurance have been shown to affect cancer screening participation [64]. Poverty tends to delay detection of breast cancer worldwide [65], and low income tends to lead to lower breast cancer screening rates [23, 24, 66]. Free or discount vouchers for mammography have been shown to significantly improve the breast cancer screening rate in Japan [67-70]. However, there are costs involved in screening, so the provision of free or low-cost screening to targeted women should be carefully considered. Individual screening recommendations and free vouchers would likely be effective in encouraging breast cancer screening participation [71]. Strategies and educational programs should be prepared that will help women recognize breast cancer risk and the significance of attending screenings so that they can proactively participate in the screenings regardless of cost. However, as discussed above, support is urgently needed for women with low income and limited education. Unfortunately, Japan has no nationwide program for breast cancer screening, which is particularly disadvantageous for Page 13/20 Page 13/20 women in the lower socioeconomic groups and those with limited education. This study indicates that employment status and income might be triggers for non-participation in breast cancer screening. Conclusions This study used representative data from a panel survey of Japanese households and showed that women who recognize the actual risk of developing breast cancer and women with a high level of awareness of the need to prevent breast cancer are likely to attend for breast cancer screening. Healthy preventive behaviors were also associated with participation in breast cancer screening. However, being self-employed, being unemployed, and living in relative poverty would be barriers to participation in screening. Relationships between demographic factors and breast cancer screening participation Women with these risk factors need to be identified so that they are not left out of breast cancer prevention programs. Empirical studies of the associations of breast cancer screening participation with economic status and education level are needed so that effective measures can be implemented. Moreover, the findings of this study suggest that lower income might delay participation in breast cancer screening. Further research is needed to understand the confounders of the association between income and participation in breast cancer screening, such as educational level, accuracy of knowledge about breast cancer, accessibility of information about breast cancer screening, the difficulties in taking time off work for cancer screening and the cost burden of screening, after which effective strategies have to be built. For example, an education program about breast cancer could be implemented in primary care to ensure that no woman is without accurate knowledge of breast cancer screening. Alternatively, a public awareness campaign could be developed to ensure that all women know that screening can help prevent breast cancer. Finally, this study found that having children is associated with participating in breast cancer screening. Women with children have childcare responsibilities, so it is likely that their health awareness is already high and that they undertake preventive health behaviors. This study has several limitations. First, it was preliminary in nature and used cross-sectional data to investigate the associations of breast cancer screening participation with decision-making tendencies under conditions of uncertainty and preventive health behaviors. As such, causality cannot be inferred. Second, the survey data were all self-reported, so the findings may have been affected by response bias [72, 73]. Further investigations are needed using objective data. Third, the study participants were from various regions of Japan, and more factors related to participants’ health status should be considered by region. Fourth, risk aversion and time preference can be measured from various perspectives, but the survey used only one questionnaire to address each of these variables. Further examination of the relationship between these tendencies and breast cancer screening participation should use several types of questionnaires to explore risk aversion and time preference. Acknowledgments We are grateful to the Keio University Panel Data Research Center for providing the KHPS data. Abbreviations Page 14/20 ASR: adjusted standardized residual; CI: confidence interval; KHPS: Keio Household Panel Survey; OR: odds ratio; SD: standard deviation; SRH: self-rated health ASR: adjusted standardized residual; CI: confidence interval; KHPS: Keio Household Panel Survey; OR: odds ratio; SD: standard deviation; SRH: self-rated health Declarations Ethics approval and consent to participate Consent for publication Not applicable. Availability of data and materials Ethics approval and consent to participate We received approval from the Keio University Panel Data Research Center to use the KHPS data for our research (Approval No. 1322: Data ID 156-JHPS/KHPS2004-2018). The center also provided the data. According to Japan’s Ethical Guidelines for Epidemiological Research, this study did not require ethical approval. The study complied with Keio University’s data use policy and Japan’s Ethical Guidelines for Epidemiological Research. Author contributions MS conceptualized and carried out the study and wrote the manuscript. NS provided important scientific comments on the study design, data analysis, and manuscript content. All authors contributed to and have approved the final manuscript. Competing interests Competing interests The authors declare that they have no competing interests. The authors declare no significant financial support for this work that could have influenced its outcome. Author contributions Availability of data and materials The data are available from the corresponding author upon reasonable request. The data set supporting the conclusions of this article is available from the Panel Data Research Center at Keio University with the approval of this organization (Approval No. 1322: Data ID 156-JHPS/KHPS2004-2018). References Page 15/20 1. Siegel R, Miller KD, Jemal A. Cancer statistics, 2018. CA Cancer J Clin. 2018; doi: 10.3322/caac.21442. Page 15/20 Page 15/20 2. 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Participatory evaluation guides the development and selection of farmers’ preferred rice varieties for salt- and flood-affected coastal deltas of South and Southeast Asia
Field crops research
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Rothamsted Research is a Company Limited by Guarantee Registered Office: as above. Registered in England No. 2393175. Registered Charity No. 802038. VAT No. 197 4201 51. g y Founded in 1843 by John Bennet Lawes. Patron: Her Majesty The Queen Rothamsted Research Harpenden, Herts, AL5 2JQ Telephone: +44 (0)1582 763133 Web: http://www.rothamsted.ac.uk/ Rothamsted Repository Download A - Papers appearing in refereed journals Burman, D., Maji, B., Singh, Sudhanshu, Mandal, Subhasis, Sarangi, Sukanta K., Bandyopadhyay, B. K., Bal, A. R., Sharma, D. K., Krishnamurthy, S. L., Singh, H. N., delosReyes, A. S., Villanueva, D., Paris, T., Singh, U. S., Haefele, S. M. and Ismail, Abdelbagi M. 2018. Participatory evaluation guides the development and selection of farmers’ preferred rice varieties for salt- and flood-affected coastal deltas of South and Southeast Asia. Field Crops Research. 220, pp. 67-77. Rothamsted Research Harpenden, Herts, AL5 2JQ Telephone: +44 (0)1582 763133 Web: http://www.rothamsted.ac.uk/ Rothamsted Research Harpenden, Herts, AL5 2JQ Patron: Her Majesty The Queen Harpenden, Herts, AL5 2JQ Telephone: +44 (0)1582 763133 Web: http://www.rothamsted.ac.uk/ Rothamsted Repository Download A - Papers appearing in refereed journals Burman, D., Maji, B., Singh, Sudhanshu, Mandal, Subhasis, Sarangi, Sukanta K., Bandyopadhyay, B. K., Bal, A. R., Sharma, D. K., Krishnamurthy, S. L., Singh, H. N., delosReyes, A. S., Villanueva, D., Paris, T., Singh, U. S., Haefele, S. M. and Ismail, Abdelbagi M. 2018. Participatory evaluation guides the development and selection of farmers’ preferred rice varieties for salt- and flood-affected coastal deltas of South and Southeast Asia. Field Crops Research. 220, pp. 67-77. The publisher's version can be accessed at: • https://dx.doi.org/10.1016/j.fcr.2017.03.009 The output can be accessed at: https://repository.rothamsted.ac.uk/item/8v655/participatory-evaluation-guides-the- development-and-selection-of-farmers-preferred-rice-varieties-for-salt-and-flood- affected-coastal-deltas-of-south-and-southeast-asia. © 2018. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 14/01/2020 09:23 repository.rothamsted.ac.uk library@rothamsted.ac.uk g g g Registered Charity No. 802038. VAT No. 197 4201 51. Rothamsted Repository Download A - Papers appearing in refereed journals Burman, D., Maji, B., Singh, Sudhanshu, Mandal, Subhasis, Sarangi, Sukanta K., Bandyopadhyay, B. K., Bal, A. R., Sharma, D. K., Krishnamurthy, S. L., Singh, H. N., delosReyes, A. S., Villanueva, D., Paris, T., Singh, U. S., Haefele, S. M. and Ismail, Abdelbagi M. 2018. Participatory evaluation guides the development and selection of farmers’ preferred rice varieties for salt- and flood-affected coastal deltas of South and Southeast Asia. Field Crops Research. 220, pp. 67-77. A B S T R A C T Keywords: Farmers’ preference Participatory varietal selection Salinity tolerance Sundarbans Waterlogging Rice is the staple food and provides livelihood for smallholder farmers in the coastal delta regions of South and Southeast Asia. However, its productivity is often low because of several abiotic stresses including high soil salinity and waterlogging during the wet (monsoon) season and high soil and water salinity during the dry season. Development and dissemination of suitable rice varieties tolerant of these multiple stresses encountered in coastal zones are of prime importance for increasing and stabilizing rice productivity, however adoption of new varieties has been slow in this region. Here we implemented participatory varietal selection (PVS) processes to identify and understand smallholder farmers’ criteria for selection and adoption of new rice varieties in coastal zones. New breeding lines together with released rice varieties were evaluated in on-station and on-farm trials (researcher-managed) during the wet and dry seasons of 2008–2014 in the Indian Sundarbans region. Significant correlations between preferences of male and female farmers in most trials indicated that both groups have similar criteria for selection of rice varieties. However, farmers’ preference criteria were different from researchers’ criteria. Grain yield was important, but not the sole reason for variety selection by farmers. Several other factors also governed preferences and were strikingly different when compared across wet and dry seasons. For the wet season, farmers preferred tall (140–170 cm), long duration (160–170 d), lodging resistant and high yielding rice varieties because these traits are required in lowlands where water stagnates in the field for about four months (July to October). For the dry season, farmers’ preferences were for high yielding, salt tolerant, early maturing (115–130 d) varieties with long slender grains and good quality for better market value. Pest and disease resistance was important in both seasons but did not rank high. When farmers ranked the two most preferred varieties, the ranking order was sometimes variable between locations and years, but when the top four varieties that consistently ranked high were considered, the variability was low. This indicates that at least 3–4 of the best-performing entries should be considered in succeeding multi-location and multi-year trials, thereby increasing the chances that the most stable varieties are selected. These findings will help improve breeding programs by providing information on critical traits. Participatory evaluation guides the development and selection of farmers’ preferred rice varieties for salt- and flood-affected coastal deltas of South and Southeast Asia D. Burmana,1, B. Majia,1, Sudhanshu Singhb,1, Subhasis Mandala,1, Sukanta K. Sarangia,1, B.K. Bandyopadhyaya, A.R. Bala, D.K. Sharmac, S.L. Krishnamurthyc, H.N. Singhd, A.S. delosReyese, D. Villanuevae, T. Parise, U.S. Singhb, S.M. Haefelef, Abdelbagi M. Ismaile,⁎ a Indian Council of Agricultural Research-Central Soil Salinity Research Institute, Regional Research Station, Canning Town, India b International Rice Research Institute, Delhi Office, India c Indian Council of Agricultural Research-Central Soil Salinity Research Institute, Karnal, India d Govind Ballabh Pant University of Agriculture and Technology, Uttarakhand, India e International Rice Research Institute, Los Baños, Philippines f University of Adelaide, Adelaide, Australia ⁎ Corresponding author at: Genetics and Biotechnology Division, International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines. 1 Authors contributed equally to manuscript writing E-mail addresses: burman.d@gmail.com (D. Burman), b.maji57@gmail.com (B. Maji), sud.singh@irri.org (S. Singh), subhasis2006@gmail.com (S. Mandal), sksarangicanning@gmail.com (S.K. Sarangi), bimalbkb@gmail.com (B.K. Bandyopadhyay), asitbal@yahoo.com (A.R. Bal), DK.Sharma@icar.gov.in (D.K. Sharma), krishnagene@gmail.com (S.L. Krishnamurthy), hns_eco@rediffmail.com (H.N. Singh), a.cueno@irri.org (A.S. delosReyes), d.villanueva@irri.org (D. Villanueva), t.paris@irri.org (T. Paris), u.singh@irri.org (U.S. Singh), stephan.haefele@acpfg.com.au (S.M. Haefele), a.ismail@irri.org (A.M. Ismail). The publisher's version can be accessed at: The publisher's version can be accessed at: The publisher's version can be accessed at: • https://dx.doi.org/10.1016/j.fcr.2017.03.009 The output can be accessed at: https://repository.rothamsted.ac.uk/item/8v655/participatory-evaluation-guides-the- development-and-selection-of-farmers-preferred-rice-varieties-for-salt-and-flood- affected-coastal-deltas-of-south-and-southeast-asia. © 2018. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 14/01/2020 09:23 repository.rothamsted.ac.uk library@rothamsted.ac.uk The publisher's version can be accessed at: • https://dx.doi.org/10.1016/j.fcr.2017.03.009 The output can be accessed at: https://repository.rothamsted.ac.uk/item/8v655/participatory-evaluation-guides-the- development-and-selection-of-farmers-preferred-rice-varieties-for-salt-and-flood- affected-coastal-deltas-of-south-and-southeast-asia. © 2018. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 14/01/2020 09:23 repository.rothamsted.ac.uk library@rothamsted.ac.uk • https://dx.doi.org/10.1016/j.fcr.2017.03.009 Field Crops Research 220 (2018) 67–77 Contents lists available at ScienceDirect http://dx.doi.org/10.1016/j.fcr.2017.03.009 Received 1 August 2016; Received in revised form 7 March 2017; Accepted 23 March 2017 Available online 23 April 2017 0378-4290/ © 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecom m ons.org/licenses/BY/4.0/). 1. Introduction Rice is the staple food for about half of the world’s population, and about 90% of the world’s rice is produced and consumed in Asia (Mackill et al., 2012). Rice is a major crop in tropical coastal deltas in South and Southeast Asia. However, several climatic adversities including abiotic stresses like soil and water salinity in both wet (Kharif) and dry (Rabi) seasons, waterlogging or flash floods, coastal storms and cyclones during the wet season, often affects its productivity (Burman et al., 2013; Islam and Gregorio, 2013; Ismail and Tuong, 2009; Ismail et al., 2010; Singh et al., 2010). Globally, about 230 million ha of coastal areas are saline (Li et al., 2014) of which about 27 million ha are in coastal zones of South and Southeast Asia (Ismail et al., 2010). In India alone, 3.1 million ha are affected by salinity in coastal regions (Yadav et al., 1983). g g y y Rice is grown in about 98% of the cultivated area as a rainfed crop during the wet season. Growing other crops during this season is difficult due to excessive wetting and waterlogging of low-lying fields (Sarangi et al., 2015, 2016). In the dry (winter) season, rice is grown on a limited area (20%), and most of the remaining fields are left fallow due to lack of good quality water for irrigation and high soil salinity. Cultivation of rice during the dry season in coastal saline soils is challenging and requires careful choices of suitable rice varieties and good management practices (Sarangi et al., 2014). In both seasons, the productivity of rice is low due to high soil and water salinity, water- logging, and submergence, besides other problems. Mostly traditional rice varieties are grown during the wet season with little adoption of modern varieties. Evaluation of breeding material in actual field conditions in such diverse ecosystem, and with input from farmers, will ensure selection of proper types and subsequent uptake and adoption. Rice farming is the primary source of livelihood for millions of poor and smallholder farmers in these areas, despite being sensitive to salt stress, with an upper threshold limit of 3 dS m−1 (Maas and Hoffmann, 1977). Above this threshold, rice yield decreases by 12% for every additional unit increase in salinity (ECe, dS m−1) (Maas and Grattan, 1999). 2.1. Characterization of the experimental sites It comprises 102 islands of which 54 are inhabited, spreading across 19 blocks of the two southern most districts of West Bengal, North 24 Parganas (6 blocks) and South 24 Parganas (13 blocks). While the Sundarbans region is one of the richest ecosystems in the world, the inhabitants often face severe poverty, which both contributes to and arises from the vulnerability of the population to a range of natural The PVS approach has been successfully implemented in marginal lands before, considering the social context of end users during evaluation, validation, and promotion of new varieties (Joshi et al., 2007, 2012; Manzanilla et al., 2011; Morris and Bellon, 2004; Ortiz- Ferrara et al., 2007; Paris et al., 2001, 2002; Singh et al., 2010, 2014, 2016). It is designed to include viewpoints of resource-limited farmers, identify varieties suitable for different stress conditions and incorporate a wider range of traits that match specific client preferences (Dorward et al., 2007; Morris and Bellon, 2004; Sperling et al., 2001). Participa- tory research approaches involving farmers in decision making pro- cesses help improve the effectiveness of technology development, increase the speed of adoption and payoffs to agricultural research (Atlin et al., 2002; Ceccarelli and Grando, 2007; Ceccarelli et al., 2000; Freeman, 2001; Gregorio et al., 2004; Joshi et al., 2005; Knox and Lilza, 2004; Tiwari et al., 2009; Trouche et al., 2011; Witcombe et al., 1996). The climate at the experimental sites is characterized by high monsoon rainfall in a hot and humid summer and a dry, mild winter. Data on weather conditions, hydrology, and salinity were collected from the ICAR-CSSRI-RRS station. The seasonal variability in mean rainfall, surface soil salinity and depth of field water (stagnant water) during 2008–2014, along with the timing of rice cultural operations are presented in Fig. 2. The monthly depth of field water during the wet season and monthly topsoil salinity (0–15 cm depth) were recorded from the experimental fields. Most rainfall is received during May to October. An average of 1630 mm (range of 1140–2160 mm) rainfall The Sundarbans represent typical salt-affected areas of the coastal delta regions of South Asia, located between 21° 32′ and 21° 40′ N, and 88° 05′ and 89° 00′ E, in the Ganges delta of eastern coastal part of India. A B S T R A C T Selected varieties through PVS are also more likely to be adopted by farmers and will ensure higher and more stable productivity in the salt- and flood- affected coastal deltas of South and Southeast Asia. D. Burman et al. Field Crops Research 220 (2018) 67–77 hazards (World Bank, 2014). The percentage of households below the poverty line in the 19 Sundarbans blocks of South and North 24 Pargans districts is 43.5%, compared with about 23% in the remaining non- Sundarbans blocks of these two districts (CSE, 2012). Farmers in the region are mostly smallholders, resource poor and dominated by scheduled castes and tribes (Mandal et al., 2011). About 90% of the farmers are marginal (< 1 ha landholding) and small (1–2 ha land- holding). High monsoon rainfall, poor soil, and water quality and natural adversaries like coastal storms and cyclones make agriculture in this region highly non-remunerative, complex and risky. 1. Introduction This sensitivity to salt stress is one of the reasons why the average productivity of rice in coastal delta regions is far below the national average in several countries. For example, in India, the average productivity of rice in this area is less than 1.5 t ha−1, which is considerably below the 2.8 t ha−1 of the country’s average rice yield (Singh, 2006). High-yielding and stress-tolerant rice varieties are expected to provide a yield increase of about 2 t ha−1 in these areas (Ponnamperuma, 1994). However, limited progress has been made in developing and disseminating suitable varieties that can enhance and sustain the productivity of this ecosystem to exploit its considerable potential for food supply. In this study, we conducted PVS trials on a research station and on- farm locations over seven years in these coastal saline regions. The objectives were to identify stress-tolerant rice varieties suitable for the target areas for use during the wet and dry seasons and to understand smallholder farmers’ preferences and bases of their cultivars’ selection. We also conducted critical analyses to understand the effectiveness of the PVS process, to guide necessary adjustments to ensure selection of appropriate varieties for the spatially and temporally variable produc- tion conditions encountered in these regions. There is a need to develop stress tolerant varieties adapted to local conditions and also meet the preferences of local farming communities to ensure adoption. Conventionally, breeders evaluate breeding lines and make decisions on release for commercial use, a process that ignores farmers’ preferences. This approach has probably contributed to the slow adoption of new rice varieties, as breeders mostly prefer traits that do not always match the needs of local farmers, such as agronomic and eating quality traits. The participatory varietal selection (PVS; Witcombe et al., 1996) process, where farmers and other stakeholders are involved in the selection of desired breeding lines before their formal commercialization may address this issue to a great extent. 2.1. Characterization of the experimental sites The experiments were conducted at the Indian Council of Agricultural Research-Central Soil Salinity Research Institute, Regional Research Station (ICAR-CSSRI-RRS), Canning Town, located in the South 24 Parganas district in the coastal Sundarbans region, and also in numerous farmers’ fields at locations spreading across both South 24 Parganas and North 24 Parganas districts (Fig. 1). Trials were conducted in the wet (Kharif) and dry (Rabi) seasons during 2008–2014. Soil characteristics were initially determined using stan- dard methods before the start of the experiments. Soil samples were analyzed for organic carbon (Walkley and Black, 1934), available N (Subbiah and Asija, 1956), available P (Olsen et al., 1954), and available K (Hanway and Heidel, 1952). The soil at the experimental sites was mostly heavy textured, varying from silty clay to clay. The top (0–15 cm depth) soil pH ranged from 6.85 to 7.68, and average organic carbon was 0.62%. Available N, P, and K concentrations in the topsoil were 243 kg ha−1, 10.5 kg ha−1 and 482 kg ha−1, respectively. y g The PVS approach has been successfully implemented in marginal lands before, considering the social context of end users during evaluation, validation, and promotion of new varieties (Joshi et al., 2007, 2012; Manzanilla et al., 2011; Morris and Bellon, 2004; Ortiz- Ferrara et al., 2007; Paris et al., 2001, 2002; Singh et al., 2010, 2014, 2016). It is designed to include viewpoints of resource-limited farmers, identify varieties suitable for different stress conditions and incorporate a wider range of traits that match specific client preferences (Dorward et al., 2007; Morris and Bellon, 2004; Sperling et al., 2001). Participa- tory research approaches involving farmers in decision making pro- cesses help improve the effectiveness of technology development, increase the speed of adoption and payoffs to agricultural research (Atlin et al., 2002; Ceccarelli and Grando, 2007; Ceccarelli et al., 2000; Freeman, 2001; Gregorio et al., 2004; Joshi et al., 2005; Knox and Lilza, 2004; Tiwari et al., 2009; Trouche et al., 2011; Witcombe et al., 1996). The Sundarbans represent typical salt-affected areas of the coastal delta regions of South Asia, located between 21° 32′ and 21° 40′ N, and 88° 05′ and 89° 00′ E, in the Ganges delta of eastern coastal part of India. 2.1. Characterization of the experimental sites It comprises 102 islands of which 54 are inhabited, spreading across 19 blocks of the two southern most districts of West Bengal, North 24 Parganas (6 blocks) and South 24 Parganas (13 blocks). While the Sundarbans region is one of the richest ecosystems in the world, the inhabitants often face severe poverty, which both contributes to and arises from the vulnerability of the population to a range of natural 68 D. Burman et al. Field Crops Research 220 (2018) 67–77 Fig. 1. Experimental sites in different locations in North and South 24 Parganas districts in Sundarbans region, West Bengal, India. Fig. 1. Experimental sites in different locations in North and South 24 Parganas districts in Sundarbans region, West Bengal, India. Fig. 2. Seasonal variability in soil salinity, depth of floodwater in the field and rainfall during 2008–2014, averaged across sites and months. Arrows indicate the time of different rice cultural operations during the wet and dry seasons. Fig. 2. Seasonal variability in soil salinity, depth of floodwater in the field and rainfall during 2008–2014, averaged across sites and months. Arrows indicate the time of different rice cultural operations during the wet and dry seasons. 69 D. Burman et al. Field Crops Research 220 (2018) 67–77 was 15 cm x 20 cm in all trials. Recommended management practices were used as described in all on-station and on-farm trials. was received during the experimental years. Of this, around 85% (range of 80–89%) was during the wet season; and only 6% (1–12%) occurred during the dry season. The mean monthly maximum temperature varied from 24.6 °C in January to 35.4 °C in April and mean monthly minimum temperature was lowest in January (12.9 °C) and highest (26.8 °C) in June. Field waterlogging was deepest in August and September, reaching 40–50 cm at the trials sites. The lowest salinity in the topsoil was about 2 dS m−1 reached in October, and the highest was 9 dS m−1 in May. Nurseries were established at the start of each season and 40 and 30 days old seedlings were transplanted during the wet and dry seasons, respectively, with 1–2 seedlings per hill. Fertilizers were applied at rates of 120-20-0 and 50-20–10 kg of N-P2O5-K2O ha−1 during the wet and dry seasons, respectively. Table 1 Table 1 Overview of rice genotypes included in various researcher-managed trials in different years during the wet and dry seasons of 2008–2014. No. Wet season Dry season Variety/breeding line No. of trials Variety/breeding line No. of trials 1 Sabita* 17 Lal Minikit (WGL 20471) 13 2 Amal-Mana [CSRC(S) 7- 1-4] 17 Khitish 6 3 SR 26B 13 Lalat* 13 4 Geetanjali 15 Satabdi (IET 4786) 13 5 CST 7-1 4 Gontra Bidhan-2 (IET 19571) 13 6 Sumati 2 Boby 13 7 Bhutnath 2 Canning 7 10 8 Utpala 2 CSR 4 7 9 Pankaj 2 CSR 36 3 10 Dinesh 6 CSR 22 6 11 Patnai 23 2 CSR38 3 12 Manasswarabar 5 Annada 13 13 Swarna-Sub1 2 Rasi (IET 1444) 3 14 BINA 8 2 Sankarsaru 3 15 CSRC(S) 21-2-5-B-1-1** 17 Super Minikit 3 16 CSRC(D) 7-0-4** 9 N Sankar 3 17 CSRC(D) 2-17-5** 4 Super Sankar 3 18 CSRC(D) 13-16-9** 4 Parijat 3 19 CSRC(D) 12-8-12** 4 BRRIdhan47 3 20 CN 1039-9** 8 BINA 8 3 21 CN 1233-39-9** 4 IR 64 Saltol** 3 22 CN 12133-3-9** 4 IR 72593-B-18-2-2-2** 3 23 CSRC(S) 47-7-B-B** 1 IR 72593-B-3-2-3-3** 3 24 NC 678 5 IR 76346-B-B-10-1-1- 1** 3 25 CR 2006-71-2** 1 IR 76393-2B-7-1-1-3- 1** 3 26 CR 2095-181-1** 1 27 CR 2070-52-2** 1 28 CR 2094-46-3** 1 29 IR 76393-28-7** 1 30 IR 206-29-2-1-1** 1 Table 1 Overview of rice genotypes included in various researcher-managed trials in different years during the wet and dry seasons of 2008–2014. 2.2. On-station and on-farm participatory varietal selection trials Promising rice varieties and new breeding lines were evaluated following the PVS approach in on-station and on-farm trials, both managed by researchers (Paris et al., 2011), and in both wet and dry seasons. A total of 17 wet season trials (4 on-station and 13 on-farm) and 16 dry season trials (3 on-station and 13 on-farm) were conducted, each using a randomized complete block design (RCBD) with three replications. The entries included released varieties and new breeding lines recommended from different breeding programs and networks, with 8–13 entries evaluated in each trial. Within a year, the set of entries were the same for all on-station and on-farm trials but were different between years. The entries selected as the best in a preceding year were included in subsequent years along with newly added entries. The entries and the total number of times they have been included in the trials in different years and seasons are presented in Table 1. Plot size varied from 30 m2 (5 m x 6 m) to 40 m2 (5 m x 8 m), and spacing 2.1. Characterization of the experimental sites All P and K were incorporated before transplanting, whereas N in the form of urea was applied in three equal splits at seven days after transplanting (DAT), maximum tillering (45 DAT) and at the initiation of flowering (75 DAT) during the wet season. During the dry season, all of P, K and 25% N were applied before land leveling. Half of N was broadcasted 21 DAT, and the remaining 25% was broadcasted at 60 DAT. Hand weeding was accomplished twice (30 and 60 DAT) during both seasons. Chloropyriphos at 2 ml l−1 water and tricyclazole at 0.6 g l−1 water were used to control insects and diseases, respectively. 2.3. Preference analysis A Preference analysis (PA) through casting votes was conducted during a pre-harvest period when most varieties reached around 80% maturity (Paris et al., 2011). At each PA, a group of male and female farmers and researchers were invited to vote for two most preferred (positive vote) and two least preferred (negative votes) entries, using paper ballots and envelopes placed at the head of each plot. Names of entries were kept anonymous with codes used for each entry through- out the voting process. Votes were then used to identify the most and least preferred rice genotypes and farmers interviewed to understand the reasons behind their choices. This resulted in two types of data; (i) the quantitative preference score (PS) for each variety generated by expressing the number of positive votes minus the negative votes divided by the total number of votes, and (ii) a list of characteristics considered by farmers as the basis for selecting the most and least preferred entries. *, ** represent ‘check’ and ‘breeding line’, respectively. 2.4. Data collection and analysis Ten hills were randomly selected at harvest from each plot, and plant height was measured from stem base to the tip of the longest leaf or panicle, whichever was longer. The depth of water in the field was monitored using a meter stick installed in each plot. Soil salinity was measured using an electrical conductivity meter in 1:2 (soil: water) and converted to ECe by multiplying with an appropriate conversion factor. At maturity, plants were harvested, then sun dried and weighed to determine grain yield, which was then adjusted to 14% grain moisture content. The Pearson correlation coefficient (r) was used for correlation analysis between male and female farmers’ PS, between all farmers’ and researchers’ PS and between yield and all farmers’ PS. The ‘r’ value was used for comparing the extent of agreement or disparity in preference choices between any two groups. Growth parameters and yield data were collected from the inner rows, leaving 50 cm border rows at both ends of the plot. Descriptive statistical methods were then used for analyzing the effects of waterlogging and salt stress on rice grain yield. To rank the various preference criteria, the Rank Based Quotient (RBQ) analysis was employed (Mandal et al., 2013). The criteria used by farmers for their selection of the most preferred lines through PVS trials were listed first, and then they were asked to rank them according to their individual priority on a scale of 1–5. The most preferred criteria were ranked as 1, the least preferred as 5. The analysis allowed ranking farmers’ preferences based on RBQ. A total of 60 farmers (30% females) from 2 locations were interviewed for this preference analysis during the wet season. A similar methodology was used in the dry season. Farmers’ choice of a particular rice variety was influenced by several attributes like salinity tolerance, capacity to withstand waterlogging (plant height), resistance to pests and diseases, grain and straw quality, The Pearson correlation coefficient (r) was used for correlation analysis between male and female farmers’ PS, between all farmers’ and researchers’ PS and between yield and all farmers’ PS. The ‘r’ value was used for comparing the extent of agreement or disparity in preference choices between any two groups. Growth parameters and yield data were collected from the inner rows, leaving 50 cm border rows at both ends of the plot. 2.4. Data collection and analysis Descriptive statistical methods were then used for analyzing the effects of waterlogging and salt stress on rice grain yield. To rank the various preference criteria, the Rank Based Quotient (RBQ) analysis was employed (Mandal et al., 2013). The criteria used by farmers for their selection of the most preferred lines through PVS trials were listed first, and then they were asked to rank them according to their individual priority on a scale of 1–5. The most preferred criteria were ranked as 1, the least preferred as 5. The analysis allowed ranking farmers’ preferences based on RBQ. A total of 60 farmers (30% females) from 2 locations were interviewed for this preference analysis during the wet season. A similar methodology was used in the dry season. Farmers’ choice of a particular rice variety was influenced by several attributes like salinity tolerance, capacity to withstand waterlogging (plant height), resistance to pests and diseases, grain and straw quality, 70 Field Crops Research 220 (2018) 67–77 D. Burman et al. resistance to lodging and duration of the crop. These attributes were identified from the feedback of farmers during multi-location PVS trials across both seasons and over the years (2008–2014). These attributes were ranked through RBQ analysis during the 2013-14 season. RBQ is a problem identification technique, mathematically presented as follows: preference for farmers in the wet season included tall plants, long panicles with more grains, less infestation with pest and diseases, numerous tillers, good grain type, overall good crop performance, enough straw for fodder, thatching and fuel, apparent suitability for parboiling, optimum maturity period, suitability for specific field situations (low/medium land), good lodging resistance, and high grain yield. RBQ fi n i N n = Σ ( + 1 −)*100 * j n =1 The analyses reflected significant correlation at 5% and 1% levels between preference scores of male and female farmers in 59% of the sites. However, for other sites, a positive but non-significant correlation was observed. Correlations between the preference scores of all farmers versus researchers were strong and significant at 5% and 1% levels in 47% of the trial sites, which is 12% lower than that of male and female farmers. Significant correlations between preference scores of all farm- ers and yield were observed in only 41% of the sites. 3.1. Preferences of varieties and breeding lines in the wet season 3.1. Preferences of varieties and breeding lines in the wet season Waterlogging was a major limiting factor for rice growth and productivity at the experimental sites in the monsoon season (Fig. 2 and Table 2). Due to poor or non-existence of drainage facilities, flat topography, low-lying lands and heavy rainfall, the experimental fields remained waterlogged with a depth of more than 30 cm from July to October (Fig. 2). Soil salinity was high (> 4.0 dS m−1) during early seedling stages (Table 2) when rice is highly sensitive; but lower during reproductive stage, which is also highly sensitive to salt stress. The performance of different rice varieties and breeding lines tested in 17 on-station and on-farm trials during the wet seasons between 2008 and 2013 is summarized in Table 3. The entries were of medium to long duration (140–170 d) and are medium to tall (90–175 cm). Grain yield varied between 2.58–4.39 t ha−1. Chi-square test was applied to assess associations between the preference criteria and ranks assigned by respondents. For the wet season, the calculated chi-square was 40.925, with P value = 0.055. With P > 0.05, we accepted the null hypothesis and concluded that there was no association between preference criteria and ranks assigned by the respondents (Table 5). Hence preference criteria and ranks assigned by the respondents are independent for the wet season. Cyclones with strong winds are common in these coastal areas and can cause considerable damage to standing rice crop. Farmers also selected some genotypes with medium height (100–115 cm) like Sumati, CSRC(S) 21-2-5-B-1-1, and Swarna-Sub1, as in their opinion, they are suitable for medium lands (water depth of 20–30 cm for most of the wet season with no flooding problem). The duration of the crop was another important consideration by farmers. They preferred long duration varieties (160–170 d) like CSRC(D) 12-8-12, Geetanjali, Amal- Mana, CSRC(D) 7-0-4, CSRC(D) 13-16-9, Sabita, SR 26B, and Patnai 23 for lowlands to avoid harvesting in standing water. Medium duration (140–150 d) rice varieties like Sumati, CSRC(S) 21-2-5-B-1-1, and Swarna-Sub1 were targeted for medium-lands. Farmers did not prefer early maturing varieties because they are not suitable for local land conditions and are often damaged by rats and rodents as was evidenced in the trials. 2.4. Data collection and analysis This variation indicates more frequent mis-matches between characteristics of highest yielding rice varieties and breeding lines selected by breeders versus farmers’ preferences. Thus, grain yield was not the most preferred trait for farmers in the wet season; other traits seem equally important for the selection of best rice varieties for this region. Where N = total number of farmers, n = total number of ranks (there are five ranks, n = 5), i = the rank for which the RBQ is calculated (for a problem), and f = number of farmers reporting the rank i (for the problem). Since RBQ scores are index values, tests of significance are not applicable. The data consists of numbers of persons assigning the different ranks, so is considered discrete data, and Chi-square test was used for testing associations between the preference criteria and ranks assigned by the respondents. The null hypothesis “there is no association between preference criteria and ranks assigned by the respondents,” was tested against the alternative hypothesis, i.e. “there exists an association between preference criteria and ranks assigned by the respondents”. Farmers’ preference traits were ranked using the RBQ analysis (Table 5) and the results revealed that the taller genotypes were most preferred, followed by high grain yield. The quality and quantity of straw constituted the third most important trait. Farmers prefer tall varieties (140–170 cm; Table 4) because they are more suited for the lowland situations in coastal areas where land remains waterlogged (> 30 cm depth) for about 4 months (July to October). Taller varieties also provide sufficient straw for fodder, for roof thatching and for use as fuel. Resistance to lodging is another important trait preferred by farmers, as they perceive that lodging of tall varieties might cause yield losses. 3.1. Preferences of varieties and breeding lines in the wet season Criteria such as pest and disease resistance, grain quality The participants in the PA for rice variety selection (681 persons during 2008–2013) were 44% male farmers, 36% female farmers, and 20% researchers. Correlation analysis was performed between the preference scores of male and female farmers, between all farmers and researchers, and all farmers versus grain yield, using Pearson’s correlation (Table 4). Based on farmers’ preferences scores, the four top ranking varieties or breeding lines in each year from the PA are also presented in Table 4. Out of a total of 30 entries, 11 entries were ranked at least once among the top 4, in the 17 on-station and on-farm trials. However the most preferred entries differed across locations and years, and the most preferred entries in a preceding year were not always the most preferred in subsequent years. The most preferred varieties and breeding lines during the six wet seasons (2008–2013) included Geetanjali, Amal-Mana, CSRC(S)21-2-5-1-1, and Sabita. The traits of Table 3 Table 3 Agronomic characteristics of rice varieties and breeding lines evaluated in multi-location trials during the wet and dry seasons of 2008–2014. Grain yield represents mean values across the number of trials in which particular entry was used ± SE. Table 3 Grain yield represents mean values across the number of trials in which particular entry was used ± SE. *, **, *** significant at P < 0.05, 0.01 and 0.001, respectively. Table 3 Wet Season Dry season Varieties/breeding lines Plant height (cm) Duration (d) Grain yield ± SE Varieties/breeding lines Plant height (cm) Duration (d) Grain yield ± SE CSRC(D) 12-8-12 145–165 165–170 4.39 ± 0.08 Gontra Bidhan-2 105–110 120–125 4.77 ± 0.09 Geetanjali 150–170 165–170 4.12 ± 0.12 BINA 8 90–100 115–120 4.62 ± 0.11 Amal-Mana 150–162 160–170 4.11 ± 0.12 Boby 100–110 125–130 4.34 ± 0.14 Swarna-Sub1 105–110 145–150 4.02 ± 0.33 Annada 95–105 120–125 4.28 ± 0.07 CSRC(D) 7-0-4 140–155 165–170 3.99 ± 0.14 N Sankar 95–105 120–125 4.25 ± 0.06 CSRC(D) 2-17-5 145–155 165–170 3.85 ± 0.06 BRRI dhan47 105–110 120–125 4.25 ± 0.06 CSRC(S) 21-2-5-B-1-1 115–125 140–145 3.84 ± 0.09 IR 76393-2B-7-1-1-3-1 85–95 120–125 4.18 ± 0.23 CSRC(D) 13-16-9 155–165 165–170 3.82 ± 0.07 Lal Minikit 90–105 120–125 4.15 ± 0.15 Manasswarabar 140–145 160–165 3.80 ± 0.32 Parijat 85–95 115–120 4.15 ± 0.04 Sabita 150–165 165–170 3.78 ± 0.14 Super Sankar 90–100 115–120 4.08 ± 0.07 CN 12133-3-9 105–115 3.76 ± 0.10 Rasi 95–105 115–120 4.03 ± 0.08 SR 26B 140–160 165–170 3.71 ± 0.18 Canning 7 95–105 125–130 3.98 ± 0.14 Patnai 23 140–155 160–165 3.63 ± 0.11 Satabdi 85–95 120–125 3.96 ± 0.09 NC 678 155–165 165–170 3.53 ± 0.032 IR 76346-B-B-10-1-1-1 95–1005 120–125 3.92 ± 0.022 BINA 8 90–110 145–150 3.52 ± 0.04 IR 64 Saltol 95–105 120–125 3.91 ± 0.05 CST 7-1 110–120 140–150 3.49 ± 0.13 CSR 4 95–100 125–130 3.82 ± 0.06 CN 1039-9 105–115 3.42 ± 0.02 CSR38 95–100 125–130 3.70 ± 0.12 Sumati 100–105 140–145 3.33 ± 0.17 CSR 22 95–105 125–130 3.65 ± 0.12 CSRC(S) 47-7-B-B 90–100 145–150 3.27 Lalat 100–105 125–130 3.58 ± 0.26 Utpala 105–115 140–145 3.25 ± 0.20 Super Minikit 90–100 115–120 3.54 ± 0.15 CN 1233-39-9 125–135 3.17 ± 0.35 IR 72593-B-3-2-3-3 120–125 3.45 ± 0.23 CR 2094-46-3 150–160 160–165 3.17 Sankarsaru 95–105 110–115 3.36 ± 0.34 CR 2095-181-1 150–160 160–165 3.13 Khitish 85–95 115–120 3.34 ± 0.26 Bhutnath 95–105 130–135 3.13 ± 0.16 IR 72593-B-18-2-2-2 120–125 3.21 ± 0.25 Pankaj 130–150 140–145 3.10 ± 0.14 CSR 36 95–105 125–130 2.19 ± 0.16 IR 206-29-2-1-1 105–115 145–150 3.10 Dinesh 145–175 165–170 3.09 ± 0.38 CR 2006-71-2 150–165 2.98 CR 2070-52-2 145–155 160–165 2.87 IR 76393-28-7 90–110 145–150 2.58 Table 3 Agronomic characteristics of rice varieties and breeding lines evaluated in multi-location trials during the wet and dry seasons of 2008–2014. a Numbers in parenthesis in the second column are on-farm trials. Table 2 Year Trials Sites Ranking of most preferred varieties/breeding lines Correlations between preferences 1st 2nd 3rd 4th Male vs. Female Farmers vs. Researchers Farmers vs. yield 2008 On-station Site 1 Amal-Mana Sabita SR 26 B Sumati 0.61** 0.84*** 0.35 On-farm Site 1 SR 26B Amal-Mana CSRC(S) 21-2-5-B-1-1 Sumati 0.75** 0.92*** 0.15 2009 On-station Site 1 CSRC(S) 21-2-5- 1-1 Geetanjali Amal-Mana Sabita 0.85*** 0.90*** 0.86*** On-farm (3)a Site 1 CSRC(S) 21-2-5- 1-1 Geetanjali Amal-Mana SR 26 B/ Sabita 0.10 0.48 0.96*** Site 2 CSRC(S) 21-2-5- 1-1 Geetanjali Amal-Mana SR 26 B 0.62 0.91*** 0.19 Site 3 CSRC(S) 21-2-5- 1-1 Geetanjali Amal-Mana Sabita 0.85** 0.90*** 0.73** 2010 On-station Site 1 Geetanjali Amal-Mana Sabita CSRC(S) 21-2-5-1-1/ SR 26B 0.91*** 0.94*** 0.72* On-farm (3) Site 1 Geetanjali Amal-Mana CSRC(D) 7-0-4/ CSRC(S) 21-2-5-1-1 – 0.94*** 0.91*** 0.50 Site 2 Amal-Mana Geetanjali CSRC(S) 21-2-5-1-1/ Sabita – 0.85** 0.81** 0.74* Site 3 Geetanjali Amal-Mana CSRC(S) 21-2-5-1-1 CSRC(D) 7-0-4 0.93*** 0.78* 0.74 2011 On-station Site 1 CSRC(D) 7-0-4 SR 26 B Amal-Mana/Sabita – 0.65* 0.48 0.79** 2012 On-farm (3) Site 1 Sabita CSRC(S) 21-2-5- 1-1 CSRC(D) 7-0-4 CSRC(D) 13-16-9 0.73* 0.71* 0.51 Site 2 Sabita CSRC(D) 12-8-12 Amal-Mana Geetanjali 0.37 0.44 0.67 Site 3 CSRC(D) 12-8-12 Sabita Geetanjali Amal-Mana 0.74* 0.57 0.49 Site 4 CSRC(D) 12-8-12 Sabita Amal-Mana CSRC(D) 13-16-9 0.81* 0.72* 0.88** 2013 On-farm Site 1 CSRC(S) 21-2-5- 1-1 Swarna-Sub1 Patnai-23 Geetanjali 0.81*** 0.56* 0.66** Sabita Amal-Mana Swarna-Sub1 CSRC (S) 21-2-5-1-1 0.75** 0.15 0.87*** *, **, *** significant at P < 0.05, 0.01 and 0.001, respectively. a Numbers in parenthesis in the second column are on-farm trials. D. Burman et al. Field Crops Research 220 (2018) 67–77 Field Crops Research 220 (2018) 67–77 D. Burman et al. D. Burman et al. Table 2 Table 2 The depth of water in the field and top soil salinity (0–15 cm depth) at the two salt sensitive growth stages of rice (early seedling and reproductive stages) during the wet and dry seasons. Values are averages across seasons and years during 2008–2014. Sensitive growth stages of rice Wet season (Kharif) Dry season (Rabi) Depth of field water (cm) Soil salinity (ECe, dS m−1) Soil salinity (ECe, dS m−1) Range Mean ± SE Range Mean ± SE Range Mean ± SE Early seedling 26.0–37.0 31.4 ± 0.60 4.10–5.80 4.58 ± 0.11 3.80–5.60 4.45 ± 0.11 Reproductive 27.0–42.0 33.2 ± 1.00 1.80–2.80 2.05 ± 0.06 4.90–6.90 5.73 ± 0.11 The depth of water in the field and top soil salinity (0–15 cm depth) at the two salt sensitive growth stages of rice (early seedling and reproduct Values are averages across seasons and years during 2008–2014. 71 Table 3 Agronomic characteristics of rice varieties and breeding lines evaluated in multi-location trials during the wet and dry seasons of 2008–2014. Grain yield represents mean values across the number of trials in which particular entry was used ± SE. Table 2 Wet Season Dry season Varieties/breeding lines Plant height (cm) Duration (d) Grain yield ± SE Varieties/breeding lines Plant height (cm) Duration (d) Grain yield ± SE CSRC(D) 12-8-12 145–165 165–170 4.39 ± 0.08 Gontra Bidhan-2 105–110 120–125 4.77 ± 0.09 Geetanjali 150–170 165–170 4.12 ± 0.12 BINA 8 90–100 115–120 4.62 ± 0.11 Amal-Mana 150–162 160–170 4.11 ± 0.12 Boby 100–110 125–130 4.34 ± 0.14 Swarna-Sub1 105–110 145–150 4.02 ± 0.33 Annada 95–105 120–125 4.28 ± 0.07 CSRC(D) 7-0-4 140–155 165–170 3.99 ± 0.14 N Sankar 95–105 120–125 4.25 ± 0.06 CSRC(D) 2-17-5 145–155 165–170 3.85 ± 0.06 BRRI dhan47 105–110 120–125 4.25 ± 0.06 CSRC(S) 21-2-5-B-1-1 115–125 140–145 3.84 ± 0.09 IR 76393-2B-7-1-1-3-1 85–95 120–125 4.18 ± 0.23 CSRC(D) 13-16-9 155–165 165–170 3.82 ± 0.07 Lal Minikit 90–105 120–125 4.15 ± 0.15 Manasswarabar 140–145 160–165 3.80 ± 0.32 Parijat 85–95 115–120 4.15 ± 0.04 Sabita 150–165 165–170 3.78 ± 0.14 Super Sankar 90–100 115–120 4.08 ± 0.07 CN 12133-3-9 105–115 3.76 ± 0.10 Rasi 95–105 115–120 4.03 ± 0.08 SR 26B 140–160 165–170 3.71 ± 0.18 Canning 7 95–105 125–130 3.98 ± 0.14 Patnai 23 140–155 160–165 3.63 ± 0.11 Satabdi 85–95 120–125 3.96 ± 0.09 NC 678 155–165 165–170 3.53 ± 0.032 IR 76346-B-B-10-1-1-1 95–1005 120–125 3.92 ± 0.022 BINA 8 90–110 145–150 3.52 ± 0.04 IR 64 Saltol 95–105 120–125 3.91 ± 0.05 CST 7-1 110–120 140–150 3.49 ± 0.13 CSR 4 95–100 125–130 3.82 ± 0.06 CN 1039-9 105–115 3.42 ± 0.02 CSR38 95–100 125–130 3.70 ± 0.12 Sumati 100–105 140–145 3.33 ± 0.17 CSR 22 95–105 125–130 3.65 ± 0.12 CSRC(S) 47-7-B-B 90–100 145–150 3.27 Lalat 100–105 125–130 3.58 ± 0.26 Utpala 105–115 140–145 3.25 ± 0.20 Super Minikit 90–100 115–120 3.54 ± 0.15 CN 1233-39-9 125–135 3.17 ± 0.35 IR 72593-B-3-2-3-3 120–125 3.45 ± 0.23 CR 2094-46-3 150–160 160–165 3.17 Sankarsaru 95–105 110–115 3.36 ± 0.34 CR 2095-181-1 150–160 160–165 3.13 Khitish 85–95 115–120 3.34 ± 0.26 Bhutnath 95–105 130–135 3.13 ± 0.16 IR 72593-B-18-2-2-2 120–125 3.21 ± 0.25 Pankaj 130–150 140–145 3.10 ± 0.14 CSR 36 95–105 125–130 2.19 ± 0.16 IR 206-29-2-1-1 105–115 145–150 3.10 Dinesh 145–175 165–170 3.09 ± 0.38 CR 2006-71-2 150–165 2.98 CR 2070-52-2 145–155 160–165 2.87 IR 76393-28-7 90–110 145–150 2.58 Table 4 Results of the preference analyses conducted during the wet seasons of 2008–2013. Table 5 Table 5 Ranking of farmers’ preference criteria for selection of rice entries in the wet and dry seasons. Preference traits were established based on farmers’ feedback during 2008–2013, and ranking was done in 2014 wet and dry seasons, each involving 60 farmers. selection of rice entries in the wet and dry seasons. Preference traits were established based on farmers’ feedback during 2008–2013, and asons, each involving 60 farmers. Table 5 Ranking of farmers’ preference criteria for selection of rice entries in the wet and dry seasons. Preference traits were established based on farmers’ feedback during 2008–2013, and ranking was done in 2014 wet and dry seasons, each involving 60 farmers. Wet season Dry season Preference Criteria Ranks as assigned by respondents (5 years) RBQa Score Rank Ranks as assigned by respondents (5 years) RBQ Score Rank 1 2 3 4 5 1 2 3 4 5 Yield 18 12 10 6 6 62.00 2 24 12 12 10 10 78.00 1 Tolerant to salinity 2 0 2 4 8 10.67 8 22 14 8 6 8 70.00 2 Capacity to withstand waterlogging (height) 10 14 14 14 12 62.67 1 0 0 4 8 8 12.00 8 Pest and disease resistant 4 6 4 10 6 27.33 6 2 8 8 6 4 27.33 5 Quality of straw for thatching/fodder/ fuel 10 10 10 8 6 47.33 3 2 2 4 6 8 16.67 7 Resistance to lodging 12 8 8 6 6 44.67 4 0 6 4 8 6 19.33 6 Grain quality for better market price 2 2 4 6 8 16.67 7 2 8 8 6 8 28.67 4 Duration of crop 2 8 8 6 8 28.67 5 8 10 12 10 8 48.00 3 Chi Square Statistic 40.925 76.082*** RBQa, Rank Based Quotient. *** P < 0.001. of all farmers and grain yields were recorded in 69% of all trials, which was much higher than in the wet season (41%). This variation indicates that yield was the most preferred criteria for farmers in the dry season. The RBQ analysis (Table 5) also reflected that ranking of preference criteria was different compared to that of the wet season. The yield was the most critical trait for choosing suitable rice varieties and breeding lines in the dry season. 3.2. Preferences of varieties and breeding lines in the dry season During the monsoon season, excess salt is mostly washed away with drainage water and pushed down the soil profile. Therefore, salinity stress was lowest in September-October (Fig. 2). However, in the dry season, rice is grown using irrigation, and soil salinity is the major abiotic stress. Salinity built up at the study sites due to gradual drying of the soil after the monsoon season and capillary rise of salinity from the subsoil. Starting in December, topsoil salinity increased progres- sively and reached a maximum in April-May (Fig. 2). Therefore, soil salinity was high (> 4.0 dS m−1) during both sensitive growth stages of rice at the experimental sites (Table 2). The plant height, duration and grain yield of different rice varieties and breeding lines evaluated in sixteen on-station and on-farm trials during the dry seasons of 2008–09 to 2013-14 are presented in Table 3. Entries were mostly of short duration (115–130 d), short to medium height (85–110 cm) and with grain yield of 2.19–4.77 t ha−1. High grain quality for better market value was identified as another critical trait for selection. Farmers grow rice in the dry season mostly for marketing because the productivity is higher compared with the wet season. Farmers have better control over crop management, especially water and nutrients. During field days, participating farmers indicated that about 90% of their rice production in the dry season was sold. Consequently, farmers in the study areas preferred rice with long slender grains (Tables 4 and 6). Farmers in general do not keep seeds of the dry season rice varieties for the succeeding dry season because good storage facilities are required to maintain optimum humidity, especially during the hot and humid monsoon months. However, this is less of an issue for storage for the wet season’s rice seed because of the dry conditions after harvest. To select suitable rice genotypes, 384 farmers participated in the evaluation process from 2008 to 2014. Out of these, 45% were male farmers, 40% were females, and the rest were researchers. A total of 25 entries were included in these trials, 13 of them were favored by farmers and ranked from 1st to 4th (Table 6). The most frequently selected genotypes were Gontra Bidhan-2, Boby, Lal Minikit, and Annada. Pest and disease resistance and resistance to lodging were also considered important for selection of rice lines for the dry season. Table 5 After yield, the tolerance to salinity was 2nd because salinity is a major constraint for farming in coastal saline areas in the dry season. Crop growth duration was another important criterion for selection. Farmers in the region preferred short duration (115–130 d) varieties, because of freshwater scarcity, and together with high soil salinity, most of the land (about 80%) remains fallow during the dry season. Where possible, farmers irrigate their crops from shallow tube wells. Farmers are mostly smallholders with limited resources to invest in purchasing water from neighboring farmers who own shallow tube wells. Soil salinity also builds up gradually as the dry season progresses. Short duration rice varieties will have fewer irrigation requirements and will mature before the build up of high soil salinity towards the end of the season. for better market price, and tolerance to salinity ranked least according to the PA. Farmers in this region are mostly small and marginal, growing rice during the wet season for their consumption. They keep some seed for the next wet season planting and sell the only remaining surplus. Farmers preferred bold grain types as they feel that bold types satiate longer after cooking and are also more suitable for parboiling. Because salinity is not a major stress except at early seedling stage, salinity tolerance was not considered important for farmers during the wet season. Table 4 Table 4 Results of the preference analyses conducted during the wet seasons of 2008–2013. Year Trials Sites Ranking of most preferred varieties/breeding lines Correlations between preferences 1st 2nd 3rd 4th Male vs. Female Farmers vs. Researchers Farmers vs. yield 2008 On-station Site 1 Amal-Mana Sabita SR 26 B Sumati 0.61** 0.84*** 0.35 On-farm Site 1 SR 26B Amal-Mana CSRC(S) 21-2-5-B-1-1 Sumati 0.75** 0.92*** 0.15 2009 On-station Site 1 CSRC(S) 21-2-5- 1-1 Geetanjali Amal-Mana Sabita 0.85*** 0.90*** 0.86*** On-farm (3)a Site 1 CSRC(S) 21-2-5- 1-1 Geetanjali Amal-Mana SR 26 B/ Sabita 0.10 0.48 0.96*** Site 2 CSRC(S) 21-2-5- 1-1 Geetanjali Amal-Mana SR 26 B 0.62 0.91*** 0.19 Site 3 CSRC(S) 21-2-5- 1-1 Geetanjali Amal-Mana Sabita 0.85** 0.90*** 0.73** 2010 On-station Site 1 Geetanjali Amal-Mana Sabita CSRC(S) 21-2-5-1-1/ SR 26B 0.91*** 0.94*** 0.72* On-farm (3) Site 1 Geetanjali Amal-Mana CSRC(D) 7-0-4/ CSRC(S) 21-2-5-1-1 – 0.94*** 0.91*** 0.50 Site 2 Amal-Mana Geetanjali CSRC(S) 21-2-5-1-1/ Sabita – 0.85** 0.81** 0.74* Site 3 Geetanjali Amal-Mana CSRC(S) 21-2-5-1-1 CSRC(D) 7-0-4 0.93*** 0.78* 0.74 2011 On-station Site 1 CSRC(D) 7-0-4 SR 26 B Amal-Mana/Sabita – 0.65* 0.48 0.79** 2012 On-farm (3) Site 1 Sabita CSRC(S) 21-2-5- 1-1 CSRC(D) 7-0-4 CSRC(D) 13-16-9 0.73* 0.71* 0.51 Site 2 Sabita CSRC(D) 12-8-12 Amal-Mana Geetanjali 0.37 0.44 0.67 Site 3 CSRC(D) 12-8-12 Sabita Geetanjali Amal-Mana 0.74* 0.57 0.49 Site 4 CSRC(D) 12-8-12 Sabita Amal-Mana CSRC(D) 13-16-9 0.81* 0.72* 0.88** 2013 On-farm Site 1 CSRC(S) 21-2-5- 1-1 Swarna-Sub1 Patnai-23 Geetanjali 0.81*** 0.56* 0.66** Sabita Amal-Mana Swarna-Sub1 CSRC (S) 21-2-5-1-1 0.75** 0.15 0.87*** * ** *** significant at P < 0 05 0 01 and 0 001 respectively f the preference analyses conducted during the wet seasons of 2008–2 72 Field Crops Research 220 (2018) 67–77 D. Burman et al. D. Burman et al. 3.3. Selection of best varieties based on farmers’ preferences During the PA, farmers were asked to cast their votes for the two most preferred and two least preferred entries, which allowed ranking of all entries based on cumulative farmers’ preference scores. The ranking of most preferred entries changed from year to year in both seasons (Tables 4 and 6), and even between sites within same years. For example, Amal-Mana, which was included in all 17 wet season trials, had quite different rankings between trial locations and years (Table 4). Similarly, in the dry season, Annada, which was included in 13 out of the 16 trials, had different rankings in different locations and years (Table 6). Based on farmers’ preference scores across locations and years, eight entries in both seasons were ranked 1st or 2nd, while 11 and 13 entries were ranked among the top 4 in the wet and dry seasons, respectively (Table 7). 3.2. Preferences of varieties and breeding lines in the dry season Farmers preferred varieties with sturdy culms to avoid lodging and yield losses because of occasional rain and storms that occur during the dry season. Quality of straw and height were ranked as least important compared to other criteria. Farmers preferred medium height (85–100 cm) because they believe it is associated with resistance to lodging. Similar to the wet season, a ranking of the most preferred entries in the dry season varied across locations and years. Preferred entries were selected based on a range of traits includingd tolerance of soil salinity, long panicles with more grains, no or minimum infestation by pest and diseases, more tillers, good grain type, overall good crop performance, sufficient straw for fodder, thatching and for use as fuel, suitability for parboiling, optimum maturity period, and high yield. Correlations between preferences of male and female farmers were significant in 75% of the trials (Table 6). Significant correlations were also observed between preference scores of all farmers versus researchers in 56% of the trials. Similarly, significant correlations between preference scores Chi-square tests were applied to assess the associations between preference criteria and ranks assigned by the respondents. Based on preference ranking under dry season, the calculated Chi-square was 76.082 (P < 0.001). This indicated a stronger association between preference criteria and ranks assigned by the respondents in the dry 73 Field Crops Research 220 (2018) 67–77 D. Burman et al. season than during the wet season. season than during the wet season. Year Trials Sites No. of entries Ranking of most preferred varieties/lines Correlation between preferences 1st 2nd 3rd 4th Male vs. Female Farmers vs. Researchers Farmers vs. 3.2. Preferences of varieties and breeding lines in the dry season yield 2008−09 On-station Site 1 12 Gontra Bidhan-2 Canning 7 Lal Minikit Satabdi 0.81** 0.85** 0.86* On-farm (2)a Site 1 12 Gontra Bidhan-2 Canning 7 Satabdi Lalat 0.73** 0.86** 0.69* Site 2 12 Gontra Bidhan-2 Canning 7 Satabdi Khitish 0.93** 0.79** 0.66* 2009−10 On-station Site 1 8 Gontra Bidhan-2 Annada Rasi Boby 0.81** 0.85*** 0.86* On-farm (2) Site 1 8 Gontra Bidhan-2 Boby Rasi Canning 7 0.93** 0.08 0.72** Site 2 8 Canning 7 Annada Rasi Boby 0.97** 0.86** 0.16 2011−12 On-station Site 1 8 Boby Bidhan-2 Lal Minikit Annada 0.91* 0.82* 0.89* On-farm (3) Site 1 8 Lal Minikit Annada Gontra Bidhan-2 Boby 0.83 0.51 0.66 Site 2 8 Gontra Bidhan-2 Boby Annada Lal Minikit 0.83 0.64 0.70 Site 3 8 Gontra Bidhan-2 Annada Lal Minikit Satabdi 0.93* 0.82 0.73 2012−13 On-farm (3) Site 1 10 Gontra Bidhan-2 Boby Annada Parijat/Super Sankar 0.46 0.39 0.80** Site 2 10 Super Sankar Gontra Bidhan-2 Boby N Sankar 0.78** 0.52 0.84** Site 3 10 Boby Gontra Bidhan-2 Super Sankar Annada 0.94** 0.90*** 0.32 2013−14 On-farm (3) Site 1 10 Lal Minikit Lalat Satabdi Boby 0.83** 0.95** 0.65* Site 2 10 Lal Minikit Lalat Annada/IR 64 Saltol – 0.20 0.95** 0.68* Site 3 10 Lal Minikit Lalat/IR 64 Saltol Boby – 0.83** 0.92** 0.65* 4. Discussion Most rural people living in coastal tropical deltas of south and southeast Asia are dependent on agriculture for their livelihood. The majority of them are smallholders, facing severe poverty and regular hunger periods because of low land productivity and limited alter- natives. Rice productivity in the coastal region is low and unstable because of the persistence of several abiotic stresses like waterlogging in the wet season and soil and water salinity in both wet and dry seasons (Sarangi et al., 2014; Sarangi et al., 2016). The assumption in this study is that the rice productivity in these areas can be enhanced by developing and disseminating suitable stress tolerant varieties presently not accessible to farmers. Participation of local smallholder farmers in the process of selecting suitable rice varieties is crucial to understand their preferences and to select material adapted to these difficult conditions for subsequent use (Gyawali et al., 2007). The need for participatory approaches to identify such material is highlighted by the fact that conventional breeding has not brought significant crop improvement to smallholder farmers in these marginal environments (Kerr and Kolavalli, 1999; Lipton and Longhurst, 1989; Tiwari et al., 2009). The PVS trial approach is a simple and cost-effective method to understand farmer’s selection process and to identify their most preferred varieties for these less favorable areas. This approach was found successful especially in remote and marginal areas where farmers had limited resources and opportunities (Belay et al., 2005; Joshi and Witcombe, 2002; van Asten et al., 2009; Witcombe et al., 1996). Consequently, germplasm evaluation and selection and knowledge of varietal preferences of farmers were the primary reasons behind this PVS study. Farmers are the key stakeholders for the adoption of new technologies (Antle and Crissman, 1990). The feedback will help rationalize breeding strategies by targeting necessary traits and will also contribute to optimizing resource use (Bhuiyan et al., 2004; Waldman et al., 2014). Table 6 Results of the preference analyses conducted during the dry seasons of 2008–2014. *, ** significant at P < 0.05, and 0.01, respectively. a Numbers in parenthesis in the second column are on-farm trials. *, ** significant at P < 0.05, and 0.01, respectively. a Numbers in parenthesis in the second column are on-farm trials. a conducted during the wet and dry seasons of 2008 −2014, in the coastal regions of the Indian Sundarbans. Trial details presented in Table 1. b Frequency of preferences indicates the number of times a particular variety was ranked as 1st to 4th. preferred varieties, but also other traits necessary for their local farming conditions (Joshi et al., 1997; Mandal et al., 2002; Manzanilla et al., 2011; Singh et al., 2010, 2014, 2016). Site-specific characteristics other than grain yield are expressly important in the wet season, as reflected by the significant correlations with yield in fewer trials than in the dry season (Tables 4 and 6). The ranking of preference criteria following the RBQ analysis showed that plant height rather than grain yield is the most critical trait for farmers in the wet season, because of the predominance of waterlogging (Table 5). importance for various uses, including construction of homes, fodder for livestock, and fuel for cooking. The fourth-ranked trait was resistance to lodging, which is of particular importance for taller varieties to maintain their high yield, thus a consequence of the first and second preferences. Least important traits for wet season varieties were market- driven grain quality and salinity tolerance, though the latter might have been ranked higher if the PA was conducted at the sensitive seedling stage. importance for various uses, including construction of homes, fodder for livestock, and fuel for cooking. The fourth-ranked trait was resistance to lodging, which is of particular importance for taller varieties to maintain their high yield, thus a consequence of the first and second preferences. Least important traits for wet season varieties were market- driven grain quality and salinity tolerance, though the latter might have been ranked higher if the PA was conducted at the sensitive seedling stage. In the dry season, there is no flooding risk, but salinity increases as the season progresses, and most of the crop is sold rather than used for household consumption. Consequently, farmers prefer high yielding varieties with at least some salinity tolerance, short duration, and good grain quality for high market value. Least important were plant height and straw characteristics. Farmers' overall germplasm choices as well as their preference rankings were, therefore seamlessly rational and emulated the constraints and opportunities of their production envir- onment. *, ** significant at P < 0.05, and 0.01, respectively. a Numbers in parenthesis in the second column are on-farm trials. p gg g ( ) Another outcome of these PVS trials is that farmers’ preferences are seldom uniform across different locations or years. In this process, farmers select and rank varieties based on visual traits of the standing crop during a field day, usually organized between flowering and maturity (at about 80% maturity in the present study). If for some reasons, any variety does not perform well in a given year or at a specific site, farmers will not select it even if it was ranked high in previous seasons. Misiko (2013) pointed out this problem as a funda- mental dilemma for PVS. The farmers who participate in PVS and the final selection mostly see these entries only once during the field day without prior knowledge of their performance throughout the season or in previous years. They also have no information about the environ- mental conditions at the specific site, e.g., presence/absence of diseases or abiotic stresses like floods or salinity. Farmers might also select entries because of some attractive traits like high yield or profuse tillering, neglecting other important traits. This ‘impulse buying’ (Misiko, 2013) can adversely affect the proper selection of germplasm. Misiko (2013) proposed involvement of participating farmers at different crop development stages to give them a better grasp of the characteristics and performance of the germplasm being evaluated, which then would enable farmers to more accurately select appropriate genotypes for their local conditions. Other studies (Bellon and Reeves, 2002; Paris et al., 2011; Sperling et al., 1993), pointed that selection of rice varieties by smallholder farmers is also influenced by other factors, such as farmers’ socio-economic conditions, available resources, spe- cific needs, and preferences. In-depth discussions with farmers during PA field days at various locations, seasons and years provided similar evidence. Although the exact ranking of entries was not always consistent between sites and years (Tables 4 and 6), several entries were repeatedly among the top four at different sites and in different years. And farmers’ preference criteria (Table 5) did not indicate much ‘impulse buying’ but rather a good understanding of their environment without the necessity for a particular stress occurring at the trial site. *, ** significant at P < 0.05, and 0.01, respectively. a Numbers in parenthesis in the second column are on-farm trials. Our study showed that characteristics of suitable rice varieties for smallholder farmers in this coastal salt and waterlogging troubled region are variable, as farmers need to adjust to the complex production systems and stresses typical of their local conditions. Preferential traits were obviously different for the wet versus the dry season (Table 5). Sarangi et al. (2016) observed that preference for new varieties by farmers during the wet season largely depends on traits necessary for survival and higher yields in flood-affected areas of the Sundarbans region of West Bengal. In the wet season, uncontrolled waterlogging and poor drainage are the dominant risks that require taller varieties or varieties capable of elongation with rising water (Singh et al., 2011; Kato et al., 2014); therefore, plant height at maturity was the most preferred trait. Because the wet season crop is the main source of food supply for most farmers, grain yield ranked second. The third pre- ference rank is quality and quantity of straw, demonstrating its 74 74 Field Crops Research 220 (2018) 67–77 D. Burman et al. D. Burman et al. Table 7 Farmers’ most preferred varieties and breeding lines in trialsa conducted during the wet and dry seasons of 2008 −2014, in the coastal regions of the Indian Sundarbans. Most preferred varieties/lines Wet season Most preferred varieties/lines Dry season Frequency of preferences (no.)b Frequency of preferences (no.) 1st rank 2nd rank 3rd rank 4th rank 1st rank 2nd rank 3rd rank 4th rank Amal-Mana 2 5 7 1 Gontra Bidhan-2 8 3 1 – SR 26B 1 1 1 2 Boby 2 3 2 4 CSRC(S) 21-2-5-1-1 5 1 3 2 Lal Minikit 4 3 1 Geetanjali 3 5 1 2 Super Sankar 1 1 1 CSRC(D) 7-0-4 1 2 1 Canning 7 1 3 Sabita 3 3 3 3 Annada – 4 3 2 CSRC(D) 12-8-12 2 1 Lalat – 3 1 Swarna-Sub1 – 1 1 – IR64 Saltol – 1 1 Patnai-23 – – 1 – Satabdi – – 3 2 Sumati – – – 2 Rasi – – 3 – CSRC(D) 13-16-9 – – – 2 Khitish – – – 1 Parijat – – – 1 N Sankar – – – 1 a Trial details presented in Table 1. b Frequency of preferences indicates the number of times a particular variety was ranked as 1st to 4th. References Antle, J.M., Crissman, C.C., 1990. Risk, efficiency, and the adoption of modern crop varieties: evidence from the Philippines. Econ. Dev. Cult. Chang. 38, 517–537. A standard second step in PVS involves further evaluation of one or two top preferred lines in farmer-managed trials in subsequent season (s). However, when only a few top-ranked entries are selected for these trials, there are chances of losing some promising lines early in the process. As was observed in our study, the 2 top ranked entries were not selected as most preferred varieties every year (Table 7). Therefore we would propose that more than two entries (e.g., 4), should be tested in follow-up multi-year researcher and farmer managed trials, to increase the chances of selecting the best and stable varieties that are most preferred by farmers, as some preferences might be site- or condition- specific. Our PVS results indicated that choices of farmers were not always consistent because the preference criteria (also the performance of a particular variety) vary across groups. Increasing the number of most preferred rice varieties while providing more options for the farmers will also contribute to increasing farmers’ acceptance. Addi- tional entries will also increase varietal diversity and provide more stability in these unfavorable areas, as one of the benefits of PVS trial system (Tshewang and Ghimiray, 2010; Subedi et al., 2011; Witcombe et al., 2001). Atlin, G., Paris, T., Courtois, B., 2002. Sources of variation in participatory varietal selection trials with rainfed rice: implications for the design of mother-baby trial networks. In: Bellon, M.R., Reeves, J. (Eds.), Quantitative Analysis of Data From Participatory Methods in Plant Breeding. Centro Internacional de Mejoramiento de Maíz y Trigo, Mexico, D.F, pp. 36–43. Belay, G., Tefera, H., Tadesse, B., Metaferia, G., Jarra, D., Tadesse, T., 2005. Participatory variety selection in the Ethiopian cereal. Tef. Exp. Agric. 42, 91–101. Bellon, M.R., Reeves, J. (Eds.), 2002. Quantitative Analysis of Data From Participatory Methods in Plant Breeding. International Maize and Wheat Improvement Center, Mexico, DF (143 p). Rice Research and Development in the Flood-Prone Ecosystem. In: Bhuiyan, S.I., Abedin, M.Z., Singh, V.P., Hardy, B. (Eds.), Proceedings of the International Workshop on Flood-Prone Rice Systems Held in Gazipur, Bangladesh. 9–11 January 2001. International Rice Research Institute, Los Baños, Philippines. pp. 283. Burman, D., Bandyopadhyay, B.K., Mandal, S., Mandal, U.K., Mahanta, K.K., Sarangi, S.K., Maji, B., Rout, S., Bal, A.R., Gupta, S.K., Sharma, D.K., 2013. 5. Conclusions I l M R G i G B 2013 P f li i l i i d l i Islam, M.R., Gregorio, G.B., 2013. Progress of salinity tolerant rice variety development in Bangladesh. SABRAO J. Breed. Genet. 45, 21–30. Ismail, A.M., Tuong, T.P., 2009. Brackish water coastal zones of the monsoon tropics: challenges and opportunities. In: Haefele, S.M., Ismail, A.M. (Eds.), Natural Resource Management for Poverty Reduction and Environmental Sustainability in Fragile Rice- based Systems. Proceedings No. 15. International Rice Research Institute, Los Baños, Laguna, Philippines, pp. 113–121. Ismail, A.M., Thomson, M.J., Vergara, G.V., Rahman, M.A., Singh, R.K., Gregorio, G.B., Mackill, D.J., 2010. Designing resilient rice varieties for coastal deltas using modern tools. In: Hoanh, C.T., Szuster, B.W., Suan-Pheng, K., Ismail, A.M., Nobel, A.D. (Eds.), Tropical Deltas and Coastal Zones: Food Production, Communities and Environment at the Land-Water Interface. CAB International, Nosworthy Way, Wallingford,Oxfordeshire, UK, pp. 166–182. Joshi, K.D., Witcombe, J.R., 2002. Participatory varietal selection in rice in Nepal in favorable agricultural environments—a comparison of two methods assessed by varietal adoption. Euphytica 127, 445–458. Joshi, K., Subedi, M., Rana, R., Kadayat, K., Sthapit, B., 1997. Enhancing on farm varietal diversity through participatory varietal selection: a case study for Chaiterice in Nepal. Exp. Agric. 33, 335–344. Joshi, K.D., Biggs, S., Gauchan, D., Devkota, K.P., Devkota, C.K., Shrestha, P.K., Sthapit, B.R., 2005. Institutional innovations in the Nepal rice improvement system. Asp. Appl. Biol. 75, 93–97. Joshi, K.D., Musa, A.M., Johansen, C., Gyawali, S., Harris, D., Witcombe, J.R., 2007. Highly client-oriented breeding: using local preferences and selection produces widely adapted rice varieties. Field Crops Res. 100, 107–116. References Land Shaping – a Unique Technology for Improving Productivity of Coastal Land. CSSRI/Canning Town/Bulletin/2013/02. ICAR-Central Soil Salinity Research Institute, Regional Research Station, Canning Town, West Bengal, India (38 p). Town/Bulletin/2013/02. ICAR-Central Soil Salinity Research Institute, Regional Research Station, Canning Town, West Bengal, India (38 p). CSE, 2012. In: Chaudhuri, J. (Ed.), Living with Changing Climate: Impact, Vulnerability and Adaptation Challenges in Indian Sundarbans. Centre for Science and Environment Centre for Science and Environment, New Delhi, India (108 p). Ceccarelli, S., Grando, S., 2007. Decentralized-participatory plant breeding: an example of demand driven research. Euphytica 155, 349–360. Ceccarelli, S., Grando, S., Tutwiler, R., Baha, J., Martini, A.M., Salahieh, H., Goodchild, A., Michael, M., 2000. A methodological study on participatory barley breeding I. Selection phase. Euphytica 111, 91–104. 5. Conclusions y Chi, T.T.N., Liem, P.V., Paris, T.R., 2007. Farmers’ participation in rice variety selection. Omonrice 15, 159–163. The study assessed smallholder farmers’ criteria when selecting new rice varieties suitable for salt-affected coastal delta regions of tropical Asia. The data showed that farmers have different preference criteria for rice varieties for the wet and dry seasons. Farmers’ assessment was multivariate and involves multiple traits, including agronomic char- acteristics, tolerance of prevailing abiotic stresses, and socio-economic conditions. Farmers have a clear understanding of their rice environ- ment, and the major traits that the new varieties must possess. In addition to grain yield, conventional breeding programs should obviously consider these important and sometimes site and season specific traits such as tolerance to waterlogging, quality and quantity of straw, and lodging resistance for wet season rice varieties. Besides grain yield in the dry season, preferred traits include salinity tolerance, medium to short duration, and good grain quality for better market value. Comparative analyses of the order of ranking of top-preferred entries in these complex ecosystems suggested the need for testing entries over multiple locations and years to select the best performing and stable genotypes. Furthermore, the study suggests preferably more (e.g. four rather than two) better-performing varieties should be retained over the years and promoted through farmers’ managed PVS trials, thereby increasing the chances of the best entries being selected. These findings highlighted the importance and effectiveness of PVS process in these less favorable, marginal, and complex environments in providing useful feedback to inform breeding programs and facilitate the development of adapted varieties that meet farmers’ expectations in coastal regions of tropical South and Southeast Asia. Dorward, P., Craufurd, P., Marfo, K., Dogbe, W., Bam, R., 2007. Improving participatory varietal selection processes: Participatory varietal selection and the role of informal seed diffusion mechanisms for upland rice in Ghana. Euphytica 155, 315–327. Freeman, H.A., 2001. Comparison of Farmer-participatory Research Methodologies: Case Studies in Malawi and Zimbabwe. Working Paper Series No. 10, Socio-economics and Policy Program. International Crops Research Institute for the Semi-Arid Tropics, Nairobi, Kenya (28 p.). Gregorio, G.B., Salam, M.A., Karim, N.H., Seraj, Z.I., 2004. Evaluation Report of Sub- project on Development of High-yielding Varieties for Coastal Wetlands of Bangladesh. International Rice Research Institute, Dhaka, Bangladesh. Gyawali, S., Sunwar, S., Subedi, M., Tripathi, M., Joshi, K.D., Witcombe, J.R., 2007. Collaborative breeding with farmers can be effective. Field Crops Res. 101, 88–95. *, ** significant at P < 0.05, and 0.01, respectively. a Numbers in parenthesis in the second column are on-farm trials. Moreover, our results imply that PVS should be conducted over multiple years and locations to be able to judge consistency in farmers’ Correlation between preference scores of male and female farmers, an indicative of consensus, reflected a strong to very strong agreement in most of the trials across locations and years, especially in the dry season (Tables 4 and 6). This agreement showed that male and female smallholder farmers in the target areas had similar requirements in new varieties. Women and men often work together for a range of different agricultural operations in the Sundarbans region (Mandal et al., 2011). And because of the common cases of male labor migration in this region, women become the major working force for rice production (Mondal, 2013), reflecting that they have full knowledge of the rice production system and needs. Nevertheless, there remain some dis- crepancies between male and female preferences. In addition to agronomic characters of new varieties, the PVS process can also be extended to include post-harvest characteristics, where obviously women will have some additional preference criteria such as good eating quality, softness after cooking, and high market value (Chi et al., 2007; Paris et al., 2011). The relatively low correlation in preference criteria between farm- ers and researchers during both wet and dry seasons indicated that farmers often had different priorities than the researchers, as also reported before (Singh et al., 2010). Farmers’ preferences and their reasoning are important criteria for breeders to consider when devel- oping new varieties to ensure they will be acceptable by farmers and adapted to local conditions. This concern is particularly highlighted by the weak correlation between the preference scores of all farmers and grain yield, with the latter being a central criterion in most breeding programs. 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Innovative solid desiccant dehumidification using distributed microwaves
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Innovative solid desiccant dehumidification using distributed microwaves Doskhan Ybyraiymkul 1*, Qian Chen 2, Muhammad Burhan 1, Faheem Hassan Akhtar 3, Raid AlRowais4, Muhammad Wakil Shahzad5, M. Kum Ja1 & Kim Choon Ng1 OPEN Doskhan Ybyraiymkul 1*, Qian Chen 2, Muhammad Burhan 1, Faheem Hassan Akhtar 3, Raid AlRowais 4, Muhammad Wakil Shahzad 5, M. Kum Ja 1 & Kim Choon Ng 1 Dehumidification is one of the key challenges facing the air conditioning (AC) industry in the treatment of moist air. Over many decades, the dual role of heat exchangers of AC chillers for the sensible and latent cooling of space has hindered the thermal-lift reduction in the refrigeration cycle due to the requirements of water vapor removal at dew-point and heat rejection to the ambient air. These practical constraints of AC chillers have resulted in the leveling of energy efficiency of mechanical vapor compressors (MVC) for many decades. One promising approach to energy efficiency improvement is the decoupling of dehumidification from sensible processes so that innovative but separate processes can be applied. In this paper, an advanced microwave dehumidification method is investigated in the laboratory, where the microwave (2.45 GHz) energy can be irradiated onto the dipole structure of water vapor molecules, desorbing rapidly from the pores of adsorbent. Results show a significant improvement in performance for microwave dehumidification, up to fourfold, as compared to data available in the literature. Dehumidification is water vapor removal from the air to maintain human comfort and a healthy environment (relative humidity (RH) at 40%-60%)1–4. Presently, dehumidification is provided by cooling the airstream to its dew-point to condense water vapor using a dual-role AC ­chiller5; and air-cooled AC chillers have reached their asymptotic performance limit, 0.7–0.85 kW/Rton (equivalent to a coefficient of performance (COP)6 of 4–5)7. Much literature on chiller manufacturers cites low kW/Rton is attributed to acceptance test conditions ignoring electricity consumption incurred by long chilled water piping ­losses5. One of the solutions to improve the performance of AC is to decouple dehumidification from sensible cooling, thus permitting the incorporation of new dehumidification methods. It is well known that microwaves can desorb water molecules from adsorbent or sorbent. Therefore, the mechanism is used in microwave dehumidification, which is an emerging environmental- friendly method. In microwave dehumidification, the air becomes dehumidified due to the attraction of water molecules onto a solid adsorbent (desiccant) pore surface by physio-sorption (physical adsorption)8–11, a characteristic of weak Van der Walls ­forces12–15. When adsorbent pores are saturated with water, microwave- assisted desorption (removal of water) initiates, and highly humid air purges out. The process is schematically presented in Fig. www.nature.com/scientificreports www.nature.com/scientificreports 1BESE Division, Water Desalination and Reuse Center, King Abdullah University of Science and Technology, Thuwal  23955, Saudi Arabia. 2Institute for Ocean Engineering, Shenzhen International Graduate School, Tsinghua University, Tsinghua Campus, University Town, Shenzhen  518055, China. 3Department of Chemistry and Chemical Engineering, Lahore University of Management Sciences, Lahore  54792, Pakistan. 4Department of Civil Engineering, College of Engineering, Jouf University, Sakakah  72388, Saudi Arabia. 5Mechanical and Construction Engineering Department, Northumbria University, Newcastle Upon Tyne  NE1 8ST, UK. *email: doskhan.ybyraiymkul@kaust.edu.sa Figure 1. Schematically representation of microwave dehumidification. (a) Dehumidification of airstream by adsorption of water vapor from the moist air onto the pore surface of the adsorbent due to Van der Walls forces. Nitrogen and oxygen molecules in the air are very little attracted at ambient pressure and temperatures; (b) Desorption of water vapor from pores of adsorbent due to irradiation by microwave energy, where microwave energy (energized by oscillating) is directly delivered to polar adsorbed water molecules. Desorbed water molecules and air molecules are almost not adsorbing microwave energy as they can freely move in a gas state. Microwave desorption is needed to regain the adsorbent’s ability to attract water molecules. Figure 1. Schematically representation of microwave dehumidification. (a) Dehumidification of airstream by adsorption of water vapor from the moist air onto the pore surface of the adsorbent due to Van der Walls forces. Nitrogen and oxygen molecules in the air are very little attracted at ambient pressure and temperatures; (b) Desorption of water vapor from pores of adsorbent due to irradiation by microwave energy, where microwave energy (energized by oscillating) is directly delivered to polar adsorbed water molecules. Desorbed water molecules and air molecules are almost not adsorbing microwave energy as they can freely move in a gas state. Microwave desorption is needed to regain the adsorbent’s ability to attract water molecules. Figure 1. Schematically representation of microwave dehumidification. (a) Dehumidification of airstream by adsorption of water vapor from the moist air onto the pore surface of the adsorbent due to Van der Walls forces. Nitrogen and oxygen molecules in the air are very little attracted at ambient pressure and temperatures; (b) Desorption of water vapor from pores of adsorbent due to irradiation by microwave energy, where microwave energy (energized by oscillating) is directly delivered to polar adsorbed water molecules. Desorbed water molecules and air molecules are almost not adsorbing microwave energy as they can freely move in a gas state. Microwave desorption is needed to regain the adsorbent’s ability to attract water molecules. systems. MCOP can be calculated using microwave power, microwave exposure duration, and water desorbed amount. The calculated values of MCOP for other authors were extremely low, as summarized in Table 1. The system’s performance depends on the uniform propagation of the electric ­field25, the geometry of the microwave chamber, microwave irradiation time, the type of irradiation (continuous, pulsed), and the reflected power amount. Innovative solid desiccant dehumidification using distributed microwaves Doskhan Ybyraiymkul 1*, Qian Chen 2, Muhammad Burhan 1, Faheem Hassan Akhtar 3, Raid AlRowais4, Muhammad Wakil Shahzad5, M. Kum Ja1 & Kim Choon Ng1 OPEN 1a,b.ii p g From the available literature, Roussy and Chenot demonstrated the first microwave dehumidification process with a single-mode waveguide in ­198116. They presented the dependence of desiccant temperature on the electric ­field16. Moreover, Roussy et al. proposed a model to represent the fast kinetics of microwave ­desorption17. Most of the research within 40 years has been focused on developing the microwave-assisted desorption method with small ­volumes16–22. Notably, the investigation was extended with different adsorbents (activated alumina, zeolite, and silica gel)18. Many advantages of microwave desorption were revealed, such as transferring energy more efficiently than convection energy ­transport23 and desorbing at low temperatures due to direct energy ­transport24. However, a critical parameter such as COP was usually omitted in the literature. In addition, no electrical power values were provided; instead, microwave power was shown. Therefore, the microwave coefficient of performance (MCOP) was introduced, which can be the platform for comparing different microwave dehumidification 1BESE Division, Water Desalination and Reuse Center, King Abdullah University of Science and Technology, Thuwal  23955, Saudi Arabia. 2Institute for Ocean Engineering, Shenzhen International Graduate School, Tsinghua University, Tsinghua Campus, University Town, Shenzhen  518055, China. 3Department of Chemistry and Chemical Engineering, Lahore University of Management Sciences, Lahore  54792, Pakistan. 4Department of Civil Engineering, College of Engineering, Jouf University, Sakakah  72388, Saudi Arabia. 5Mechanical and Construction Engineering Department, Northumbria University, Newcastle Upon Tyne  NE1 8ST, UK. *email: doskhan.ybyraiymkul@kaust.edu.sa 1BESE Division, Water Desalination and Reuse Center, King Abdullah University of Science and Technology, Thuwal  23955, Saudi Arabia. 2Institute for Ocean Engineering, Shenzhen International Graduate School, Tsinghua University, Tsinghua Campus, University Town, Shenzhen  518055, China. 3Department of Chemistry and Chemical Engineering, Lahore University of Management Sciences, Lahore  54792, Pakistan. 4Department of Civil Engineering, College of Engineering, Jouf University, Sakakah  72388, Saudi Arabia. 5Mechanical and Construction Engineering Department, Northumbria University, Newcastle Upon Tyne  NE1 8ST, UK. *email: doskhan.ybyraiymkul@kaust.edu.sa | https://doi.org/10.1038/s41598-023-34542-9 | https://doi.org/10.1038/s41598-023-34542-9 Scientific Reports | (2023) 13:7386 www.nature.com/scientificreports/ Figure 1. Schematically representation of microwave dehumidification. (a) Dehumidification of airstream by adsorption of water vapor from the moist air onto the pore surface of the adsorbent due to Van der Walls forces. Nitrogen and oxygen molecules in the air are very little attracted at ambient pressure and temperatures; (b) Desorption of water vapor from pores of adsorbent due to irradiation by microwave energy, where microwave energy (energized by oscillating) is directly delivered to polar adsorbed water molecules. A multi-mode chamber system similar to a home oven was used for ­desorption19. Furthermore, the fixed zeolite-coated desiccant rotor was regenerated using microwave and temperature swing desorption methods, but the performance was low, with MCOP around 0.2221,22.i p Despite many decades of studying microwave dehumidification, we have discovered a knowledge gap that hindered its development. It is associated with narrowing research focus on methods advancement with small samples over the past half-century. The bounding of research by small single-mode cavities is due to the uniform electric field distribution, which led to impeding factor neglect, such as low performance. So far, most experiments have been carried out on scales less than 160 g with a volume of 1 ­L16–22. Consequently, all bench-scale studies were not developed to the pilot level to dehumidify the air. Scaling up the bench-scale system can lead to an uneven distribution of the electric field, heating of narrow sections (close to the surface), and, consequently, a drop in ­efficiency25. For achieving a high-performance pilot system, the following critical limitations had to be solved: (I) Internal entropy generation due to unheated areas (the non-uniformity of the electric field); (II) Enormous energy wasting (a high reflection of microwaves power); (III) Excess microwave irradiating time. g In this paper, we experimentally demonstrated that 97–99.5% of irradiated microwave power could be efficiently utilized, increasing performance by fourfold at a laboratory-scale pilot for the first time. Optimization allowed us to reduce a reflected power to 0.5–3% of input microwave power and distribute microwaves homogeneously. The key novelties are as follows: (a) A rotating reflector with attached honeycomb desiccant was proposed; it makes a more uniform distribution of the electric field and prevents overheating of desiccant; (b) a new approach of optimization was proposed to decrease reflected microwave power and unheated areas; (c) The effects of microwave irradiation time and heat recovery on COP were experimentally evaluated. The proposed dehumidification system with an optimized structure and operating parameters will help overcome all of the above limitations and achieve sustainable green dehumidification goals. We hope our work will help Table 1. A comparison of microwave dehumidification systems that are available in the literature. MCOP refers to the ratio of desorbed enthalpy of water vapor to irradiated microwave energy. MCOP values were calculated or obtained from the literature. Calculation of MCOP for literature is provided in supplementary materials. *calculated values. Innovative solid desiccant dehumidification using distributed microwaves Doskhan Ybyraiymkul 1*, Qian Chen 2, Muhammad Burhan 1, Faheem Hassan Akhtar 3, Raid AlRowais4, Muhammad Wakil Shahzad5, M. Kum Ja1 & Kim Choon Ng1 OPEN Desorbed water molecules and air molecules are almost not adsorbing microwave energy as they can freely move in a gas state. Microwave desorption is needed to regain the adsorbent’s ability to attract water molecules. Results Microwave dehumidification was developing with negligence of performance hitherto, so numerical optimization and experiments were highly focused on performance. The RD-type silica gel-coated desiccant wheel with a honeycomb structure was used as a desiccant wheel. The diameter of the wheel was 0.448 m, the height was 0.4 m, the composite material density was 668 kg/m3, the volume, including voids, was 0.063 ­m3, and the dry mass of the desiccant wheel was 11.8 kg. The thickness of honeycomb channel wall’s average thickness (including coating + cellulose + binder) was obtained from a cross-sectional SEM image (thickness was 208 µm), which is shown in Fig. 2a. Figure 2b depicts a fractured desiccant coating surface bound with fiber material. These fractures increase mass diffusion and flow of moisture. The selection of honeycomb structure and RD-type silica gel was due to its high-water adsorption capacity and high microwave penetration depth. Additionally, adsorption isotherms of the desiccant wheel, which is a combined (honeycomb cellulose, adsorbent, and binder) desiccant structure, were obtained, as shown in Fig. 2c. According to the results, the composite structure can adsorb moisture up to 30% of its dry bone mass at higher humidity. However, it can adsorb around 20% of its dry bone mass within a moderate humidity region. Besides, Fig. 2c demonstrates a composite desiccant sample on the crucible of the dynamic vapor sorption analyzer "Aquadyne DVS," which operates on the gravimetric principle and is fully automated for measuring adsorption isotherms. An Agilent impedance analyzer was used to measure the complex permittivity of composite desiccant material that was uniformly sampled without voids, and then effective values were determined according to Eq. (2). Figure 2d illustrates the complex permittivity Figure 2. Characteristics of composite (honeycomb cellulose, adsorbent, and binder) desiccant structure. (a) SEM image of desiccant cross-section; (b) SEM image of desiccant surface; (c) Isotherms of composite desiccant structure. A composite desiccant sample on the crucible of dynamic vapor sorption analyzers "Aquadyne DVS"; (d) Dependency of effective complex permittivity and penetration depth of microwaves on adsorption water uptake at 2.45 GHz. Figure 2. Characteristics of composite (honeycomb cellulose, adsorbent, and binder) desiccant structure. (a) SEM image of desiccant cross-section; (b) SEM image of desiccant surface; (c) Isotherms of composite desiccant structure. References MCOP Irradiation duration (min) Desorbed water mass (g) Microwave energy (kJ) Roussy et al.17 0.09* 1.66 0.67 17.5 Polaet et al.18 0.018* 130 2.73 390 Kubota et al.22 0.22 – 4 47 References MCOP Irradiation duration (min) Desorbed water mass (g) Microwave energy (kJ) Roussy et al.17 0.09* 1.66 0.67 17.5 Polaet et al.18 0.018* 130 2.73 390 Kubota et al.22 0.22 – 4 47 Table 1. A comparison of microwave dehumidification systems that are available in the literature. MCOP refers to the ratio of desorbed enthalpy of water vapor to irradiated microwave energy. MCOP values were calculated or obtained from the literature. Calculation of MCOP for literature is provided in supplementary materials. *calculated values. https://doi.org/10.1038/s41598-023-34542-9 Scientific Reports | (2023) 13:7386 | www.nature.com/scientificreports/ further model microwave processes and develop emerging technologies. A detailed explanation of the system is provided in the following sections. further model microwave processes and develop emerging technologies. A detailed explanation of the system is provided in the following sections. www.nature.com/scientificreports/ (dielectric properties) of a composite desiccant material with different adsorption uptakes. Penetration depth is the depth at which the electric field is reduced to 37% of its entrance value in the medium. It should be noted that microwaves can penetrate to the center of the desiccant wheel as the wheel’s radius (0.224 m) is lower than the penetration depth at the operating range of adsorption uptake (0.05–0.2). As the amount of adsorbed water reduces, the microwave’s penetration depth is increased with decreasing in the desiccant wheel’s complex permittivity. Moreover, it reveals that the penetration depth varies little at the operating range of adsorption uptake (0.05–0.02), which helps in simulation simplification. Chamber design and optimization. Three global optimizations were done for 3 cases (cylindrical, rectangular, and hopper shape) with random initial control variables, as explained in the section “Methods” (subsection “Multi-objective optimization of the microwave chamber”). Figure  3a shows the evolution of reflected power and low electric field ratio for the most optimal 3 cases of global optimization with a random initial control variable. Moreover, within 3 cases, case-3 was the most optimal. The optimum value of the control variable (illustrated in Fig. 6b) for case-3 was equal to 0.14 or c = 0.14. The difference in low electric field ratio was insignificant in 3 cases at optimal values. Results show that microwave emitting waveguide should be placed at the central part of the chamber than aside to get a more homogeneous (uniform) propagation of electric field, which corresponds to the lowest electric field ratio. The reflected power for case-3 is 44 W (0.7% of emitted power), which is the lowest value among other cases. The low reflection cannot be explained by 4 Reports | (2023) 13:7386 | https://doi.org/10.1038/s41598-023-34542-9 Figure 3. Optimization results of a microwave dehumidification system. (a) Reflected power and low electric field ratio for the most optimal 3 cases of global optimization with random initial control variable; (b) Streamline of the “Poynting” vector of microwaves at the cross-section. The desiccant wheel is embedded in the cylindrical microwave chamber. The wavelength of the microwave is 0.122 m. The radius of the cylinder is r = 0.5 m. The color scale refers to the normalized value of S. (c) Objective functions space for multi-objective global optimization with random initial control values. Figure 3. Optimization results of a microwave dehumidification system. Results A composite desiccant sample on the crucible of dynamic vapor sorption analyzers "Aquadyne DVS"; (d) Dependency of effective complex permittivity and penetration depth of microwaves on adsorption water uptake at 2.45 GHz. Scientific Reports | (2023) 13:7386 | https://doi.org/10.1038/s41598-023-34542-9 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Experiments were carried out in two modes: without heat recovery and with heat recovery (from the outflow of purged air).hfif Experimental results. Based on the optimization results, the system with an efficient multi-mode chamber was built by the authors. Experiments were carried out in two modes: without heat recovery and with heat recovery (from the outflow of purged air).hfif l The most efficient one (maximum COP) within the experimental results (different microwave irradiation, air flow rate) were demonstrated in Fig. 4 a,b for each mode. Figure 4a shows temperature and humidity ratio profiles at the inlet and outlet of the system without heat recovery. Microwave radiation time was equal to 17 min. However, the desorption time was longer than the radiation time due to the residual energy (thermal mass of the desiccant wheel). Inlet air temperature was constant during the adsorption and desorption cycles, and it was equivalent to 24 °C. Similarly, the inlet humidity ratio (ω) was equal to 10.3 g/kg throughout the experiment. As presented in Fig. 4a, the temperature of the desiccant wheel increased sharply at the start of microwave emission. The outlet air temperature increased over time, but its temperature was lower than the wheel temperature. h p p p It proved that energy was transported by microwaves directly to adsorbed water. Consequently, pressure on the surface of the adsorbent increased, raising the outlet value of the humidity ratio to 43 g/kg. Nevertheless, the humidity ratio started declining after 500 s and reached 40 g/kg at the stopping time of microwave irradiation. As the airflow rate during the desorption was constant and equal to 185 ­m3/h, decreasing the outlet humidity ratio evidently reduced the system’s performance. The outlet humidity ratio was meager due to the thermal mass of adsorbed water and desiccant at the starting time of microwave emission. The thermal mass effect needs more microwave emission time. However, the decreasing trend of the humidity ratio demonstrates that it cannot be very long. 2 kg of water was desorbed for the current mode during the desorption cycle, showing that many water vapors can be captured and turned into potable water or used to run IEC systems. The COP of the system was 0.55, and MCOP was 0.83. The temperature of the desiccant wheel did not go very high, which proves the excellent distribution of the electric field obtained by numerical results. www.nature.com/scientificreports/ (a) Reflected power and low electric field ratio for the most optimal 3 cases of global optimization with random initial control variable; (b) Streamline of the “Poynting” vector of microwaves at the cross-section. The desiccant wheel is embedded in the cylindrical microwave chamber. The wavelength of the microwave is 0.122 m. The radius of the cylinder is r = 0.5 m. The color scale refers to the normalized value of S. (c) Objective functions space for multi-objective global optimization with random initial control values. Figure 3. Optimization results of a microwave dehumidification system. (a) Reflected power and low electric field ratio for the most optimal 3 cases of global optimization with random initial control variable; (b) Streamline of the “Poynting” vector of microwaves at the cross-section. The desiccant wheel is embedded in the cylindrical microwave chamber. The wavelength of the microwave is 0.122 m. The radius of the cylinder is r = 0.5 m. The color scale refers to the normalized value of S. (c) Objective functions space for multi-objective global optimization with random initial control values. Scientific Reports | (2023) 13:7386 | https://doi.org/10.1038/s41598-023-34542-9 www.nature.com/scientificreports/ the position of the waveguide as the reflected power increases during optimization to 914 W (15% of emitted power). Nevertheless, circular geometry had the lowest microwave power reflection during the optimization. Optimization results demonstrate that the shape of the chamber has a significant impact on performance than the position of the waveguide. Figure 3b illustrates streamlines of the Poynting vector of microwaves at the cross- section of the chamber. These streamlines indicate the propagation path of microwaves. The color scale refers to the normalized value of S (power flow). According to the results, most of the microwave power was adsorbed during the reflection (bouncing) of the microwaves inside the chamber. Owing to the parabolic form (which can be seen at the cross-section), the metal cover of the chamber in case-3 prevents returning of microwave rays back to the magnetron. Figure 3c shows objective function space, where we can also spot the optimum values for 3 cases located near the optimum front line, which is also called the Pareto front. The sum of objective functions at case-3 (cylindrical shape) reached extremum value, so the global optimum condition belongs to case-3 with c = 0.14 m. Experimental results. Based on the optimization results, the system with an efficient multi-mode chamber was built by the authors. www.nature.com/scientificreports/ Hot spots or decreasing performance of the system were not observed due to the continuous operation of the stirrer at the center of the desiccant wheel and wheel rotation that made the system safe and sustainable. Moreover, the desiccant temperature did not exceed 80 °C. Nevertheless, some transported energy is wasted by heating the outlet temperature to 51 °C, so it is motivated to consider the mode with heat recovering from hot outlet air to inlet air by the heat exchanger. Figure 4b presents temperature and humidity ratio profiles for microwave desorption with a heat recovering mode. A schematic diagram of the mode is shown in Fig. 7b. Microwaving time was equal to 12 min 20 s, and the airflow rate was 140 ­m3/h. Inlet air temperature increased due to heat recovering from the outlet temperature. Besides, the temperature profile was different from the temperature profile without heat recovery; notably, the outlet air temperature reached 51 °C in a shorter time than the previous mode. As a result, the system has Figure 4. Experimental results of a microwave dehumidification system. (a) Humidity ratio and temperature for non-heat recovery mode; (b) Humidity ratio and temperature for heat recovery mode. Experimental results correspond to the maximum COP value of each mode. Figure 4. Experimental results of a microwave dehumidification system. (a) Humidity ratio and temperature for non-heat recovery mode; (b) Humidity ratio and temperature for heat recovery mode. Experimental results correspond to the maximum COP value of each mode. Scientific Reports | (2023) 13:7386 | https://doi.org/10.1038/s41598-023-34542-9 www.nature.com/scientificreports/ the highest COP than other modes, and COP is equal to 0.58, and MCOP is equivalent to 0.87. Moreover, high performance can be seen from the humidity ratio profile that increased with time until microwave irradiation was stopped. Compared with the non-heat recovering mode, some wasted heat is used efficiently, thus increasing system performance. Around 1.54 kg of water vapor was desorbed from the desiccant wheel during the desorption process. Figure 5b demonstrates system performance and amount of desorbed water for the different duration (3.5–17 min) of microwave emission for both modes. The desorbed amount of water had almost linear dependence from time. Results show that COP increases with the duration increasing of microwave irradiation for non-heat recovery modes because of the thermal mass of saturated composite desiccant. www.nature.com/scientificreports/ At the beginning of microwave radiation, some portion of energy was used for rapid heating of the saturated desiccant wheel from 24 °C to 48 °C (Fig. 4a), so COP at the short time was low. Running the microwave longer, we can reduce the effect of thermal mass and increase the COP of the system. However, microwave irradiation was not more than 17 min as most of the water was desorbed (adsorption uptake was 0.03). www.nature.com/scientificreports/ www.nature.com/scientificreports/ recovery modes was more elevated than for non-heat recovery modes. The performance of microwave desorption should be considered as the general coefficient of performance (COP) and microwave power-based coefficient of performance (MCOP) by the following equation: (1) MCOP = m ∗hfg Emw (2) COP = m ∗hfg Pelec (1) MCOP = m ∗hfg Emw MCOP = m ∗hfg Emw (1) (2) COP = m ∗hfg Pelec (2) where m is desorbed (water) moisture mass in [kg], hfg is a heat of evaporation in [J/kg], Emw is microwave energy emitted from the magnetron in [J], and Pelec is electrical energy consumed in [J]. Dehumidification by desiccant always works as a cycle (adsorption and desorption), so the amount of adsorbed and desorbed water mass are the same. To focus on the performance of microwave dehumidification in the calculation, desorbed water mass was used. Hence the conversion efficiency η is 0.7, Fig. 5a illustrates the difference between MCOP and COP in the methodology of estimation. Figure 5c shows a comparison in MCOP for different authors with microwave desorption. It can be seen that the current system that was developed, designed, and built by authors at King Abdullah University of Science and Technology (KAUST) has the highest COP and MCOP. It verifies that numerical optimization of chamber shape and obtaining the best operating conditions can overcome both limitations. Quantum jump was achieved in microwave dehumidification, with a fourfold increase in the MCOP, up to 0.87, as compared with available literature. Improving the microwave-based MCOP allowed us to cross- compare the microwave performances of assorted authors. Another issue was not considering microwaving time on the performance of the system.h p y The system was improved to commercial large system performance. It shows that building a microwave system needs an understanding of the physics and mechanics of electromagnetic waves, which can help design a system with high safety standards and high performance. The role of numerical optimization methods is crucial, as well as experiments. Conclusion A laboratory-scale microwave dehumidifier pilot was successfully tested to achieve a fourfold improvement in the MCOP up to 0.87 compared with available literature. These dehumidification improvements were attributed to the better waveguide and chamber design, demonstrating the sensitivity of water molecules desorption (dehumidification) to microwave energy delivery. However, the parasitic losses of electricity conversion to microwave, around 30% of electricity input, have resulted in an overall COP of 58%. Hence, there is much room for the electrical technology of microwave generation research, should a sustainable microwave dehumidifier COP of 75% is desired. Methods h f Theory of microwave dehumidification. Van der Waals force (or energy sites) attract water molecules onto the surface of sold desiccant material during dehumidification (Fig. 1a). Adsorbed water can be removed by pressure ­swing26,27 and thermal ­swing12,28. It is the most energy-consuming process in ­dehumidification29. On the other hand, attraction forces have electrostatic behavior, so oscillating dipole-structured water molecules with electromagnetic waves (microwaves) could detach from the surface faster and with less energy compared to the above-mentioned methods. Microwave-assisted desorption is an emerging method (Fig. 1b), where two desorption mechanisms are applied: the direct microwave effect on molecules (selective energy transport) and the thermal microwave ­effect23. It does not require heating of purged air stream to transport energy as in thermal swing; instead, the energy is transported directly to the water ­molecule23. Microwaves are electromagnetic waves ranging from about 1 m to 0.001 m (with frequencies between 0.3 GHz and 300 GHz)30, and like all electromagnetic waves, it obeys Maxwell’s equation systems. The time-harmonic electromagnetic field can be represented by the following differential equation that is obtained from Maxwell equation systems by applying a frequency-domain approach: (3) ∇×  ∇× E  −k2 0  εr,eff  E = 0, (3) where ∇ (nabla) is a vector differential operator, −→ E (Ex, Ey, Ez) is the vector field of an electric field in [V/m], k0 is wavenumber in [rad/m]. εr,eff is effective complex permittivity, and it has real and complex components, as shown by the following equation: where ∇ (nabla) is a vector differential operator, −→ E (Ex, Ey, Ez) is the vector field of an electric field in [V/m], k0 is wavenumber in [rad/m]. εr,eff is effective complex permittivity, and it has real and complex components, as shown by the following equation: (4) εr,eff= ε ′ r,eff+ iε ′′ r,eff (4) where ε" r,eff is the real part of effective complex permittivity (dielectric constant), ε" r,eff is the imaginary part of effective complex permittivity (dielectric loss factor). In simulations, averaged microwave power consumed by the desiccant wheel is calculated according to the Poynting equation: Pmw = Vπf ε0ε" r,effE2 , where Pmw is the microwave power in [W], V is the desiccant wheel’s volume in ­[m3], f is the microwave’s frequency in [Hz], ε0 is the free space permittivity in [F/m]. Another important parameter is the time-averaged vector field ( −→S  ), which showed the power flow and microwave direction. Discussion Not focusing on the performance of microwave dehumidification was the gap in research hitherto. Since simulation and experiment were targeted at getting the maximum performance, the most efficient microwave dehumidification system was developed with COP of 0.58 and MCOP of 0.87. MCOP of 0.87 means most of the microwave energy was delivered to water molecules directly, with the least internal entropy generation and low reflected power to the magnetron. Internal entropy generation is attributed to uneven heating within the volume. It occurs in high values region of “low electric field ratio.” Reflected microwave power was at the lowest value (0.5–3% of input microwave power), which has a tremendous impact on microwave dehumidification performance. The microwave generator’s conversion efficiency, which turns electrical energy into microwave energy, was 0.7, which was accountable for the COP of 0.58 when MCOP was as high as 0.87. However, COP was the highest within the available literature. The highest COP (0.58) for heat recovery modes corresponds to the time when the humidity ratio reaches the highest value. Recovering heat can increase performance, but heat recovery has less effect for a short time or a long time. Meanwhile, the desorbed amount of water for heat Figure 5. Performance of microwave dehumidification system. (a) Methodology for calculation of MCOP to compare the efficiency of different authors’ microwave dehumidification systems and their difference from COP calculation concept. (b) COP and desorbed amount of water vs. duration of microwave emission. (c) Comparison of dehumidification performance with reference authors with current advance, which is 4 times higher than other authors. Figure 5. Performance of microwave dehumidification system. (a) Methodology for calculation of MCOP to compare the efficiency of different authors’ microwave dehumidification systems and their difference from COP calculation concept. (b) COP and desorbed amount of water vs. duration of microwave emission. (c) Comparison of dehumidification performance with reference authors with current advance, which is 4 times higher than other authors. https://doi.org/10.1038/s41598-023-34542-9 Scientific Reports | (2023) 13:7386 | www.nature.com/scientificreports/ cellulose) are almost transparent to microwave radiation; hence, microwave energy is solely focused on ejecting the water molecules from the pores of the adsorbent. cellulose) are almost transparent to microwave radiation; hence, microwave energy is solely focused on ejecting the water molecules from the pores of the adsorbent. Effective complex permittivity and penetration depth. The honeycomb-based adsorbent wheel permits airflow through its channeled voids. For accurate modeling, it is necessary to obtain the effective complex permittivity of the honeycomb wheel, which is a function of complex permittivity of air and desiccant materials (silica gel, binder, cellulose), that ­is31: (5) εr,eff= fopεr,air + fcdεr,cd = fop  ε ′ r,air  + fcd  ε ′ r,cd + iε ′′ r,cd (5) where fop is the volume fraction of air in the openings (honeycomb), and fcd = 1-fop is the volume fraction of composite desiccant. The penetration depth of microwaves is also calculated with effective complex permittivity by the following ­formula23: where fop is the volume fraction of air in the openings (honeycomb), and fcd = 1-fop is the volume fraction of composite desiccant. The penetration depth of microwaves is also calculated with effective complex permittivity by the following ­formula23: (6) Dp = 0 2π  2ε ′ r,eff  1         1 +  ε′′ r,eff ε′ r,eff 2 −1   (6) where Dp is the penetration depth of microwaves in [m] and 0 is the wavelength of microwaves in [m]. Multi‑objective optimization of the microwave chamber. Optimization was conducted, minimizing the reflected microwave power and unheated areas to increase heating performance within the honeycomb wheel. Three multi-objective optimizations with the weighted sum of objectives were carried out as three shapes were considered, namely, (i) the first is a rectangular block chamber with a pyramidal hopper-shaped side, (ii) the second is a rectangular block, and (iii) a cylindrical chamber. The control variables were denoted by a,b,c,d and displayed in Fig. 6a. Global optimum value was obtained with random initial control values within the constraining range. Moreover, the optimization was constrained by the chamber’s geometry, with wheel dimensions kept constant at a radius of 0.224 m and height of 0.4 m (Fig. 6b). www.nature.com/scientificreports/ For mathematical modeling, the following assumptions were used: 1) The complex permittivity and the effective complex permittivity of honeycomb material are homogeneous and isotropic; 2) The perforated metal sheet was assumed to have the same reflective characteristics as the non-perforated one due to the much smaller perforation hole diameter (4 mm) than the wavelength of microwaves (124 mm). For the design of the waveguide and chamber, Eq. (1) was solved to obtain the electric field (V/m) subjected to the boundary conditions. At the entrance of the waveguide (from the magnetron), the electric field of the x-direction is designed according to Eq. (7) whilst the corresponding values in the y and z directions are ­zero32: (7) (7) Ex =  Pin/Pmode∗sin  w −y  /w∗pi  /w, Ey = 0, Ez = 0. Equation (7) would satisfy the microwave irradiation classified as the ­TE10 mode under the standard industrial waveguide (WR340) at a frequency (f) of 2.45 GHz. The assumption of the perfect electrical conductor was applied for all walls and perforated sheets, where tangential components of the electric field were set equal to zero: Equation (7) would satisfy the microwave irradiation classified as the ­TE10 mode under the standard industrial waveguide (WR340) at a frequency (f) of 2.45 GHz. The assumption of the perfect electrical conductor was applied for all walls and perforated sheets, where tangential components of the electric field were set equal to zero: (8) (8) n × E = 0 n × E = 0 The computation region consists of two domains (Fig. 6b) because air (gray) and the desiccant wheel (red) have different effective complex permittivity. The Nelder-Mead algorithm was used for optimization calculations. Nelder–Mead algorithm is a nonlinear optimization method that uses the simplex concept. At each iteration, a new vertex is defined by the four operations known as reflection, expansion, contraction, and shrinkage. The value of the objective function at n + 1 vertex of a simplex is calculated as it is moved toward the minimum ­point33. Two objective functions were defined such as reflected power ratio and low electric field ratio: (9) fp = wc ∗Prefl/Pin and flow = ∫ Vhmpz ElowdV/Vhmpz (9) eighting coefficient ( wc ) was equal to 5 as usually 20% of microwave power was reflected back. where the weighting coefficient ( wc ) was equal to 5 as usually 20% of microwave power was reflected back. Methods h f −→S = −→ E × −→ H∗ , where −→ H∗ is the vector field of the magnetic field and complex conjugate. Silica gel was chosen among adsorbents and coated on a cellulose-based honeycomb structured wheel to achieve a high surface area per unit volume of the wheel. These materials (silica gel and where ε" r,eff is the real part of effective complex permittivity (dielectric constant), ε" r,eff is the imaginary part of effective complex permittivity (dielectric loss factor). In simulations, averaged microwave power consumed by the desiccant wheel is calculated according to the Poynting equation: Pmw = Vπf ε0ε" r,effE2 , where Pmw is the microwave power in [W], V is the desiccant wheel’s volume in ­[m3], f is the microwave’s frequency in [Hz], ε0 is the free space permittivity in [F/m]. Another important parameter is the time-averaged vector field ( −→S  ), which showed the power flow and microwave direction. −→S = −→ E × −→ H∗ , where −→ H∗ is the vector field of the magnetic field and complex conjugate. Silica gel was chosen among adsorbents and coated on a cellulose-based honeycomb structured wheel to achieve a high surface area per unit volume of the wheel. These materials (silica gel and https://doi.org/10.1038/s41598-023-34542-9 Scientific Reports | (2023) 13:7386 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ (10) Elow =  1, if (Enorm < Ethreshold); 0, otherwise, (10) where Ethreshold was equal to 3000 V/m, which was discovered from the authors’ waveguide experiment. A low electric field ratio is needed to maintain uniform heating by microwaves. Control variables and their bounds for all cases are presented below: where Ethreshold was equal to 3000 V/m, which was discovered from the authors’ waveguide experiment. A low electric field ratio is needed to maintain uniform heating by microwaves. Control variables and their bounds for all cases are presented below: p Case-1: 0.5[m] > h > 0.005[m]; Case-2: 0.2[m] > a > 0, 0.25[m] > b > 0; Case-3: 0.2[m] > c > 0.h COMSOL Multiphysics computational platform was used to perform optimization. The system of equations was solved with FGMRES Iterative Solver, which uses the restarted flexible generalized minimum residual method. Mesh built of (minimum) 2,251,507 tetrahedral domain elements and 73,048 triangular boundary elements. https://doi.org/10.1038/s41598-023-34542-9 Scientific Reports | (2023) 13:7386 | www.nature.com/scientificreports/ Figure 6. Numerical optimization domains and geometry of microwave chamber. (a) Microwave chambers with 3 different shapes, namely, a rectangular block shape with a pyramidal hopper-shaped side, a rectangular block shape, and a cylindrical shape. Microwave emitting waveguide positions variables a,b,c,d. which are used as control variables in optimization; (b) Positioning of the desiccant wheel in the 3 different chambers, where the air is colored gray, and the desiccant wheel is colored red to show different domains with different properties for simulation in 3 cases. Figure 6. Numerical optimization domains and geometry of microwave chamber. (a) Microwave chambers with 3 different shapes, namely, a rectangular block shape with a pyramidal hopper-shaped side, a rectangular block shape, and a cylindrical shape. Microwave emitting waveguide positions variables a,b,c,d. which are used as control variables in optimization; (b) Positioning of the desiccant wheel in the 3 different chambers, where the air is colored gray, and the desiccant wheel is colored red to show different domains with different properties for simulation in 3 cases. Experimental apparatus. A microwave generator (Fricke und Mallah, Germany) with an efficiency value of 0.7 for converting alternative current (AC) electrical (9 kW) power to microwave power was used to generate the microwaves. A magnetron head (with a circulator and a directional coupler) was used to generate microwaves and measure forwarded and reflected microwave power. www.nature.com/scientificreports/ A three-stub tuner (Fricke und Mallah, Germany) was used to tune the microwave phase to perform impedance matching for maximizing energy transfer. Frame and equipment were grounded with protective grounding to prevent users from high voltage electrical hazards. Experimental apparatus. A microwave generator (Fricke und Mallah, Germany) with an efficiency value of 0.7 for converting alternative current (AC) electrical (9 kW) power to microwave power was used to generate the microwaves. A magnetron head (with a circulator and a directional coupler) was used to generate microwaves and measure forwarded and reflected microwave power. A three-stub tuner (Fricke und Mallah, Germany) was used to tune the microwave phase to perform impedance matching for maximizing energy transfer. Frame and equipment were grounded with protective grounding to prevent users from high voltage electrical hazards. q p g p g g p g g A specially made double Faraday cage was used in order to avoid microwave leakage to the surrounding area. The first cage is a multi-mode chamber where a desiccant wheel was placed, and its geometry was built based on numerically optimized results (more detailed information about numerical optimization is provided in Sects. 4 and 5). The first cage was placed in the second cage, and both cages are made of a 3 mm thick aluminum sheet. Aluminum has low resistance and high microwave reflective characteristics. An electrical motor (DKM motors, Korea) was used to rotate a desiccant wheel with a preset speed to control the microwave process. A three-phase electrical power meter PowerLogic PM5110 (Schneider Electric, UK), with a measuring range of 3–30 kW and with (0.5% FS) accuracy, was used to measure electrical power. A calibrated aluminum nozzle was used for airflow measurements that were made according to the ISO/ANSI standards with high accuracy (RecoV, Italy). A differential pressure sensor Model 264 (Setra, USA) with high (± 0.25% FS) accuracy and with a range of 0–250 Pa was used to measure differential pressure across the nozzle. Pt100 RTD temperature sensors (Omega, UK) were used to measure dry and wet bulb temperatures with an accuracy of ± 0.1 °C and with a range of − 20–350 °C. An infrared temperature sensor OS-MINIUSB (Omega, UK) with an accuracy of 1 °C was used to measure a rotating desiccant wheel temperature that can measure at the range of 0–250 °C. www.nature.com/scientificreports/ An automated data logging system was created by authors on the software LabVIEW that logs data from Agilent 34970A. An air heat exchanger was used to recover energy from outlet air, and air-flowing channels, ducts, and microwaving chamber were thermally insulated with foam rubber. A microwave leak detector was running all the time for safety reasons. A specially made double Faraday cage was used in order to avoid microwave leakage to the surrounding area. The first cage is a multi-mode chamber where a desiccant wheel was placed, and its geometry was built based on numerically optimized results (more detailed information about numerical optimization is provided in Sects. 4 and 5). The first cage was placed in the second cage, and both cages are made of a 3 mm thick aluminum sheet. Aluminum has low resistance and high microwave reflective characteristics. An electrical motor (DKM motors, Korea) was used to rotate a desiccant wheel with a preset speed to control the microwave process. A three-phase electrical power meter PowerLogic PM5110 (Schneider Electric, UK), with a measuring range of 3–30 kW and with (0.5% FS) accuracy, was used to measure electrical power. A calibrated aluminum nozzle was used for airflow measurements that were made according to the ISO/ANSI standards with high accuracy (RecoV, Italy). A diff i l M d l 264 (S USA) i h hi h ( 0 25% FS) d i h f l A differential pressure sensor Model 264 (Setra, USA) with high (± 0.25% FS) accuracy and with a range of 0–250 Pa was used to measure differential pressure across the nozzle. Pt100 RTD temperature sensors (Omega, UK) were used to measure dry and wet bulb temperatures with an accuracy of ± 0.1 °C and with a range of − 20–350 °C. An infrared temperature sensor OS-MINIUSB (Omega, UK) with an accuracy of 1 °C was used to measure a rotating desiccant wheel temperature that can measure at the range of 0–250 °C. An automated data logging system was created by authors on the software LabVIEW that logs data from Agilent 34970A. An air heat exchanger was used to recover energy from outlet air, and air-flowing channels, ducts, and microwaving chamber were thermally insulated with foam rubber. A microwave leak detector was running all the time for safety reasons. Scientific Reports | (2023) 13:7386 | https://doi.org/10.1038/s41598-023-34542-9 www.nature.com/scientificreports/ Figure 7. View of experimental microwave dehumidification pilot system. References Saputra, D. A. et al. Experimental investigation of desiccant wheel dehumidification control method for changes in regeneration h t i t E 205 118109 (2020) f g y 27. Carter, J. W. & Wyszynski, M. L. The pressure swing adsorption drying of compressed air. Chem. Eng. Sci. 38, 1093–1099 (1983). 28 Saputra D A et al Experimental investigation of desiccant wheel dehumidification control method for changes in regeneration 27. Carter, J. W. & Wyszynski, M. L. The pressure swing adsorption drying of compressed air. Chem. Eng. Sci. 38, 1093–1099 (1983).i 8. Saputra, D. A. et al. Experimental investigation of desiccant wheel dehumidification control method for changes in regeneration heat input. Energy 205, 118109 (2020). gy 29. Bathen, D. Physical waves in adsorption technology: An overview. Sep. Purif. Technol. 33, 163–177 (2003). 0. Lidström, P., Tierney, J., Wathey, B. & Westman, J. Microwave assisted organic synthesis: A review. Tetrahedron 57, 9225–9283 (2001).f 1. Zheng, Y., Meyer, G., Lanagan, M., Agrawal, D. & Cheng, J. A study of water sorption effects on the microwave dielectric properties of calcium chloride/silica gel composites. Mater. Lett. 95, 157–159 (2013). g p 32. Cheng, D. K. Field and Wave Electromagnetics (Addison-Wesley, 1991).f g p 32. Cheng, D. K. Field and Wave Electromagnetics (Addison-Wesley, 1991). 33. Ybyraiymkul, D., Ng, K. C. & Кaltayev, A. Experimental and numerical study of effect of thermal management on storage capacity of the adsorbed natural gas vessel. Appl. Therm. Eng. 125, 523–525 (2017). Data availability The presented data is available from the corresponding author on reasonable request. The presented data is available from the corresponding author on reasonable request Received: 20 January 2023; Accepted: 3 May 2023 www.nature.com/scientificreports/ Mode with heat recovery is similar to mode without heat recovery; When the inlet and outlet Scientific Reports | (2023) 13:7386 | https://doi.org/10.1038/s41598-023-34542-9 www.nature.com/scientificreports/ temperatures became the same, air damper-1 and air damper-3 were closed, and air damper-2 and air damper-4 were opened to recover heat from outlet air. www.nature.com/scientificreports/ (a) Pictorial view of the microwave dehumidification system, composed of the control panel, magnetron head (where microwaves are generated), 3-stub tuner, microwave chamber, and desiccant wheel. Enlarged images show pitch in honeycomb structure; (b) Schematic diagram of the microwave dehumidification system. Heat recovery is affected by a heat exchanger between exhaust and inlet air. Figure 7. View of experimental microwave dehumidification pilot system. (a) Pictorial view of the microwave dehumidification system, composed of the control panel, magnetron head (where microwaves are generated), 3-stub tuner, microwave chamber, and desiccant wheel. Enlarged images show pitch in honeycomb structure; (b) Schematic diagram of the microwave dehumidification system. Heat recovery is affected by a heat exchanger between exhaust and inlet air. Desiccant (silica gel) captures moisture from the air (Fig. 1a). Then, the moisture in the desiccant is desorbed by microwaves (Fig. 1b). The key feature of microwaves is that they can oscillate water molecules and desorb from the adsorbent’s surface (silica gel). The lab-scale pilot microwave dehumidification system is illustrated in Fig. 7a, and its schematic diagram is shown in Fig. 7b. A microwave generator (Fricke und Mallah, Germany) was used to generate the microwaves. Frame and equipment were grounded with protective grounding to prevent users from high voltage electrical hazards. Two modes were considered: the mode without heat recovering and the mode with heat recovering from outlet air. Temperatures and differential pressure readings were logged continuously by software Labview and Agilent 34970A for both modes. The desiccant wheel rotating motor speed was set to the desired value, running only during desorption. Figure 7b demonstrates a setup diagram. The study performed the following procedure without heat recovery: Air damper-1 and air damper-3 were opened, and air damper-2 and air damper-4 were closed, letting the air bypass the heat-recovering device. Then, the honeycomb- structured desiccant wheel was saturated with moisture at constant relative humidity and temperature at a regular airflow rate until the inlet and outlet temperatures were the same: the same temperature and humidity indicated equilibrium conditions. Consequently, microwaves were switched on for the preset time and preset power from the control panel; the desorption process finishes when the outlet humidity ratio becomes lower than the inlet humidity ratio. References 1. Davis, R. E., McGregor, G. R. & Enfield, K. B. Humidity: A review and primer on atmospheric moisture and human health. Environ Res. 144, 106–116 (2016).ih 2. Mazzei, P., Minichiello, F. & Palma, D. HVAC dehumidification systems for thermal comfort: A critical review. Appl. Therm. Eng 25, 677–707 (2005).h 3. Standard 55 – Thermal Environmental Conditions for Human Occupancy. https://​www.​ashrae.​org/​techn​ical-​resou​rces/​books​tore/ stand​ard-​55-​therm​al-​envir​onmen​tal-​condi​tions-​for-​human-​occup​ancy. and​ard-​55-​therm​al-​envir​onmen​tal-​condi​tions-​for-​human-​occup​a p y 4. ISO - ISO 7730:2005 - Ergonomics of the thermal environment — Analytical determination and interpretation of thermal comfort using calculation of the PMV and PPD indices and local thermal comfort criteria. https://​www.​iso.​org/​stand​ard/​39155.​html.hhi g p g 5. Thu, K., Mitra, S., Saha, B. B. & Srinivasa Murthy, S. Thermodynamic feasibility evaluation of hybrid dehumidification: Mechanica vapour compression systems. Appl. Energy 213, 31–44 (2018). p p y pp gy 6. Marks, L. S. Mechanical engineers’ handbook (McGraw-Hill Book Company, 1916). 7. Jamil, M. A. et al. Experimental and normalized sensitivity based numerical analyses of a novel h indirect evaporative cooler. Int. Commun. Heat Mass Transf. 125, 105327 (2021). p f 8. Muttakin, M., Pal, A., Rupa, M. J., Ito, K. & Saha, B. B. A critical overview of adsorption kinetics for cooling and refrigeration systems. Adv. Colloid Interface Sci. 294, 102468 (2021).i p systems. Adv. Colloid Interface Sci. 294, 102468 (2021). y f 9. Sultan, M., El-Sharkawy, I. I., Miyazaki, T., Saha, B. B. & Koyama, S. An overview of solid desiccant dehumidification and air conditioning systems. Renew. Sustain. Energy Rev. 46, 16–29 (2015).i 10. Shamim, J. A., Hsu, W. L., Paul, S., Yu, L. & Daiguji, H. A review of solid desiccant dehumidifiers: Current status and near-term development goals in the context of net zero energy buildings. Renew. Sustain. Energy Rev. 137, 110456 (2021). 11. Gao, D. C., Sun, Y. J., Ma, Z. & Ren, H. A review on integration and design of desiccant air-conditioning systems for overall performance improvements. Renew. Sustain. Energy Rev. 141, 110809 (2021).i 2. Abd-Elhady, M. M., Salem, M. S., Hamed, A. M. & El-Sharkawy, I. I. Solid desiccant-based dehumidification systems: A critica review on configurations, techniques, and current trends. Int. J. Refrig. 133, 337–352 (2022). i 3. Shabir, F. et al. Recent updates on the adsorption capacities of adsorbent-adsorbate pairs for heat transformation applications Renew. Sustain. Energy Rev. 119, 109630 (2020). gy 4. Burhan, M. et al. References A universal mathematical methodology in characterization of materials for tailored design of porous surfaces Front. Chem. 8, 601132 (2021). 5. Burhan, M., Shahzad, M. W. & Ng, K. C. A universal theoretical framework in material characterization for tailored porous surface design. Sci. Rep. 9, 8773 (2019). , , , g, p design. Sci. Rep. 9, 8773 (2019). 6 Roussy G & Chenot P Selective energy supply to adsorbed water and nonclassical thermal process during microwave dehydration g p 6. Roussy, G. & Chenot, P. Selective energy supply to adsorbed water and nonclassical thermal process during microwave dehydration of zeolite. J. Phys. Chem. 85, 2199–2203 (1981).h y 7. Roussy, G., Zoulalian, A., Charreyre, M. & Thiebaut, J. M. How microwaves dehydrate zeolites. J. Phys. Chem. 88, 5702–5708 (1984).h 8. Polaert, I., Estel, L., Huyghe, R. & Thomas, M. Adsorbents regeneration under microwave irradiation for dehydration and volatile organic compounds gas treatment. Chem. Eng. J. 162, 941–948 (2010). g p g g 19. Tatsuo, O. & Akiko, W. Simple suppressing method of thermal runaway in microwave heating of zeolite and its application. PhysChemComm 4, 18–20 (2001). y 0. Ito, S. et al. Heat Transfer during microwave-assisted desorption of water vapor from zeolite packed bed. Dry. Technol. 30(15) 1707–1713. https://​doi.​org/​10.​1080/​07373​937.​2012.​714825 (2012). p g 1. Kubota, M., Hanada, T., Yabe, S., Kuchar, D. & Matsuda, H. Water desorption behavior of desiccant rotor under microwave irradiation. Appl. Therm. Eng. 31, 1482–1486 (2011). pph g 22. Kubota, M., Hanada, T., Yabe, S. & Matsuda, H. Regeneration characteristics of desiccant rotor with microwave and hot-air heating. Appl. Therm. Eng. 50, 1576–1581 (2013).fi h 3. Cherbański, R. Calculation of critical efficiency factors of microwave energy conversion into heat. Chem. Eng. Technol. 34, 2083– 2090 (2011). 4. Reuß, J., Bathen, D. & Schmidt-Traub, H. Desorption by microwaves: Mechanisms of multicomponent mixtures. Chem. Eng Technol. 25, 381–384 (2002). 5. Goyal, H., Mehdad, A., Lobo, R. F., Stefanidis, G. D. & Vlachos, D. G. Scaleup of a single-mode microwave reactor. Ind. Eng. Chem Res. 59, 2516–2523 (2020). 6. Shahzad, M. W., Ybyraiymkul, D., Burhan, M., Oh, S. J. & Ng, K. C. An innovative pressure swing adsorption cycle. in AIP Conference Proceedings vol. 2062 020057 (American Institute of Physics Inc., 2019).h f g ( y , ) 27. Carter, J. W. & Wyszynski, M. L. The pressure swing adsorption drying of compressed air. Chem. Eng. Sci. 38, 1093–1099 (1983). 28. Author contributions k d l D. Y. worked on conceptualization, methodology, analysis, investigation, modeling, validation, writing the original draft, and visualization. Q. C. worked on writing, reviewing, editing, formal analysis, and methodology. M. W. S. worked on writing, reviewing, editing, formal analysis and methodology. R. A. did a formal analysis. K. J. did a formal analysis. M. B. worked on investigation and visualization. F. H. A. worked on investigation and visualization. K. C. N. worked on conceptualization, methodology, formal analysis, writing the original draft, supervision, and funding acquisition. All authors reviewed the manuscript. Acknowledgementsh g The authors gratefully acknowledge the generous funding from (1) the KAUST Cooling Initiative (KCI) project, REP/1/3988-01-01, and REP/1/3988-04-01, (2) the Water Desalination and Reuse Center (WDRC), King Abdullah University of Science and Technology (KAUST). Figures 1a,b were drawn by D.Y. https://doi.org/10.1038/s41598-023-34542-9 Scientific Reports | (2023) 13:7386 | www.nature.com/scientificreports/ Competing interests h p g The authors declare no competing interests. © The Author(s) 2023 Additional informationh Additional information Supplementary Information The online version contains supplementary material available at https://​doi.​org/​ 10.​1038/​s41598-​023-​34542-9. Correspondence and requests for materials should be addressed to D.Y. Correspondence and requests for materials should be addressed to D.Y. 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. 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/. 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) 2023 https://doi.org/10.1038/s41598-023-34542-9 Scientific Reports | (2023) 13:7386 |
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Multi-Volume Hemacytometer
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Ravangnam Thunyaporn1, Il Doh2 & Dong Woo Lee1,3* Cell counting has become an essential method for monitoring the viability and proliferation of cells. A hemacytometer is the standard device used to measure cell numbers in most laboratories which are typically automated to increase throughput. The principle of both manual and automated hemacytometers is to calculate cell numbers with a fixed volume within a set measurement range ­(105 ~ ­106 cells/ml). If the cell concentration of the unknown sample is outside the range of the hemacytometer, the sample must be prepared again by increasing or decreasing the cell concentration. We have developed a new hemacytometer that has a multi-volume chamber with 4 different depths containing different volumes (0.1, 0.2, 0.4, 0.8 µl respectively). A multi-volume hemacytometer can measure cell concentration with a maximum of ­106 cells/ml to a minimum of 5 × ­103 cells/ml. Compared to a typical hemacytometer with a fixed volume of 0.1 µl, the minimum measurable cell concentration of 5 × ­103 cells/ml on the multi-volume hemacytometer is twenty times lower. Additionally, the Multi-Volume Cell Counting model (cell concentration calculation with the slope value of cell number in multi-chambers) showed a wide measurement range (5 × ­103 ~ 1 × ­106 cells/ ml) while reducing total cell counting numbers by 62.5% compared to a large volume (0.8 µl-chamber) hemacytometer. Cell counting is a method used to determine the concentration of cells in a sample volume. For example, in cell culture experiments counting cells is vital to monitor cell viability, proliferation rate, immortalization or transformation, seeding, or preparing cells in subsequent experiments, transfection, or ­infection1. Diagnosti- cally, the concentration of white blood cells and red blood cells can assist those in the medical field to identify specific diseases and can predict severity of disease states such as coronary ­atherosclerosis2–4, HIV detection from whole-blood5, 6, etc.hi Cell counting is a method used to determine the concentration of cells in a sample volume. For example, in cell culture experiments counting cells is vital to monitor cell viability, proliferation rate, immortalization or transformation, seeding, or preparing cells in subsequent experiments, transfection, or ­infection1. Diagnosti- cally, the concentration of white blood cells and red blood cells can assist those in the medical field to identify specific diseases and can predict severity of disease states such as coronary ­atherosclerosis2–4, HIV detection from whole-blood5, 6, etc.hi The hemacytometer was first invented over a century ago to count blood cells. Ravangnam Thunyaporn1, Il Doh2 & Dong Woo Lee1,3* Presently the hemacytometer remains the gold standard for cell counting and is used by most laboratories to count cells as it is both inexpen- sive and ­versatile7. A hemacytometer is made from optical glass for use under a microscope and consists of 2 parts: a thick glass slide and cover glass with a small gap to contain cell suspension in a 0.1 µl small grid area size 1 × 1 ­mm8. It cannot be accurately measured in high concentrations because the cells too crowned and difficult to count. The cells have to be diluted and counting ­again9. h g g Over the last two decades, automated hemacytometers have been developed to increase the throughput of counting a large number of samples in a short period of time. Automation also reduces human error in the cell counting process and automatic hemacytometers are commonly used in laboratories for these reasons. In general, hemacytometers are composed of a digital camera and a slide or cartridge containing the cell suspension to be counted. The digital camera captures cells on the slide in the fixed volume and analyzes them using specialized software for cell counters to calculate cell concentration based on the fixed ­volume6. Both manual and automated hemacytometers use a fixed volume to measure cell concentration. Though manual and automated hemacytom- eters are standard in most laboratories, they cannot measure low cell concentration samples and have a narrow measurement range ­(105 ~ ­106 cells/ml) due to the fixed volume containing cell. Most hemacytometers count cells in 0.1 µl fixed volume where one cell exists at ­104 cells/ml sample. The range of hemacytometer is approxi- mately ­105 cells/ml. For example, the Cedex HiRes Analyzer which is an automatic hemacytometer, can measure cell density ranging from 3.13 × ­105 to approximately 1.0 × ­107 cells/ml10. The optimal range of Cellometer cell counters is approximately 1 × ­105 to 1 × ­106 cells/ml11.hi pp y The previous study has shown that the size of the fixed control volume of cells in the suspension increases, the number of cells is also increased which can be extended to measure cells in a wide range of ­concentrations12, 13. The limit of the range of cell concentration measurement is due to the fixed volume of the sample which causes the minimum and maximum measurable cell concentration to decrease or increase. www.nature.com/scientificreports www.nature.com/scientificreports 1Department of Biomedical Engineering, Konyang University, Daejeon 35365, Republic of Korea. 2Korea Research Institute of Standards and Science, Daejeon, Korea. 3Central R & D Center, Medical & Bio Decision (MBD) Co., Ltd, Suwon, Republic of Korea. *email: mems@konyang.ac.kr Scientific Reports | (2021) 11:14106 Multi‑volume hemacytometer OPEN Ravangnam Thunyaporn1, Il Doh2 & Dong Woo Lee1,3* Ravangnam Thunyaporn1, Il Doh2 & Dong Woo Lee1,3* A typical 0.1 µl fixed vol- ume (most used volume in hemacytometers) contains 10 ~ 500 cells at ­105 ~ 5 × ­106 cells/ml while a 0.2 µl fixed volume contains 10 ~ 500 cells at 5 × ­104 ~ 2.5 × ­106 cells/ml. Therefore, if a sample of unknown concentration 1Department of Biomedical Engineering, Konyang University, Daejeon 35365, Republic of Korea. 2Korea Research Institute of Standards and Science, Daejeon, Korea. 3Central R & D Center, Medical & Bio Decision (MBD) Co., Ltd, Suwon, Republic of Korea. *email: mems@konyang.ac.kr | https://doi.org/10.1038/s41598-021-93477-1 www.nature.com/scientificreports/ Figure 1. Schematic view of the multi-volume hemacytometer. (a) The counting chamber was attached to a glass slide that had an inlet for pipetting and an outlet for airflow. The cell counting chamber consisted of four chambers of varying depths including 0.1, 0.2, 0.4, 0.8 mm respectively. (b) The PDMS fabrication device. (c) Counting cell number in each chamber are proportional to the volume of the chamber when a sample flowed in the chambers having different volumes. In case of low cell concentration sample, slope which mean cells per unit volume is small. However, in high cell concentration sample, the slop is large due to large different of counting cells per chamber. Figure 1. Schematic view of the multi-volume hemacytometer. (a) The counting chamber was attached to a glass slide that had an inlet for pipetting and an outlet for airflow. The cell counting chamber consisted of four chambers of varying depths including 0.1, 0.2, 0.4, 0.8 mm respectively. (b) The PDMS fabrication device. (c) Counting cell number in each chamber are proportional to the volume of the chamber when a sample flowed in the chambers having different volumes. In case of low cell concentration sample, slope which mean cells per unit volume is small. However, in high cell concentration sample, the slop is large due to large different of counting cells per chamber. is out of range of the hemacytometer, the sample must be prepared again by increasing or decreasing the cell concentration.h To solve this problem, the current study proposed the use of a multi-volume hemacytometer. This device counts cells in four chambers at volumes of 0.1, 0.2, 0.4, 0.8 µl as shown in Fig. 1. A multi-volume cell counter can measure cell concentrations ranging from ­106 cells/ml to 5 × ­103 cells/ml. Ravangnam Thunyaporn1, Il Doh2 & Dong Woo Lee1,3* Compared to a hemacytometer having a fixed volume of 0.1 µl, the minimum measurable cell concentration is 5 × ­103 cells/ml which is twenty times lower. Additionally, the Multi-Volume Cell Counting model (cell concentration calculation with the slope value of the cell number in multi-chambers) was also validated to measured cell concentration and increases the measurement speed as a sample of only 1.5 µl can measure the wide range of cell concentrations. Results To compare the accuracy and range of cell concentration measurement between the conventional hemacytom- eter and the proposed multi-volume hemacytometer, we measured five samples at ­103, 5 × ­103, ­104, ­105, and ­106 cells/ml. In the case of the multi-volume hemacytometer, there are two cell concentration calculation models necessary to determine the cell concentration. In the conventional Fixed-Volume Cell Counting (FVCC), the cell concentrations were calculated by counting cells in each chamber at different volumes. In the Multi-Volume Cell Counting (MVCC) model, the cell concentrations were calculated using the slope (cell number growth per volume) in the graph of cell number by volume as show in Fig. 1c. Fixed‑volume cell counting (FVCC) model. A hemacytometer has a ruled surface below the cover glass of 0.1 mm which limits the volume of liquid suspension to 0.1 µl per one of nine squares and restricts it to accu- Scientific Reports | (2021) 11:14106 | https://doi.org/10.1038/s41598-021-93477-1 www.nature.com/scientificreports/ Table 1. Comparison of cell concentration measurements between a standard hemacytometer and a multi- volume hemacytometer. *FVCC Model: Fixed-Volume Cell Counting Model. Results **MVCC Model: Multi-Volume Sample Concentration unit Hemacytometer (volume: 0.5 µl) Multi-volume hemacytometer FVCC Model* MVCC model ** (volume: 1.5 µl) Chamber 1 (volume: 0.5 µl) Chamber 2 (volume: 1 µl) Chamber 3 (volume: 2 µl) Chamber 4 (volume: 4 µl) Mean ± SD (%RSD) Mean ± SD (%RSD) Mean ± SD (%RSD) Mean ± SD (%RSD) Mean ± SD (%RSD) Fit values ± SD (%RSD) 1 106 cells/ml 1.08 ± 0.11 (9.7%) 1.14 ± 0.02 (1.6%) 0.99 ± 0.07 (7.0%) 1.14 ± 0.07 (5.7%) 1.02 ± 0.03 (3.3%) 1.04 ± 0.03 (2.5%) 2 1.18 ± 0.13 (10.8%) 1.14 ± 0.11 (9.3%) 0.94 ± 0.06 (6.7%) 1.08 ± 0.10 (9.6%) 1.14 ± 0.05 (4.5%) 1.12 ± 0.04 (3.6%) 3 1.05 ± 0.09 (8.5%) 1.07 ± 0.16 (14.9%) 1.09 ± 0.15 (17%) 1.08 ± 0.16 (14.4%) 1.07 ± 0.01 (1.3%) 1.07 ± 0.03 (3.1%) 4 1.16 ± 0.12 (10.5%) 0.88 ± 0.06 (6.5%) 0.94 ± 0.04 (4.6%) 1.08 ± 0.15 (14.0%) 1.12 ± 0.07 (6.5%) 1.10 ± 0.03 (3.0%) 5 1.19 ± 0.14 (11.3%) 1.12 ± 0.17 (15.2%) 0.92 ± 0.06 (6.8%) 0.94 ± 0.07 (7.2%) 1.05 ± 0.13 (13.3%) 1.03 ± 0.11 (10.4%) AVG 1.13 ± 0.06 1.07 ± 0.11 0.98 ± 0.08 1.07 ± 0.08 1.08 ± 0.06 1.07 ± 0.04 %RSD 5.61 10.51 6.89 6.86 4.54 3.64 1 105 cells/ml 1.38 ± 0.35 (25.3%) 1.26 ± 0.27 (21.4%) 1.14 ± 0.20 (17.4%) 0.94 ± 0.12 (11.5%) 1.22 ± 0.10 (8.3%) 1.17 ± 0.02 (1.5%) 2 1.24 ± 0.42 (34.0%) 1.12 ± 0.34 (30.5%) 1.07 ± 0.15 (14.3%) 1.18 ± 0.30 (25.7%) 1.26 ± 0.08 (6.4%) 1.23 ± 0.09 (7.7%) 3 1.20 ± 0.43 (35.8%) 1.16 ± 0.49 (42.1%) 1.28 ± 0.47 (36.9%) 1.20 ± 0.27 (22.3%) 1.21 ± 0.11 (9.0%) 1.21 ± 0.11 (9.4%) 4 1.22 ± 0.35 (28.6%) 1.16 ± 0.43 (37.4%) 1.21 ± 0.19 (15.3%) 1.28 ± 0.29 (22.5%) 1.26 ± 0.12 (9.6%) 1.26 ± 0.13 (10.2%) 5 1.20 ± 0.53 (44.1%) 1.00 ± 0.16 (15.8%) 1.28 ± 0.27 (21.1%) 1.13 ± 0.23 (19.9%) 1.30 ± 0.10 (8.0%) 1.26 ± 0.10 (8.1%) AVG 1.25 ± 0.08 1.14 ± 0.09 1.20 ± 0.09 1.15 ± 0.13 1.25 ± 0.04 1.23 ± 0.04 %RSD 6.06 8.23 7.63 11.10 6.06 3.17 1 104 cells/ml 1.40 ± 1.14 (81.4%) 1.40 ± 1.14 (81.4%) 1.20 ± 0.57 (47.5%) 1.15 ± 0.14 (11.9%) 1.18 ± 0.14 (12.1%) 1.17 ± 0.10 (8.7%) 2 1.00 ± 0.71 (70.7%) 1.00 ± 1.00 (100%) 1.10 ± 0.65 (59.3%) 1.15 ± 0.42 (36.4%) 1.20 ± 0.19 (15.8%) 1.22 ± 0.12 (9.5%) 3 0.60 ± 0.55 (91.3%) 1.40 ± 0.89 (63.9%) 1.20 ± 0.57 (47.5%) 1.15 ± 0.38 (33.0%) 1.15 ± 0.27 (23.6%) 1.16 ± 0.22 (19.0%) 4 1.00 ± 0.71 (70.7%) 1.20 ± 0.83 (69.7%) 1.20 ± 0.57 (47.5%) 1.10 ± 0.29 (25.9%) 1.28 ± 0.29 (22.4%) 1.24 ± 0.18 (14.3%) 5 1.00 ± 0.71 (70.7%) 0.80 ± 0.45 (55.9%) 1.00 ± 0.50 (50.0%) 1.15 ± 0.52 (45.1%) 1.18 ± 0.59 (22.1%) 1.16 ± 0.23 (19.9%) AVG 1.00 ± 0.28 1.16 ± 0.26 1.14 ± 0.09 1.14 ± 0.02 1.20 ± 0.05 1.19 ± 0.04 %RSD 28.28 22.48 7.85 1.96 4.02 3.19 1 5 × ­103 cells/ml 4.00 ± 5.58 (136.9%) 8.00 ± 4.47 (55.9%) 2.00 ± 2.74 (136.9%) 7.00 ± 3.26 (46.6%) 5.50 ± 1.43 (25.9%) 5.65 ± 1.27 (22.4%) 2 6.00 ± 5.48 (91.3%) 4.00 ± 8.94 (223.6%) 9.00 ± 9.62 (106.9%) 6.00 ± 2.85 (47.5%) 5.25 ± 2.85 (54.3%) 5.54 ± 3.06 (55.3%) 3 2.00 ± 4.47 (223.6%) 4.00 ± 8.94 (223.6%) 4.00 ± 4.18 (104.6%) 7.50 ± 2.50 (33.3%) 6.00 ± 1.63 (27.2%) 6.16 ± 1.17 (19.0%) 4 2.00 ± 4.47 (223.6%) 6.00 ± 8.94 (149.1%) 7.00 ± 8.37 (119.5%) 6.00 ± 1.37 (22.8%) 6.25 ± 1.77 (28.3%) 6.24 ± 1.67 (28.8%) 5 8.00 ± 8.37 (104.6%) 2.00 ± 4.47 (223.6%) 6.00 ± 2.24 (37.3%) 5.50 ± 2.09 (38.0%) 5.25 ± 2.05 (39.1%) 5.29 ± 1.90 (36.0%) AVG 4.40 ± 2.61 4.80 ± 2.28 5.60 ± 2.70 6.40 ± 0.82 5.65 ± 0.45 5.78 ± 0.41 %RSD 59.27 47.51 48.25 12.84 8.04 7.06 1 103 cells/ml 0 0 0 1.50 ± 1.37 (91.3%) 1.50 ± 0.56 (37.3%) 1.41 ± 0.33 (23.6%) 2 2.00 ± 4.47 (223.6%) 0 0 1.50 ± 1.37 (91.3%) 1.25 ± 0.88 (70.7%) 1.22 ± 0.85 (69.9%) 3 4.00 ± 5.48 (136.9%) 2.00 ± 4.47 (223.6%) 1.00 ± 2.24 (223.6%) 2.00 ± 1.77 (70.7%) 2.00 ± 0.68 (34.2%) 2.05 ± 0.83 (40.6%) 4 2.00 ± 4.47 (223.6%) 2.00 ± 4.47 (223.6%) 1.00 ± 2.24 (223.6%) 2.50 ± 3.26 (163%) 1.75 ± 1.43 (81.4%) 1.76 ± 0.70 (39.4%) 5 0 0 1.00 ± 2.24 (223.6%) 2.50 ± 3.06 (122%) 1.75 ± 2.43 (81.4%) 1.84 ± 0.89 (48.3%) AVG 1.60 ± 1.67 0.80 ± 1.10 0.60 ± 0.55 2.00 ± 0.50 1.65 ± 0.29 1.66 ± 0.33 %RSD 104.58 136.93 91.28 25.00 17.28 20.10 Table 1. Table 1.   Comparison of cell concentration measurements between a standard hemacytometer and a multi- volume hemacytometer. *FVCC Model: Fixed-Volume Cell Counting Model. **MVCC Model: Multi-Volume Cell Counting Model. Bold values Cell concentration measurement quality control pass (%RSD < 15%). Results Microscopic cell images in multiple volumes (0.1, 0.2, 0.4, and 0.8 µl) in (a) low concentration at ­10 cells/ml, and (b) high concentration at ­106 cells/ml liquid sample (under 40 × magnification). Figure 2. Microscopic cell images in multiple volumes (0.1, 0.2, 0.4, and 0.8 µl) in (a) low concentration at ­104 cells/ml, and (b) high concentration at ­106 cells/ml liquid sample (under 40 × magnification). chamber 1 in area 1 ­mm2 contained a volume of 0.1 µl equal to the volume of the standard hemacytometer. In the low volume chamber, the number of cells was not great enough to calculate the cell concentration. In con- trast, as the volume of the chamber increased, the cells also increased enough to allow the cell concentration to be calculated. Therefore, chamber 4 has a wide measurement range and was able to be measured at the lowest concentration at 5 × ­103 cells/ml. However, in the maximum cell concentration of ­106 cells/ml, it was difficult to count a high number of cells in a large volume (0.8 µl) and repeat the measurement five times. Therefore, the user must select the appropriate chamber volume according to the sample. chamber 1 in area 1 ­mm2 contained a volume of 0.1 µl equal to the volume of the standard hemacytometer. In the low volume chamber, the number of cells was not great enough to calculate the cell concentration. In con- trast, as the volume of the chamber increased, the cells also increased enough to allow the cell concentration to be calculated. Therefore, chamber 4 has a wide measurement range and was able to be measured at the lowest concentration at 5 × ­103 cells/ml. However, in the maximum cell concentration of ­106 cells/ml, it was difficult to count a high number of cells in a large volume (0.8 µl) and repeat the measurement five times. Therefore, the user must select the appropriate chamber volume according to the sample. Multi‑volume cell counting (MVCC) model. Figure 3 shows the cell number measurement by chamber volume and the slope in the Multi-Volume Cell Counting (MVCC) model. The slope indicates the cell number per volume, reflecting the cell concentration. The slope (cell numbers per volume) was calculated by the cell number in 0.1, 0.2, 0.4, and 0.8 µl chambers. Dotted lines indicate a fit for standard error. As the sample cell concentration decreased, the spacing between the dotted lines increased. Results Comparison of cell concentration measurements between a standard hemacytometer and a multi- volume hemacytometer. *FVCC Model: Fixed-Volume Cell Counting Model. **MVCC Model: Multi-Volum Cell Counting Model. Bold values Cell concentration measurement quality control pass (%RSD < 15%). rately measure the concentration of cells in the range of 2 × ­105 ~ 2.5 × ­106 cells/ml9. As shown in Table 1, in the current experiment the hemacytometer shows a large standard deviation (more than 15%) in the samples lower than ­105 cells/ml. The proposed device has several chambers with different volumes such as 0.1, 0.2, 0.4, and 0.8 µl to contain the multi-volume of cells in suspension. Figure 2 shows that the number of cells varied in con- junction with the volume. The number of cells increased with the volume of the chamber. As shown in Table 1, chamber 1, 2, 3, 4, and the MVCC model were used to measure cell concentration. In the current experiments, rately measure the concentration of cells in the range of 2 × ­105 ~ 2.5 × ­106 cells/ml9. As shown in Table 1, in the current experiment the hemacytometer shows a large standard deviation (more than 15%) in the samples lower than ­105 cells/ml. The proposed device has several chambers with different volumes such as 0.1, 0.2, 0.4, and 0.8 µl to contain the multi-volume of cells in suspension. Figure 2 shows that the number of cells varied in con- junction with the volume. The number of cells increased with the volume of the chamber. As shown in Table 1, chamber 1, 2, 3, 4, and the MVCC model were used to measure cell concentration. In the current experiments, https://doi.org/10.1038/s41598-021-93477-1 Scientific Reports | (2021) 11:14106 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ ntificreports/ Figure 2. Microscopic cell images in multiple volumes (0.1, 0.2, 0.4, and 0.8 µl) in (a) low concentration at ­104 cells/ml, and (b) high concentration at ­106 cells/ml liquid sample (under 40 × magnification). chamber 1 in area 1 ­mm2 contained a volume of 0.1 µl equal to the volume of the standard hemacytometer Figure 2. Microscopic cell images in multiple volumes (0.1, 0.2, 0.4, and 0.8 µl) in (a) low concentration at ­1 cells/ml, and (b) high concentration at ­106 cells/ml liquid sample (under 40 × magnification). Figure 2. Results In the repeated cell concentration measurement of 5 × ­103 cells/ml sample, the MVCC model indicates the standard deviation of cell concentration is lower than 15%, while the FVCC model indicates a higher standard deviation. As shown in Fig. 4, measure- https://doi.org/10.1038/s41598-021-93477-1 Scientific Reports | (2021) 11:14106 | www.nature.com/scientificreports/ Figure 3. The cell numbers according to chamber volume in different cell concentration. (a) ­106 cells/ml, (b) ­105 cells/ml, (c) ­104 cells/ml, (d) 5 × ­103 cells/ml, and (e) ­103 cells/ml. Each point is the average of cell number and error bar is standard deviation in five replications. The slopes from the linear equations were calculated for measuring cell concentration. Figure 3. The cell numbers according to chamber volume in different cell concentration. (a) ­106 cells/ml, (b) ­105 cells/ml, (c) ­104 cells/ml, (d) 5 × ­103 cells/ml, and (e) ­103 cells/ml. Each point is the average of cell number and error bar is standard deviation in five replications. The slopes from the linear equations were calculated for measuring cell concentration. ment using the conventional hemacytometer shows that when the sample concentration was low, the relative standard deviation of measuring cell concentration increased significantly. The measurement range of the con- ventional hemacytometer is ­106 ~ ­105 cells/ml, and using a 0.8 µl chamber showed a wide measurement range of ­106 ~ 5 × ­103 cells/ml. However, in ­106 cells/ml, the hemacytometer using a 0.8 µl chamber had too many cells to count manually (approximately 4000). Figure 4 shows how the proposed MVCC model reduced the num- ber of cell counts needed to measure cell concentration without reducing the measurement range. The MVCC model measured 5 × ­103 ~ ­106 cells/ml within a 10% standard deviation and reduced the counting cell number by about 62.5%. The MVCC model reduced the counting burden in large volumes as a total volume of only 1.5 µl (0.1 + 0.2 + 0.4 + 0.8 µl) was needed to measure the cell concentration at a wide range of concentrations. Discussion As well known, the hemacytometer is a basic device used to count cells in most laboratories. Many times a cell sample needs to be re-prepared by either increasing or decreasing the cell concentration because the unknown cell concentration is outside the range of the hemacytometer. Thus, our study proposed a novel device to measure cell concentration using a multi-volume hemacytometer. This device is designed to have a multi-chamber that can contain multi-volume such as 0.1, 0.2, 0.4, and 0.8 μl, expanding the contained volume for a hemacytometer. https://doi.org/10.1038/s41598-021-93477-1 Scientific Reports | (2021) 11:14106 | www.nature.com/scientificreports/ Figure 4. Comparison of the percent of relative standard deviation of cell number in each concentration between a standard hemacytometer and a multi-volume hemacytometer with four chambers and the MVCC model. Figure 4. Comparison of the percent of relative standard deviation of cell number in each concentration between a standard hemacytometer and a multi-volume hemacytometer with four chambers and the MVCC model. Compared to a standard hemacytometer with a fixed volume of 0.1 μl, the minimum measurable cell concentra- tion for the multi-chamber hemacytometer is 5 × ­103 cells/ml, which is twenty times lower.hf Compared to a standard hemacytometer with a fixed volume of 0.1 μl, the minimum measurable cell concentra- tion for the multi-chamber hemacytometer is 5 × ­103 cells/ml, which is twenty times lower. Th b h l h b h h h b h h ld d ff f ll This is because the multi-chamber hemacytometer has 4 chambers that can hold different amounts of cell samples from low to high volume (0.1, 0.2, 0.4, and 0.8 µl). Cell concentration in each chamber can be calcu- lated by the average number of cells in the area divided by the fixed volume. The number of cells increases as the chamber volume increases, i.e. at fixed volume 0.1 μl, there are 10 ~ 500 cells at ­105 ~ 5 × ­106 cells/ml while at fixed volumes 0.2 μl, there are 10 ~ 500 cells at 5 × ­104 ~ 2.5 × ­106 cells/ml. This multi-volume chamber expands the range of the cell concentration measurements compared to the standard hemacytometer. Discussion When unknown sample was loaded in multi-volume hemacytometer, the cell concentration can be measured by selecting an appropriate chamber with the number of cells (10 to 200 cells/chamber) out of four chambers.fh pp p In this study, phosphate buffered saline (PBS) is used to dilute the cell concentration. Therefore, the sample viscosity is similar to PBS, allowing the sample to flow easily into the chip. At low cell concentrations, high- volume chambers can provide a clearer accurate cell count cell on bottom than high cell concentrations. There- fore, at high cell concentrations in high volume chambers, we recommend changing the focus to verify that the cells are completely precipitated before taking images. Basically, we use a cancer cell line which concentrations 5 × ­103 ~ ­106 cells/ml in PBS. In the case of blood cells, RBC has a concentration of approximately 5 × ­1012 cells/L (5 × ­106 cells/µl)14. This sample must be diluted to ­104 ~ ­106 cells/ml (1:10,000 dilution) with PBS before counting the cell by using this device. y g In addition, the multi-volume hemacytometer proposed the Multi-Volume Cell Counting (MVCC) model for cell concentration measurement. Chamber 4 contains a maximum volume of 0.8 μl and has a wide measurement range that able to measure the lowest concentration at 5 × ­103 cells/ml. Nonetheless, if cell concentration was high around 1 × ­106 cells/ml, cell counting only in 0.8 μl-chamber was difficult and used a lot of time to determine the accuracy of the cell concentration. So, we proposed and validated the Multi-Volume Cell Counting (MVCC) model that cell concentration calculation with the slope value of cell numbers in multi-chambers. The MVCC showed a wide measurement range of 5 × ­103 ~ 1 × ­106 cells/ml while reducing total cell counting numbers by approximately 62.5% compared to a 0.8 μl-chamber hemacytometer. Materials and methods Trypsin (2 ml) was added, and the cells were placed in an incubator for 3 min. Cells were then washed with phosphate-buffered saline (PBS) and centrifuged. PBS was removed and 1 ml of DMEM was added. The number of cells in the suspension was determined by manual counting using a standard hemacytometer. Cell samples were diluted in a 9:1 ratio (PBS: cells suspension) to almost the target cell concentration range, such as ­106, ­105, ­104, 5 × ­103, ­103 cells/ml. Cell concentration measurement. For the multi-volume hemacytometer two cell concentration calcu- lation models are needed. The Fixed-Volume Cell Counting (FVCC) model counted cells in a fixed volume of 0.1 µl which was repeated five times. The FVCC model is the conventional method used in a hemacytometer. The Multi-Volume Cell Counting (MVCC) model counted cells in multiple different volumes at once by calculating cells over an increasing slope per volume to measuring cell concentration. A 10 µl sample containing cells were loaded in the hemacytometer to standardize samples. After direct counting using a microscope with 5 squares (4 corners and the center squares), we calculated the mean and standard deviation of cell concentrations. The number of cells is divided by the chamber’s volume of 0.1 µl using the following formula: (1) concentration (cells/ml) = Number of cells 0.1 µl (1) concentration (cells/ml) = Number of cells 0.1 µl (1) Cell suspension of 35 µl were loaded in chip and counted after 5 min. For 5 min, most of the cells descend to the bottom of the chamber. So, all cells were counted from the bottom of the chip with a microscope focused on the bottom. A microscope with a 40 × objective capture 5 images of different areas in each chamber without changing focus. Each captured image has an area of 1 ­mm2. In the current experiment, we designed the four chambers to have heights of 0.1, 0.2, 0.4, and 0.8 mm. The equation used to calculate cell concentration is below. Chamber 1 (volume: 0.1 µl): concentration (cells/ml) = average of counted cells × 1000 0.1 µl (2) (4) Chamber 3 (volume: 0.4 µl): concentration (cells/ml) = average of counted cells × 1000 0.4 µl (4) (5) Cell concentrations were calculated by taking five images in a chamber. According to the sample cell con- centrations, chambers among the four were selected to measure the cell concentration. Materials and methods Multi‑volume hemacytometer design. The multi-volume hemacytometer is designed to address the problem of loading cell samples in unknown concentrations which can fall outside the range of a standard hemacytometer. The proposed design defines multiple volumes as it consists of 4 chambers with different depths as follows: chamber 1 depth 0.1 mm, chamber 2 depth 0.2 mm, chamber 3 depth 0.4 mm, and chamber 4 depth 0.8 mm which are connected within the same sheet. Each chamber has an area of 3 × 3 ­mm2 and connected by slope channel. The slope channel has 3 mm wide 1 mm length. The height of slope channel increases gradually from the lower chamber height to the higher chamber height as shown in Fig. 1. We designed the slope channel to avoid air bubble trap when the chamber height suddenly increases. The air bubbles make the cells bad disper- sion in chambers. The slope also helps the cells spread continuously from chamber to chamber without damag- ing the flow. The counter chamber and slop channel are made using the PDMS production process after which the PDMS sheets are bonded on a slide glass (Fig. 1). https://doi.org/10.1038/s41598-021-93477-1 Scientific Reports | (2021) 11:14106 | www.nature.com/scientificreports/ Multi‑volume hemacytometer fabrication. This cell counter sheet is made using polydimethylsiloxane (PDMS) with s SYLGARD 184 silicone elastomer base and SYLGARD 184 silicone elastomer curing agent. The mold was made of aluminium by computer numerical control (CNC) machining. In the first step silicone elasto- mer base and silicone curing agent were mixed at a 10:1 ratio and a desiccator was used to burst any bubbles for 15 min before the elastomer mix was poured into a cell counter aluminium mold and placed inside a desiccator for 15 min. The mold was then baked in the oven at 65 °C for 4 h. The PDMS cell counter sheet was then placed on a glass slide and sealed to the glass slide with an oxygen plasma bonder for 2 min under a pressure of 80 W. Cell preparation. Cancer cell line A-549 was counted in the current experiment. A-549 cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% Anti-Anti (Antibiotic–Antimycotic (100x)) and incubated at 37 °C in 5% ­CO2. For harvest, cells were rinsed with 0.25% Trypsin/0.53 mM EDTA to detach the cell layers. References Huang, L.-C. et al. Validation of cell density and viability assays using Cedex automated cell counter. Biologicals 38(3), 393–400 (2010). 1. Chan, L.L.-Y. et al. A novel image-based cytometry method for autophagy detection in living cells. Autophagy 8(9), 1371–1382 (2012).l (2012). 12. Lee, D. W., Yi, S. & Cho, Y.-H. A flow rate independent cell concentration measurement chip using electrical cell counters across a fixed control volume. J. Microelectromech. Syst. 17(1), 139–146 (2008). 12. Lee, D. W., Yi, S. & Cho, Y.-H. A flow rate independent cell concentration measurement chip using electrical cell counters a a fixed control volume. J. Microelectromech. Syst. 17(1), 139–146 (2008).l a fixed control volume. J. Microelectromech. Syst. 17(1), 139 146 (2008). 13. Lee, Y., Kim, B. & Choi, S. On-chip cell staining and counting platform for the rapid detection of blood cells in cerebrospinal fluid. Sensors 18(4), 1124 (2018). i J y ( ) ( ) 13. Lee, Y., Kim, B. & Choi, S. On-chip cell staining and counting platform for the rapid detection of blood cells in cerebrospinal fluid. Sensors 18(4), 1124 (2018). i 13. Lee, Y., Kim, B. & Choi, S. On-chip cell staining and counting platform for the rapid detection of blood Sensors 18(4), 1124 (2018). 14. Dean, L. & Dean, L. Blood Groups and Red Cell Antigens Vol. 2 (NCBI Bethesda, 2005). 14. Dean, L. & Dean, L. Blood Groups and Red Cell Antigens Vol. 2 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Table 2. Total cell counts from cell concentration measurements. *FVCC Model: Fixed-Volume Cell Counting Model. **MVCC Model: Multi-Volume Cell Counting Model. Bold values Cell concentration measurement Measure-ment method concentration (cells/ ml) Hema-cytometer (volume: 0.1 ul *5 ) Multi-volume hemacytometer FVCC model* MVCC Model ** (volume: 0.1 ul + 0.2 ul + 0.4 ul + 0.8 ul) Chamber 1 (volume: 0.1 ul * 5) Chamber 2 (volume: 0.2 ul * 5) Chamber 3 (volume: 0.4 ul * 5) Chamber 4 (volume: 0.8 ul * 5) 106 500 500 1000 2000 4000 1500 105 50 50 100 200 400 150 104 5 5 10 20 40 15 5 × ­103 2.5 2.5 5 10 20 7.5 103 0.5 0.5 1 2 4 1.5 Table 2. Total cell counts from cell concentration measurements. *FVCC Model: Fixed-Volume Cell Counting Model. **MVCC Model: Multi-Volume Cell Counting Model. Bold values Cell concentration measurement range. too many cells in the high concentration cell sample to be counted. In the proposed MVCC model, the total cell count was being reduced by more than 50% while the cell concentration measurement range remained the same. too many cells in the high concentration cell sample to be counted. In the proposed MVCC model, the total cell count was being reduced by more than 50% while the cell concentration measurement range remained the same. Received: 31 January 2021; Accepted: 24 June 2021 Acknowledgementsh g This work was supported by the Korea Medical Device Development Fund grant funded by the Korea government (the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, the Ministry of Health & Welfare, the Ministry of Food and Drug Safety) (Project Number: KMDF_PR_20200901_0135-2021). This work was sup- ported by the Korea Medical Device Development Fund grant funded by the Korea government (the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, the Ministry of Health & Welfare, the Ministry of Food and Drug Safety) (Project Number: KMDF_PR_20200901_0153-2021). This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute, funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI17C2412). Author contributions R.T. performed experiment and wrote paper. I.D. advised experimental problems and performed data analysis D.W.L. supervised the experiments and wrote discussion. References 1. Au Ongena, K. et al. Determining cell number during cell culture using the scepter cell counter. JoVE 45, e2204 (2010). 2 B i C Q lit t N t i bl d ll ti I t J L b H t l 31(3) 277 297 (2009) 2. Briggs, C. Quality counts: New parameters in blood cell counting. Int. J. Lab. Hematol. 31(3), 277–297 (2009). gg Q y p g ( ) ( ) 3. Ates, A. H. et al. Total white blood cell count is associated with the presence, severity and extent of coronary atherosclerosis d by dual-source multislice computed tomographic coronary angiography. Cardiol. J. 18(4), 371–377 (2011).l gg y g 3. Ates, A. H. et al. Total white blood cell count is associated with the presence, severity and extent of coronary atherosclerosis detected by dual-source multislice computed tomographic coronary angiography. Cardiol. J. 18(4), 371–377 (2011). y p g p y g g p y 4. Lu, H. et al. High throughput single cell counting in droplet-based microfluidics. Sci. Rep. 7(1), 1366 (2017).l g g g gl 5. Phillips, E. A. et al. Microfluidic rapid and autonomous analytical device (microRAAD) to detect HIV from whole blood samples Lab Chip 19(20), 3375–3386 (2019). l 5. Phillips, E. A. et al. Microfluidic rapid and autonomous analytical device (microRAAD) to detect HIV from whole blood samples Lab Chip 19(20), 3375–3386 (2019). p 6. Adams, S. et al. Performance evaluation of the point-of-care ­INSTITM HIV-1/2 antibody test in early and established HIV infec- tions. J. Clin. Virol. 91, 90–94 (2017). p 6. Adams, S. et al. Performance evaluation of the point-of-care ­INSTITM HIV-1/2 antibody test in early and established HIV infec- tions. J. Clin. Virol. 91, 90–94 (2017). 7. Cadena-Herrera, D. et al. Validation of three viable-cell counting methods: Manual, semi-automated, and automated. Biotechnol Rep. 7, 9–16 (2015). p ( ) 8. Absher, M. Hemocytometer counting. In Tissue Culture 395–397 (Elsevier, 1973). 8. Absher, M. Hemocytometer counting. In Tissue Culture 395–397 (Elsevier, 1973). 8. Absher, M. Hemocytometer counting. In Tissue Culture 395–397 (Elsevier, 1973). b d h h ll d b h l l d 9. Vembadi, A., Menachery, A. & Qasaimeh, M. A. Cell cytometry: Review and perspective on biotechnological advances. F Bioeng. Biotechnol. 7, 147 (2019). g 10. Huang, L.-C. et al. Validation of cell density and viability assays using Cedex automated cell counter. Biologicals 38(3), 393–400 (2010). g 0. Materials and methods If the concentration was low, a larger volume chamber was needed. If the concentration was high, 0.1 µl would be enough to measure the cell concentration. However, there were too many cells to count in a 0.8 µl chamber. Therefore, the volume of the chamber was chosen according to the cell concentration. g In the proposed multi-chamber hemacytometer, the Multi-Volume Cell Counting model (MVCC model) was applied to cell concentration. The cell numbers in each chamber from four images were plotted according to chamber volume and the slope of a linear equation between counted cell numbers and volume in multi-chambers was calculated to determine the concentration. The slope indicates the number of cells per volume (ml) in the graph of cell number by volume. In the current experiment, we counted four pictures in each chamber to find the cell concentration. The multi-volume model is derived from calculating the slope of the linear equation y = mx; m = slope and setting the intercept (x, y) at 0 as the counted cell number starts at 0 cell/ml. (Fig. 1) In the FVCC model, the large volume chamber has a wide cell concentration measurement range (Table 2). However, there are Scientific Reports | (2021) 11:14106 | https://doi.org/10.1038/s41598-021-93477-1 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/. Additional information Correspondence and requests for materials should be addressed to D.W.L. Correspondence and requests for materials should be addressed to D.W.L. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. 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https://hal.inrae.fr/hal-03325692/file/09-0036.pdf
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Maternal Antibody Transfer in Yellow-legged Gulls
Emerging infectious diseases
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cc-by
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Maternal antibody transfer in yellow-legged gulls Jessica M.C. Pearce-Duvet, Michel Gauthier-Clerc, Elsa Jourdain, Thierry Boulinier Maternal antibody transfer in yellow-legged gulls Jessica M.C. Pearce-Duvet, Michel Gauthier-Clerc, Elsa Jourdain, Thierry Boulinier To cite this version: Jessica M.C. Pearce-Duvet, Michel Gauthier-Clerc, Elsa Jourdain, Thierry Boulinier. Maternal an- tibody transfer in yellow-legged gulls. Emerging Infectious Diseases, 2009, 15 (7), pp.1147-1149. ￿10.3201/eid1507.090036￿. ￿hal-03325692￿ Distributed under a Creative Commons Attribution 4.0 International License References 1. Daszak P, Berger L, Cunningham AA, Hyatt AD, Green DE, Speare R. Emerg- ing infectious diseases and amphibian population declines. Emerg Infect Dis. 1999;5:735–48. 2. Cunningham AA, Hyatt AD, Russell P, Bennett PM. Emerging epidemic dis- eases of frogs in Britain are dependent on the source of ranavirus agent and the route of exposure. Epidemiol Infect. 2007;135:1200–12. 3. Docherty DE, Meteyer CU, Wang J, Mao J, Case ST, Chinchar VG. Diagnostic and molecular evaluation of three iridovirus- associated salamander mortality events. J Wildl Dis. 2003;39:556–66. Japan is located at middle lati- tudes and has a temperate climate. This country has long been geologically iso- lated from Asia. This isolation has re- sulted in the development of many di- verse species of amphibians in Japan; 23 species of the order Caudata and 35 species of the order Anura. Of these species, 49 (84%) are native and 36 (62%) are listed by the Ministry of the Environment as threatened species (8). R. catesbeiana frogs were introduced into Japan in 1918 as a food animal, and raising them by aquaculture was widely attempted. Although they are no longer cultured, feral populations have become established throughout Japan (9). Ranavirus in R. catesbeiana frogs represents a serious threat to am- phibians throughout Japan. 4. Marsh IB, Whittington RJ, O’Rourke B, Hyatt AD, Chisholm O. Rapid differentia- tion of Australian, European and Ameri- can ranavirus based on variation in major capsid protein gene sequence. Mol Cell Probes. 2002;16:137–51. DOI: 10.1006/ mcpr.2001.0400 5. Wolf K, Bullock GL, Dunbar CE, Quimby MC. Tadpole edema virus: a viscerotropic pathogen for anuran amphibians. J Infect Dis. 1968;118:253–62. We determined whether eggs of yellow-legged gulls (Larus micha- hellis) contained antibodies against AIVs. Yellow-legged gulls can host AIVs (C. Lebarbenchon, unpub. data), are abundant, and nest in large, dense colonies in coastal areas. In April 2008, we collected 466 eggs from 2 yellow-legged gull colonies located on the Mediterranean coast: 252 eggs from Gruissan (43.1099°N, 3.1071°E; 350 breeding pairs over 1.5 hectares), and 212 from Villeneuve-lès-Mague- lone (VLM; 43.4895°N, 3.8520°E; 400 pairs over 1 hectare). Villeneuve nests formed 2 spatially clustered 6. Miller DL, Rajeev S, Gray MJ, Bald- win CA. Frog virus 3 infection, cultured American bullfrogs. Emerg Infect Dis. 2007;13:342–3. 7. Majji S, LaPatra S, Long SM, Sample R, Bryan L, Sinning A, et al. Rana catesbeia- na virus Z (RCV-Z): a novel pathogenic ranavirus. Dis Aquat Organ. Yumi Une, Akiko Sakuma, Hiroki Matsueda, Katsuki Nakai, and Masaru Murakami Author affiliations: Azabu University, Kana- gawa, Japan (Y. Ume, A. Sakuma, H. Mat- sueda, M. Murakami); and Nature Conser- vation Division of Shiga Prefecture, Shiga, Japan (K. Nakai) Yumi Une, Akiko Sakuma, Hiroki Matsueda, Katsuki Nakai, and Masaru Murakami Author affiliations: Azabu University, Kana- gawa, Japan (Y. Ume, A. Sakuma, H. Mat- sueda, M. Murakami); and Nature Conser- vation Division of Shiga Prefecture, Shiga, Japan (K. Nakai) Yumi Une, Akiko Sakuma, Hiroki Matsueda, Katsuki Nakai, and Masaru Murakami Author affiliations: Azabu University, Kana- gawa, Japan (Y. Ume, A. Sakuma, H. Mat- sueda, M. Murakami); and Nature Conser- vation Division of Shiga Prefecture, Shiga, Japan (K. Nakai) Live freshwater fish from sev- eral countries have been imported into Japan. However, large amounts (<1,300 tons in 2007) of live aquacul- ture products, including eels and other fishes, have been imported from Tai- wan into Japan (www.customs.go.jp/ tariff/2007_4/data/03.htm). Given that viruses that originate in Japan and Tai- wan are similar, the ranavirus we de- tected was likely imported into Japan in an infected aquatic organism. How- ever, an epidemiologic survey will be necessary to determine the source of the ranavirus in the pond studied. Like- wise, this virus may be endemic to Ja- pan, and a survey of native and foreign free-ranging amphibians should be conducted. Molecular analysis of rana- viruses detected in these surveys will be necessary to differentiate endemic viruses from introduced viruses. DOI: 10.3201/eid1507.081636 To the Editor: Avian influenza viruses (AIVs) are emerging patho- gens of concern because they can cause deaths in birds and humans (1). Although wild birds likely contribute to AIV emergence because they are the natural reservoir for all known influenza virus subtypes (1), our un- derstanding of AIV transmission and immunology in natural avian popula- tions is incomplete (2). In this context, the transfer of maternal antibodies is a tool that should be used more often in immunologic analysis. Because an- tibodies in eggs and hatchlings can reflect the mother’s past exposure to pathogens (3,4) and both life stages are more easily sampled than adults, quantifying antibodies found in avian young could help clarify AIV epide- miology. HAL Id: hal-03325692 https://hal.inrae.fr/hal-03325692v1 Submitted on 25 Aug 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License LETTERS Address for correspondence: Yumi Une, Laboratory of Veterinary Pathology, School of Veterinary Medicine, Azabu University, 1-17- 71 Fuchinobe, Sagamihara, Kanagawa 229- 8501, Japan; email: une@azabu-u.ac.jp Address for correspondence: Yumi Une, Laboratory of Veterinary Pathology, School of Veterinary Medicine, Azabu University, 1-17- 71 Fuchinobe, Sagamihara, Kanagawa 229- 8501, Japan; email: une@azabu-u.ac.jp and F-081) of the Ministry of the Environ- ment, Japan, 2008, and The Promotion and Mutual Aid Corporation for Private Schools of Japan, Grant in Aid for Match- ing Fund Subsidy for Private Universities. spp.) that cohabitated the pond, but showed no external signs of disease, were positive for ranavirus by PCR us- ing primers M153 and M154. Further sequence analyses are ongoing, and additional investigations of other am- phibians and fishes are needed.i Jessica M.C. Pearce-Duvet, Michel Gauthier-Clerc, Elsa Jourdain, and Thierry Boulinier Author affiliations: Centre d’Ecologie Fonc- tionnelle et Evolutive, Montpellier, France (J.M.C. Pearce-Duvet, T. Boulinier); Centre de Recherche de la Tour du Valat, Arles, France (M. Gauthier-Clerc); and Kalmar University, Kalmar, Sweden (E. Jourdain) y Overall antibody prevalence was 14% (65/466), indicating exposure to influenza A viruses in these colonies. As expected, antibody prevalence in gulls is higher than the viral preva- lence previously estimated by reverse transcription–PCR or virus isolation on fecal samples, i.e., the methods typically used by avian influenza sur- veillance networks. A spring and sum- mer survey performed on feces from gulls of the Camargue region (east of our colonies) showed that only 0.9% of gulls (2 infected of 228 sampled) were excreting AIVs (C. Lebarben- chon, unpub. data). DOI: 10.3201/eid1507.090036 References An equal volume of chloroform was added, the solution was centrifuged (6,000 × g for 15 min), and the supernatant was used in the analyses. Extracts were tested for antibod- ies against the AIV nucleoprotein by using a commercial competitive ELISA (IDEXX/Institut Pourquier, Montpellier, France). The assay has been validated by using seagull serum (IDEXX, pers. comm.) and chicken egg yolk (5). A subset of samples was tested by using a second commercial competitive ELISA (IDVet, Montpel- lier, France). Optical density values obtained in the 2 assays were signifi- cantly correlated (r = 0.90, df = 39, p<0.001), and serostatus was consis- tent across assays. Overall antibody prevalence was 14% (65/466), indicating exposure to influenza A viruses in these colonies. As expected, antibody prevalence in gulls is higher than the viral preva- lence previously estimated by reverse colonies of Villeneuve also did not dif fer: 18/129 (14%) in VLM1 compared with 12/83 (14.5%) in VLM2 (z = 0.02, p = 0.9). There was no evidence of spatial autocorrelation in the distri bution of antibody in eggs by using the Moran I spatial statistic. This similar ity of antibody prevalence across and within colonies suggests that exposure is dictated by regional rather than loca conditions, a hypothesis that should be tested by sampling across a broade range of nest densities and over time. Our study presents evidence fo the presence of antibodies agains AIVs in wild bird eggs, and the find ings have important practical implica tions. The difference in prevalence es timated from virus isolation (0.9%) and antibody detection (14%), although expected, highlights the complemen tary nature of the 2 approaches. Mos surveys estimate current infection by virus isolation, which provides infor mation about disease risk in addition to phylogeographic tracking of strains In contrast, information on antibody prevalence, which shows past and present population exposure and risk has largely been ignored with few ex ceptions (e.g., 6,7). Future work could benefit from using both approaches in tandem with modeling to develop an understanding of avian influenza ecol ogy in nature. Egg yolks were isolated and fro- zen at –20°C until analysis. Antibod- ies were obtained by using chloroform extraction (4). The yolk was diluted 1:1 in phosphate-buffered saline and homogenized. An equal volume of chloroform was added, the solution was centrifuged (6,000 × g for 15 min), and the supernatant was used in the analyses. 4. Gasparini J, McCoy KD, Haussy C, Tveraa T, Boulinier T. Induced maternal response to the Lyme disease spirochaete Borrelia burgdorferi sensu lato in a colonial sea- bird, the kittiwake Rissa tridactyla. Proc R Soc Lond B Biol Sci. 2001;268:647–50. DOI: 10.1098/rspb.2000.1411 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 15, No. 7, July 2009 Acknowledgments We thank P. Peret, P. Mayet, R. Gar- nier, and E. Charbonnier for egg collection and V. Staszewski for technical assistance. Sampling permits were obtained from the Direction Régionale de l’Environnement de Languedoc-Roussillon. Our study presents evidence for the presence of antibodies against AIVs in wild bird eggs, and the find- ings have important practical implica- tions. The difference in prevalence es- timated from virus isolation (0.9%) and antibody detection (14%), although expected, highlights the complemen- tary nature of the 2 approaches. Most surveys estimate current infection by virus isolation, which provides infor- mation about disease risk in addition to phylogeographic tracking of strains. In contrast, information on antibody prevalence, which shows past and present population exposure and risk, has largely been ignored with few ex- ceptions (e.g., 6,7). Future work could benefit from using both approaches in tandem with modeling to develop an understanding of avian influenza ecol- ogy in nature. Extracts were tested for antibod- ies against the AIV nucleoprotein by using a commercial competitive ELISA (IDEXX/Institut Pourquier, Montpellier, France). The assay has been validated by using seagull serum (IDEXX, pers. comm.) and chicken egg yolk (5). A subset of samples was tested by using a second commercial competitive ELISA (IDVet, Montpel- lier, France). Optical density values obtained in the 2 assays were signifi- cantly correlated (r = 0.90, df = 39, p<0.001), and serostatus was consis- tent across assays. This study was supported by grants from the French National Agency for Re- search, the Agency for Inter-Institutional Promotion of Research in Developing Countries, and a Chateaubriand fellowship. Jessica M.C. Pearce-Duvet, Michel Gauthier-Clerc, Elsa Jourdain, and Thierry Boulinier Author affiliations: Centre d’Ecologie Fonc- tionnelle et Evolutive, Montpellier, France (J.M.C. Pearce-Duvet, T. Boulinier); Centre de Recherche de la Tour du Valat, Arles, France (M. Gauthier-Clerc); and Kalmar University, Kalmar, Sweden (E. Jourdain) References 2006;73:1– 11. DOI: 10.3354/dao073001 8. Ota H. Current status of the threatened amphibians and reptiles of Japan. Popula- tion Ecology. 2000;42:5–9. DOI: 10.1007/ s101440050003 8. Ota H. Current status of the threatened amphibians and reptiles of Japan. Popula- tion Ecology. 2000;42:5–9. DOI: 10.1007/ s101440050003 9. Maeda N, Matsui M. Frogs and toads of Japan. Tokyo: Bun-Ichi Sogo Shuppan Publishers; 1989. p. 100–7. 9. Maeda N, Matsui M. Frogs and toads of Japan. Tokyo: Bun-Ichi Sogo Shuppan Publishers; 1989. p. 100–7. This study was supported by the Global Environment Research Fund (F-3 1147 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 15, No. 7, July 2009 LETTERS LETTERS subcolonies: VLM1 (129 eggs) and VLM2 (83 eggs). We also collected global positioning system coordinates for VLM1 nests. subcolonies: VLM1 (129 eggs) and VLM2 (83 eggs). We also collected global positioning system coordinates for VLM1 nests. mized by taking blood samples from young nestlings of a standardized age. Finally, such samples provide abundant material for the simultaneous surveil- lance of other emerging pathogens of interest, such as Campylobacter spp. (8) and West Nile virus (9). colonies of Villeneuve also did not dif- fer: 18/129 (14%) in VLM1 compared with 12/83 (14.5%) in VLM2 (z = 0.02, p = 0.9). There was no evidence of spatial autocorrelation in the distri- bution of antibody in eggs by using the Moran I spatial statistic. This similar- ity of antibody prevalence across and within colonies suggests that exposure is dictated by regional rather than local conditions, a hypothesis that should be tested by sampling across a broader range of nest densities and over time. colonies of Villeneuve also did not dif- fer: 18/129 (14%) in VLM1 compared with 12/83 (14.5%) in VLM2 (z = 0.02, p = 0.9). There was no evidence of spatial autocorrelation in the distri- bution of antibody in eggs by using the Moran I spatial statistic. This similar- ity of antibody prevalence across and within colonies suggests that exposure is dictated by regional rather than local conditions, a hypothesis that should be tested by sampling across a broader range of nest densities and over time. subcolonies: VLM1 (129 eggs) and VLM2 (83 eggs). We also collected global positioning system coordinates for VLM1 nests. Egg yolks were isolated and fro- zen at –20°C until analysis. Antibod- ies were obtained by using chloroform extraction (4). The yolk was diluted 1:1 in phosphate-buffered saline and homogenized. Prevalence of Human Bocavirus in Human Tonsils and Adenoids 5. Trampel DW, Zhou E-M, Yoon K-J, Koe- hler KJ. Detection of antibodies in serum and egg yolk following infection of chick- ens with an H6N2 avian influenza virus. J Vet Diagn Invest. 2006;18:437–42. Samples consisted of surgically removed tonsil or adenoid tissues. DNA was extracted and its concentra- tion was determined as previously de- scribed (5). Two primer sets were used for HBoV detection by using real-time PCR with SYBR Green detection and melting-point determination. We de- signed primers 3097F (5′-GTC-CAA- TTA-CAT-GAT-CAC-GCC-TAC-TC) and 3420R (5′-TGC-GTC-CAC-AGT- ATC-AGG-TTG-TTG) that targeted the viral protein 1/2 (VP1/VP2) region of HBoV. The nonstructural protein 1 (NP1) region was targeted by us- ing primers 188F and 542R from Al- lander et al. (6) Each 20-μL reaction contained SYBR Green JumpStart Taq ReadyMix (Sigma, St. Louis, MO, USA), 4 mmol/L MgCl2, 250 nmol/L primers, double-distilled H2O, and 2 μL of DNA (50–200 ng) cycled on an ABI PRISM 7900HT (Applied Biosystems, Foster City, CA, USA) instrument at 94oC for 2 min, followed by 40 cycles of 94°C for 20 s, 60°C (NP1 primers) or 68°C (VP1/VP2 primers) for 20 s, and 72°C for 14 s. Amplification and melting curves were analyzed with 7900HT version 2.2.1 software (Ap- plied Biosystems); positive samples were verified by sequence analysis. Sequenced VP- and NP1-generated amplicons were 99%–100% identical to HBoV strain ST1 (6). The detec- tion sensitivities of the VP and NP1 assays, determined by using a plasmid construct containing the full HBoV genome, were 1–5 and 5–10 gene cop- ies/reaction, respectively.i 6. Krauss S, Obert CA, Franks J, Walker D, Jones K, Seiler P, et al. Influenza in mi- gratory birds and evidence of limited in- tercontinental virus exchange. PLoS Pat- hog. 2007;3:e167. DOI: 10.1371/journal. ppat.0030167 To the Editor: Recently, Longtin et al. (1) reported a high rate (43%) of human bocavirus (HBoV) infection in a group of children chosen to serve as controls in a study of HBoV preva- lence among hospitalized children and adults. In contrast, previous reports had found low HBoV prevalence rates of 0%–1% in control groups (2,3). At- tempting to explain this surprising dif- ference in rates, Lu et al. (4) suggested that selection of control patients may be related to the difference in rates. References Our results also show the gener- alizable potential of maternal antibody transfer for tracking pathogen exposure in wild birds, notably in the case of rec- ognized emerging zoonoses. Because eggs and hatchlings are proxies of past and present adult pathogen expo- sure (3,4), the difficult and sometimes disruptive sampling of adults can be circumvented by the rapid and cost-ef- ficient sampling of their young, which will facilitate monitoring efforts. Due to the high intranest correlation in egg antibodies (4), only partial sampling of clutches (e.g., 1 of 3 eggs) is necessary to track pathogen presence and preva- lence through space and time. The sampling effects could further be mini- 1. Olsen B, Munster VJ, Wallensten A, Waldenström J, Osterhaus AD, Fouchier RA. Global patterns of influenza A virus in wild birds. Science. 2006;312:384–8. DOI: 10.1126/science.1122438 2. Boyce WM, Sandrock C, Kreuder-Johnson C, Kelly T, Cardona C. Avian influenza in wild birds: a moving target. Comp Immu- nol Microbiol Infect Dis. 2009;32:275–86. DOI: 10.1016/j.cimid.2008.01.002 2. Boyce WM, Sandrock C, Kreuder-Johnson C, Kelly T, Cardona C. Avian influenza in wild birds: a moving target. Comp Immu- nol Microbiol Infect Dis. 2009;32:275–86. DOI: 10.1016/j.cimid.2008.01.002 3. Boulinier T, Staszewski V. Maternal trans- fer of antibodies: raising immuno-ecology issues. Trends Ecol Evol. 2008;23:282–8. DOI: 10.1016/j.tree.2007.12.006 Egg antibody prevalence did not differ significantly between colonies. The antibody prevalence of 13.5% found at Gruissan (34 of 252 eggs) was comparable with the 14.5% found at Villeneuve (31 of 214 eggs) (gen- eralized linear model with binomial distribution, z = 0.4, p = 0.8). The sub- 4. Gasparini J, McCoy KD, Haussy C, Tveraa T, Boulinier T. Induced maternal response to the Lyme disease spirochaete Borrelia burgdorferi sensu lato in a colonial sea- bird, the kittiwake Rissa tridactyla. Proc R Soc Lond B Biol Sci. 2001;268:647–50. DOI: 10.1098/rspb.2000.1411 1148 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 15, No. 7, July 2009 Prevalence of Human Bocavirus in Human Tonsils and Adenoids The control group used in the Long- tin study was primarily (71%) made up of children undergoing elective surgery; previous studies had selected control groups from other sources, in- cluding well children on routine visits and outpatients with nonrespiratory symptoms. Because the Longtin study surgeries were mainly tonsillectomies, adenoidectomies, and myringotomies, Lu et al. examined the possibility that tonsillar tissues, which include the ad- enoids, are sites of persistent HBoV infection. When these researchers ex- tracted DNA from tonsillar lympho- cytes obtained from pediatric patients who had undergone tonsillectomies or adenoidectomies, they detected HBoV DNA in 32% of the samples (4). These findings strongly suggest a connec- tion between HBoV and tonsillar tis- sue. Therefore, we tested a number of tonsillar samples for the presence of HBoV DNA. 7. Stallknecht DE, Shane SM. Host range of avian influenza in free-living birds. Vet Res Commun. 1988;12:125–41. DOI: 10.1007/BF00362792 8. Sahin O, Zhang Q, Meitzler JC, Harr BS, Morishita TY, Mohan R. Prevalence, antigenic specificity, and bactericidal activity of poultry anti-Campylobacter maternal antibodies. Appl Environ Mi- crobiol. 2001;67:3951–7. DOI: 10.1128/ AEM.67.9.3951-3957.2001 9. Baitchman EJ, Tlusty MF, Murphy HW. Passive transfer of maternal antibod- ies to West Nile virus in flamingo chicks (Phoenicopterus chilensis and Phoeni- copterus ruber ruber). J Zoo Wildl Med. 2007;38:337–40. DOI: 10.1638/1042- 7260(2007)038[0337:PTOMAT]2.0.CO;2 Address for correspondence: Jessica M.C. Pearce-Duvet, Centre d’Ecologie Fonctionnelle et Evolutive, Centre National de la Recherche Scientifique–Unité de Mixte de Recherche 5175, 1919 Route de Mende, F-34293 Montpellier, France; email: jessica.pearce@cefe.cnrs.fr Letters Letters commenting on recent ar- ticles as well as letters reporting cases, outbreaks, or original re- search are welcome. Letters com- menting on articles should contain no more than 300 words and 5 references; they are more likely to be published if submitted within 4 weeks of the original article’s publi- cation. Letters reporting cases, out- breaks, or original research should contain no more than 800 words and 10 references. They may have 1 Figure or Table and should not be divided into sections. All letters should contain material not previ- ously published and include a word count. Our testing identified HBoV DNA in 5 (5.5%) of the 91 children who underwent elective tonsillecto- my/adenoidectomy. Ages ranged from 1.9 to 4.6 years (median 3.4 years). The reason for the much lower HBoV prevalence in this group of children, compared with prevalences found in studies by Longtin et al. and Lu et al., is unclear. Lu et al. (4) reported a much higher HBoV rate of lymphocytes from adenoids (56%) than from ton- sils (16%). Although we did not know Sample acquisition was approved by the Nationwide Children’s Hospi- tal Institutional Review Board. Tonsils and adenoids were obtained from 91 patients who underwent elective sur- gery at Nationwide Children’s Hos- pital from June through September 2004. Patients’ ages ranged from 1 to 16 years (median 5.9 years; age was unknown for 4 patients). 1149 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 15, No. 7, July 2009
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Irreversibility of Pressure Induced Boron Speciation Change in Glass
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OPEN SUBJECT AREAS: MATERIALS SCIENCE CHEMISTRY PHYSICS Morten M. Smedskjaer1, Randall E. Youngman2, Simon Striepe3, Marcel Potuzak2, Ute Bauer4, Joachim Deubener3, Harald Behrens4, John C. Mauro2 & Yuanzheng Yue1 1Section of Chemistry, Aalborg University, DK-9000 Aalborg, Denmark, 2Science and Technology Division, Corning Incorporated, Corning, NY 14831, USA, 3Institute of Non-Metallic Materials, Clausthal University of Technology, 38678 Clausthal-Zellerfeld, Germany, 4Institute of Mineralogy, Leibniz University Hannover, 30167 Hannover, Germany. Received 30 August 2013 Accepted 30 December 2013 Published 20 January 2014 It is known that the coordination number (CN) of atoms or ions in many materials increases through application of sufficiently high pressure. This also applies to glassy materials. In boron-containing glasses, trigonal BO3 units can be transformed into tetrahedral BO4 under pressure. However, one of the key questions is whether the pressure-quenched CN change in glass is reversible upon annealing below the ambient glass transition temperature (Tg). Here we address this issue by performing 11B NMR measurements on a soda lime borate glass that has been pressure-quenched at ,0.6 GPa near Tg. The results show a remarkable phenomenon, i.e., upon annealing at 0.9Tg the pressure-induced change in CN remains unchanged, while the pressurised values of macroscopic properties such as density, refractive index, and hardness are relaxing. This suggests that the pressure-induced changes in macroscopic properties of soda lime borate glasses compressed up to ,0.6 GPa are not attributed to changes in the short-range order in the glass, but rather to changes in overall atomic packing density and medium-range structures. A ffi A ny natural or man-made material will exhibit a significant change in microstructure and properties when subjected to high pressure1–5; particularly the coordination number (CN) of atoms or ions will generally increase with pressure6–9. It has long been known that when a glassy material or its liquid state is subjected to sufficiently high pressure, significant changes can take place in the local and medium-range structure, vibra- tional density of states, and physical properties4–8,10,11. In recent years, theoretical modeling approaches have provided insights regarding the structural response of various non-crystalline materials to composition, temper- ature, and pressure11–15. Nevertheless, the relationship between structure and dynamics in glass and glass-forming melts under high pressures remains a challenging problem in condensed matter science. The difficulties in addressing this problem are due to both experimental limitations at high pressures and the inadequacy of computer simulations under such conditions. Correspondence and requests for materials should be addressed to Y.Z.Y. (yy@bio.aau. dk) OPEN While Vickers hardness has essentially decreased to its original value prior to compression (5.0 GPa) after annealing for 6 h, density has only decreased to ,2.47 g/cm3 after annealing for 24 h, which is significantly higher than the value prior to compression (2.438 g/cm3), i.e., density has relaxed by only 43% of the total possible relaxation. In this work we investigate the microscopic and macroscopic res- ponses of the soda-lime borate glass to comparatively low pressure at an elevated temperature around the Tg, since numerous high pres- sure studies at room temperature have already been reported in literature and it is also our aim to detect the sensitivity of glass structure and properties to the low pressure. Moreover, these condi- tions are chosen to be able to prepare compressed samples, which are large enough to allow for subsequent characterization of mac- roscopic properties. The as-produced glass has a nonzero equilib- rium concentration of BO4 due to the presence of network modifiers (Na2O and CaO) that act to charge balance the tetrahedral boron species. Only recently has it been found that boron speciation (i.e. the CN) changes occur at rather low pressure (,0.6 GPa) near Tg and can be frozen-in under pressure18. Here the key questions arise: Is the pressure-quenched CN change reversible upon annealing at tem- peratures slightly below Tg at atmospheric pressure? Are the pres- sure-induced changes in the macroscopic properties also reversible upon annealing? What is the microscopic origin of the pressure- induced changes in the macroscopic properties? We answer these questions by investigating the structure and property responses of the sodium-calcium-borate glass to pressurization at ,0.6 GPa and subsequent annealing at 0.9Tg under ambient pressure. We thereby hope to obtain a better understanding of the pressure-structure- property relationship of this and other glass systems, which can be used for tailoring both microscopic and macroscopic properties of glassy materials. With increasing isostatic pressure, the overshoot in the isobaric heat capacity (Cp) above Tg increases as illustrated in Fig. 2a, where the evolution of Cp with temperature during the first DSC upscan is shown. This overshoot is considered to be a direct consequence of the nonexponentiality of the relaxation process26, i.e., due to broadening of the relaxation time distribution. OPEN fraction suddenly drops at pressures just above 4 GPa and then approaches zero as the pressure is further increased17. A structural response of vitreous B2O3 to pressure below 4 GPa was not detected. After decompression from high pressure the boron coordination reverts back from tetrahedral to trigonal, however, the densification is apparently permanent17. It has been attempted to correlate these changes in microscopic structure with the macroscopic properties. For example, the viscosity of the B2O3 liquid along the melting curve has been shown to decrease by 4 orders of magnitude upon a pressure increase up to 5.5 GPa but then remains unchanged upon further increase of the pressure22. However, a generally accepted viewpoint about the microscopic origin of the pressure-induced changes in the macroscopic properties is still lacking. The abovementioned studies were carried out on vitreous B2O3 at room temperature and rather high pressure up to 22.5 GPa. It should be noted that the structure of glasses compressed at room temper- ature (in diamond anvil cell) is different from that obtained by pres- sure-quenching liquids from above Tg 21, even though the main structural changes upon compression (i.e., decreased fraction of bor- oxol rings and increased fraction of tetrahedral boron) are identical. It should also be noted that modified borate and pure B2O3 glasses have different initial concentration of boroxol rings and may exhibit different densification mechanisms. However, Lee et al. have shown that the pressure dependent structural changes of a lithium borate glass are similar to those of vitreous B2O3 24. In particular, they showed that the pressure-induced CN change in Li2B4O7 glass from three-coordinated to four-coordinated boron at room temperature begins around 5 GPa and the BO4 fraction increases with pressure from about 50% at 1 atm to more than 95% at 30 GPa. Figure 1 | Relaxation of macroscopic properties. Evolution of density (r) and Vickers hardness (HV) with annealing duration (ta) at 0.9Tg 5 688 K of the borate glass compressed at 570 MPa. The dashed lines indicate the values of r and HV prior to compression. Inset: impact of isostatic pressure (p) on r and HV. The errors of r and HV are around 60.005 g/cm3 and 60.2 GPa, respectively. the compressed glass are relaxing during annealing towards those of the glass prior to compression. OPEN This also implies that subjecting the glass to high pressure forces the glass into a lower region of the enthalpy landscape compared to the glass under ambient pressure, even though it should be noted that the enthalpy landscape itself is changing as a function of pressure. In agreement with previous stud- ies19,27, we find that an increase in isostatic pressure enhances the nonexponentiality of the enthalpy relaxation as measured ex situ. With increasing pressure on the glass, the density of the glass increases, and at the same time the topological degree of atomic freedom decreases as well due to the BO3RBO4 conversion, i.e., the increase of network connectivity. Upon heating through the glass transition region, the glass compressed at higher pressures should exhibit a larger jump in configurational entropy in order to approach the liquid state. This is also shown in Fig. 2b, in which the pressure dependence of the fictive temperature (Tf) is plotted. The procedure for determining Tf based on DSC data is described elsewhere28,29. g f The compressed glass relaxes in the glass transition region during the first upscan and recovers back to its original state with respect to enthalpy, i.e., that of the uncompressed glass. In other words, the enthalpy level of the glass reaches that of the glass cooled under standard conditions, e.g., at 10 K/min and ambient pressure. This is seen from the second DSC upscans as shown in the inset of Fig. 2a and in Supplementary Figure S1. Enthalpy recovery of the com- pressed glass during the DSC upscan is a ‘‘structural depression’’ process, induced by thermal excitation under ambient pressure27. The structural depression leads to an increase in enthalpy, which is manifested as an enhancement of the overshoot during the first DSC upscan. This is also evident from the relaxation of refractive index before and after the first DSC upscan (Fig. 3). With increasing iso- static pressure, the refractive index at 633 nm increases due to den- sification, but the measured value on the sample following the first OPEN It is crucial to determine the link between the microscopic structure and macroscopic properties of glasses under high pressure from both scientific and technological perspectives, since the glass structures frozen-in under elevated pressure may give rise to properties unattainable under ambient pressure. Borate glass is the quintessential example of glass capable of dramatic changes in short-range order as a function of composition, pressure, and thermal history, and hence we select a simple ternary borate system, namely, sodium-calcium-borate glass, for this study in order to address the above mentioned challenging problem. Boric oxide (B2O3) is also widely used as a network forming constituent in many high-tech glass materials owing to its contribution to high glass forming ability and low melting temperature, and for its favorable impact on thermal, mechanical, and optical properties. One of the most striking structural features of borate glasses is the transformation of the coordination number (CN) of boron from three to four upon compression16–19, which is associated with anomalous pressure depend- ence of viscosity6,16 and topological disorder8,12. Densification of borate glasses can be achieved by applying isostatic pressure to glass directly at room temperature16,17, or by pressure-quenching from the molten state18–21. However, the elastic part of the densification relaxes during pressure release, i.e., the density of a compressed glass at ambient pressure decreases to the value before compression upon reheating below the glass transition tem- perature (Tg)17. For vitreous B2O3, it has been shown that the fraction of boroxol rings decreases with increasing pressure16,20,22, leading to densification of the glass16 and increase of elastic moduli20. When the pressure applied at room temperature is released, such densification is irreversible since the local structures may be topologically/ stereochemically unfavorable for the reformation of boroxol rings16. However, it is not clear whether this irre- versibility of densification is linked to the CN change of boron23. More recently, it has been found that the BO3 SCIENTIFIC REPORTS | 4 : 3770 | DOI: 10.1038/srep03770 1 Figure 1 | Relaxation of macroscopic properties. Evolution of density (r) and Vickers hardness (HV) with annealing duration (ta) at 0.9Tg 5 688 K of the borate glass compressed at 570 MPa. The dashed lines indicate the values of r and HV prior to compression. Inset: impact of isostatic pressure (p) on r and HV. The errors of r and HV are around 60.005 g/cm3 and 60.2 GPa, respectively. Results Isotropic projections fr these data (Supplementary Figure S4a) show very little change w annealing time, and thus, the estimated quadrupolar coupling pr uct (PQ) and isotropic chemical shifts (diso) for 23Na also do indicate any detectable change in sodium environment in the co pressed glass upon annealing (Supplementary Figure S4b). This s gests that the Na–O bond length does not change with anneal time, and hence it does not determine the recovery of the m roscopic properties upon annealing. Discussion Our work has shown that upon annealing at 0 9T of a soda l Figure 2 | Pressure history dependence of calorimetric glass transition. (a) Evolution of endothermic overshoot in isobaric heat capacity (Cp) at the glass transition for samples with different pressure history. DSC scans were performed at 10 K/min at ambient pressure. Inset: second DSC upscans at 10 K/min of all samples following prior up- and downscans at 10 K/min. (b) Pressure history dependence of the fictive temperature (Tf) evaluated from the first and second DSC upscan, respectively. Figure 3 | Recovery of refractive index during DSC scan. Pressure history dependence of the refractive index (n) at 633 nm before and a the first DSC scan, as shown in Fig. 2a. DSC upscan is independent of the initially applied pressure during compression. Structural response. 11B magic-angle spinning (MAS) NMR spectra obtained at 16.4 T (700 MHz) for the 0.57 GPa compressed glass Figure 2 | Pressure history dependence of calorimetric glass transition. (a) Evolution of endothermic overshoot in isobaric heat capacity (Cp) at the glass transition for samples with different pressure history. DSC scans were performed at 10 K/min at ambient pressure. Inset: second DSC upscans at 10 K/min of all samples following prior up- and downscans at 10 K/min. (b) Pressure history dependence of the fictive temperature (Tf) evaluated from the first and second DSC upscan, respectively. Figure 3 | Recovery of refractive index during DSC scan. Pressure history dependence of the refractive index (n) at 633 nm before and after the first DSC scan, as shown in Fig. 2a. remains almost constant within the error range when extending the annealing time up to 25 hours at the annealing temperature of 688 K (0.9Tg) and at the ambient pressure. In other words, the glass structure, as defined by boron coordination number, does not return to its original state prior to compression upon annealing. Results (b) Pressure history dependence of the fictive temperature (Tf) evaluated from the first and second DSC upscan, respectively. DSC upscan is independent of the initially applied pressure during compression. Structural response. 11B magic-angle spinning (MAS) NMR spectra obtained at 16.4 T (700 MHz) for the 0.57 GPa compressed glass annealed for different durations are shown in Fig. 4a. These spectra are characterised by a broad peak centered at 115 ppm, corresponding to BIII sites, and a relatively narrow peak centered around 12 ppm, corresponding to BIV sites. The spectra vary slightly with annealing time, which reflects minor changes in either the relative proportions of BIII and BIV, or changes in bond angles and distances involving boron and oxygen. We quantify these differences by accurate simulation of the spectral lineshapes and subsequent determination of the fraction of tetrahedral to total boron (N4) through integration. The simulation parameters are given in Supplementary Table S1, and two examples of the deconvolution are shown in Supplementary Figure S2. Prior to annealing, we find that with increasing isostatic pressure, N4 increases from 44.2 to 46.5 at% (inset of Fig. 4b and Supplementary Figure S3a). N4 also increases upon compression in pure B2O3 glass17 and in boron- containing multicomponent E-glass18. In Fig. 4b we observe an interesting phenomenon, viz., N4 of the compressed sample Results Physical properties and heat capacity. The glass composition under study is 25Na2O – 10CaO – 65B2O3 (mol%). As reported previously25, the density and Vickers hardness of this glass increase approximately linearly with increasing isostatic pressure (inset of Fig. 1). The glass isostatically compressed at 570 MPa is then annealed under ambient pressure at 688 K, i.e., 0.9 times its ambient Tg for various durations (ta). This results in a decrease of both density and Vickers hardness with increasing annealing duration (Fig. 1). In other words, these macroscopic properties of SCIENTIFIC REPORTS | 4 : 3770 | DOI: 10.1038/srep03770 2 Figure 3 | Recovery of refractive index during DSC scan. Pressure history dependence of the refractive index (n) at 633 nm before and after the first DSC scan, as shown in Fig. 2a. www.nature.com/scientificreports DSC upscan is independent of the initially applied pressure during compression. Structural response. 11B magic-angle spinning (MAS) NMR spectra obtained at 16.4 T (700 MHz) for the 0.57 GPa compressed glass annealed for different durations are shown in Fig. 4a. These spectra are characterised by a broad peak centered at 115 ppm remains almost constant within the error range when extend the annealing time up to 25 hours at the annealing temperatur 688 K (0.9Tg) and at the ambient pressure. In other words, the g structure, as defined by boron coordination number, does not ret to its original state prior to compression upon annealing. Altho N4 does not appear to change, the11B MAS NMR spectra do sh differences with annealing time. For example, the base of tetrahedral boron resonances in Fig. 4a exhibit some anneal time dependence, signifying a change in environment of the groups, possibly due to changes in bond angle distribution arou these boron atoms. The BIII features in Fig. 4a do not show m change with annealing time, indicating that annealing has li impact on B-O bond distance or angles for these structural eleme Supplementary Figure S3b shows 23Na MAS NMR spectra of samples prior to annealing. We see a small but systematic increas frequency shift with increasing pressure, which is due to a decreas the mean Na–O bond distance upon compression18. Fig. 4c shows 23Na MAS NMR spectra for the compressed-annealed glasses. Th MAS NMR spectra overlap completely, and to further study sodium speciation, 23Na triple-quantum magic-angle spinn (3QMAS) NMR data were also collected. Results Although N4 does not appear to change, the11B MAS NMR spectra do show differences with annealing time. For example, the base of the tetrahedral boron resonances in Fig. 4a exhibit some annealing time dependence, signifying a change in environment of the BIV groups, possibly due to changes in bond angle distribution around these boron atoms. The BIII features in Fig. 4a do not show much change with annealing time, indicating that annealing has little impact on B-O bond distance or angles for these structural elements. Supplementary Figure S3b shows 23Na MAS NMR spectra of the samples prior to annealing. We see a small but systematic increase in frequency shift with increasing pressure, which is due to a decrease in the mean Na–O bond distance upon compression18. Fig. 4c shows the 23Na MAS NMR spectra for the compressed-annealed glasses. These MAS NMR spectra overlap completely, and to further study the sodium speciation, 23Na triple-quantum magic-angle spinning (3QMAS) NMR data were also collected. Isotropic projections from these data (Supplementary Figure S4a) show very little change with annealing time, and thus, the estimated quadrupolar coupling prod- uct (PQ) and isotropic chemical shifts (diso) for 23Na also do not indicate any detectable change in sodium environment in the com- pressed glass upon annealing (Supplementary Figure S4b). This sug- gests that the Na–O bond length does not change with annealing time, and hence it does not determine the recovery of the mac- roscopic properties upon annealing. Figure 2 | Pressure history dependence of calorimetric glass transition. (a) Evolution of endothermic overshoot in isobaric heat capacity (Cp) at the glass transition for samples with different pressure history. DSC scans were performed at 10 K/min at ambient pressure. Inset: second DSC upscans at 10 K/min of all samples following prior up- and downscans at 10 K/min. (b) Pressure history dependence of the fictive temperature (Tf) evaluated from the first and second DSC upscan, respectively. Figure 2 | Pressure history dependence of calorimetric glass transition. Figure 2 | Pressure history dependence of calorimetric glass transition. (a) Evolution of endothermic overshoot in isobaric heat capacity (Cp) at the glass transition for samples with different pressure history. DSC scans were performed at 10 K/min at ambient pressure. Inset: second DSC upscans at 10 K/min of all samples following prior up- and downscans at 10 K/min. Discussion Our work has shown that upon annealing at 0.9Tg of a soda lime borate glass compressed up to ,0.6 GPa, the pressure-induced change in CN remains unchanged while the pressurised values of macroscopic properties such as density, refractive index, and hard- ness are relaxing. This suggests that the pressure-induced changes in macroscopic properties of such glasses are not attributed to changes in the short-range order, in agreement with previous studies at rela- tively low pressure30. In sodium boroaluminosilicate glasses it has been shown that while pressure-induced density changes are related to changes in boron coordination, the relatively small difference in partial molar volume of the BO4 and BO3 structural units cannot account for the dominant part of the density change18. Instead it was suggested that the shortening of the Na-O bond upon compression is a more likely factor governing the density change18,31. Such changes were observed at considerably higher pressures than the current study and thus may not in fact account for the findings reported SCIENTIFIC REPORTS | 4 : 3770 | DOI: 10.1038/srep03770 3 www.nature.com/scientificreports Figure 4 | Structural response to annealing. (a) Solid state 11B MAS NMR spectra at 16.4 T of the samples compressed at 570 MPa (solid line) and then annealed for various durations (ta) at 0.9Tg 5 688 K. (b) Evolution of fraction of tetrahedral to total boron (N4), which is obtained through integration of peaks in Fig. 4a, with annealing duration. Inset: impact of isostatic pressure on N4 in absence of annealing (corresponding 11B spectra are shown in Supplementary Figure 2a). The error of N4 is 60.5 at% for the variable pressure series (inset) and 61 at% for the variable annealing time study. The latter has higher error due to changes in lineshape and thus more difficulty in fitting the 11B MAS NMR data. (c) Solid state 23Na MAS NMR p tr t 16 4 T f th mpl mpr d t 570 MP nd th n herein. For example, Allwardt et al. reported a change in 23Na chem- ical shift of 3.5 ppm at 10 GPa, roughly corresponding to a 0.005 nm decrease in Na-O distance32. The chemical shifts for sodium in these soda-lime borates vary slightly with annealing, but within roughly 1– 2 ppm and due to measurement uncertainty, appear to indicate a negligible contribution of Na-O bond distance changes to the density change. Discussion Shannon & Prewitt33 indicate that Na-O bonds should be more compressible than Ca-O bonds. Since Na-O bonds do not change in the detectable range, the contribution of Ca-O bond con- traction under pressure could thus likely be ruled out, and therefore the expansion of these bonds does not occur upon annealing, i.e., should not be the reason for the density decrease. y Although boron speciation and modifier-oxygen bond lengths do not change with increased annealing time, density partially decreases. This might be explained as follows. In addition to coordi- nation numbers and bond distances, bond angle distributions (B-O- B and O-B-O) and superstructures (e.g., boroxol rings34) may change upon annealing16. The change in bond angle and configuration of BIV groups with annealing time could be related to the evolution of the BIV resonance around the frequency shift of 12 ppm in the 11B NMR spectra (Fig. 4a). By isostatic compression, not only the connections of neighbored BO4 tetrahedra, but possibly also the linkages between boroxol rings, become stronger or tighter, and this is accompanied by the narrowing down of the bond angle distribution. Consequently, the total potential energy in the glass decreases with increasing the compression, which is why the glass transition overshoot becomes larger upon DSC upscanning (see Fig. 2a). Once the compressed glass is subjected to annealing around Tg, the ‘‘tightened state’’ of glass will relax, leading to the decrease of density and hardness. However, the thermal energy at 0.9Tg could be insufficient for breaking the B-O bonds in favor of formation of BO3 units, despite the fact that struc- tural relaxation generally occurs even at temperatures below 0.9Tg 35. It should be noted that changes in boron speciation due to annealing at 0.92Tg have previously been reported for non-compressed boro- silicate glasses36 and the thermomechanically trapped state of the glass studied herein thus appears to be different from that of the thermally trapped glass. It is known that the a relaxation is decoupled from the b and c relaxation below Tg 37. Generally, for network glass systems, the a relaxation is dominated by the change of the network connectivity (e.g., the boron speciation), and the b relaxation is controlled by the local motion of structural units (e.g., the change of bond angles). Discussion In this context, it is understandable that the density relaxes upon annealing slightly below Tg, whereas the CN remains unchanged since the former is of the b relaxation feature requiring low temper- ature and the latter is of the a relaxation feature demanding the temperature to exceed Tg. According to literature the BIII-to-BIV ratio increases with increasing temperature above Tg 36. Thus, it is expected that the thermomechanically induced BIV units would be converted to BIII units when the temperature is well above Tg. In contrast to the density decay, the hardness is fully recovered by annealing at 0.9Tg to the original value more quickly, as shown in Fig. 1. This could be related to the dominant contribution of the c relaxation process, or to contributions of hydrated structure relaxations of a near surface volume (OH-groups and molecular water) to the relaxation of hard- ness. The latter has been found recently to proceed more rapidly than both a and b relaxations38. This implies that the hardness relaxation be governed by the fast local network relaxation of the surface layer39. Figure 4 | Structural response to annealing. (a) Solid state 11B MAS NMR spectra at 16.4 T of the samples compressed at 570 MPa (solid line) and then annealed for various durations (ta) at 0.9Tg 5 688 K. (b) Evolution of fraction of tetrahedral to total boron (N4), which is obtained through Figure 4 | Structural response to annealing. (a) Solid state 11B MAS NMR spectra at 16.4 T of the samples compressed at 570 MPa (solid line) and then annealed for various durations (ta) at 0.9Tg 5 688 K. (b) Evolution of fraction of tetrahedral to total boron (N4), which is obtained through integration of peaks in Fig. 4a, with annealing duration. Inset: impact of isostatic pressure on N4 in absence of annealing (corresponding 11B spectra are shown in Supplementary Figure 2a). The error of N4 is 60.5 at% for the variable pressure series (inset) and 61 at% for the variable annealing time study. The latter has higher error due to changes in lineshape and thus more difficulty in fitting the 11B MAS NMR data. (c) Solid state 23Na MAS NMR spectra at 16.4 T of the samples compressed at 570 MPa and then annealed for various durations at 0.9Tg 5 688 K. Methods l Rev. B 76, 014202 (2007). 20. Carini Jr, G., Gilioli, E., Tripodo, G. & Vasi, C. Structural changes and elastic characteristics of permanently densified vitreous B2O3. Phys. Rev. B 84, 024207 (2011). 21. Lee, S. K., Mibe, K., Fei, Y. W., Cody, G. D. & Mysen, B. O. Structure of B2O3 Glass at High Pressure: A 11B Solid-State NMR Study. Phys. Rev. Lett. 94, 165507 (2005). Differential scanning calorimetry. Isobaric heat capacities (Cp) of the investigated samples were determined using a differential scanning calorimeter (DSC Netzsch 404C). DSC runs included measurements of the baseline (two empty Pt–Rh crucibles, 6 mm in diameter, 0.1 mm wall thickness covered with a lid), a sapphire standard (with one crucible containing the standard and the other empty), and finally the sample (with one crucible containing the sample and the other empty). The glass samples were polished to within 1 mm to ensure an accurate fit with the bottom of the crucible and to reach a mass comparable to that of the sapphire standard (,56 mg). Calorimetry was performed under argon flow of 40 ml/min and the glasses were placed on the DSC sample holder at room temperature. Subsequently they were heated to an initial temperature of 313 K, held isothermally for 15 min, and then heated further at a rate of 10 K/min to a temperature 60 K above the Tg in order to fully relax the sample. At the next step, the sample was cooled at a rate of 10 K/min to 313 K, and then held for one hour prior to the following heating, which occurred at a rate of 10 K/min. 22. Brazhkin, V. V. et al. Structural Transformations and Anomalous Viscosity in the B2O3 Melt under High Pressure. Phys. Rev. Lett. 105, 115701 (2010). 23. Nicholas, J. D., Youngman, R. E., Sinogeikin, S. V., Bass, J. D. & Kieffer, J. Structural changes in vitreous boron oxide. Phys. Chem. Glasses 44, 249–251 (2003). 24. Lee, S. K., Eng, P. J., Mao, H.-K., Meng, Y. & Shu, J. F. Structure of Alkali Borate Glasses at High Pressure: B and Li K-Edge Inelastic X-Ray Scattering Study. Phys. Rev. Lett. 98, 105502 (2007). 25. Striepe, S. et al. Elastic and micromechanical properties of isostatically compressed soda-lime-borate glasses. J. Non-Cryst. Solids 364, 44–52 (201 26. Hodge, I. M. Enthalpy relaxation and recovery in amorphous materials. J. Non- Cryst. Solids 169, 211–266 (1994). 27. Yue, Y. Methods l 13. Klug, D. D. et al. Ab initio molecular dynamics study of the pressure-induced phase transformations in cristobalite. Phys. Rev. B 63, 104106 (2001). Sample preparation. Glass with composition (in mol%) of 25Na2O – 10CaO – 65B2O3 was prepared using melt-quenching technique in an inductively heated furnace, as described in details elsewhere25,43. Sample rods (4 3 4 3 30 mm3) were then isostatically compressed using a cold seal pressure vessel under argon gas. For details of the setup, see Ref. 25. The samples were heated under pressure to a temperature around Tg 1 20 K (Tg 5 764 K), followed by equilibration at this temperature for ,3 min, and finally cooled to room temperature at an initial cooling rate of ,3 K/min. The experiments were performed at the following pressures (p): 0.1, 100, 200, 300, 400, 500, and 570 MPa. Relaxation studies were performed on the sample compressed at 570 MPa. This was done by isothermal heat-treatment at 0.9Tg 5 688 K for the following durations (ta): 15, 30, 60, 120, 180, 360, and 1440 min. 14. Huang, L. P. et al. Transformation pathways of silica under high pressure. Nat. Mater. 5, 977–981 (2006). 15. Bauchy, M. & Micoulaut, M. Transport anomalies and adaptative pressure- dependent topological constraints in tetrahedral liquids: evidence for a reversibility window analogue. Phys. Rev. Lett. 110, 095501 (2013). 16. Grimsditch, M., Polian, A. & Wright, A. C. Irreversible structural changes in vitreous B2O3 under pressure. Phys. Rev. B 54, 152–155 (1996). 17. Lee, S. K. et al. Probing of bonding changes in B2O3 glasses at high pressure with inelastic X-ray scattering. Nat. Mater. 4, 851 (2005). 18. Wu, J. et al. Structural response of a highly viscous aluminoborosilicate melt to isotropic and anisotropic compressions. J. Chem. Phys. 131, 104504 (2009). Physical property measurements. Density (r) was measured in ethanol using Archimedes’ principle. Vickers hardness (HV) was determined using a micro- indentor (HMV2000, Shimadzu) operated at a load of 9.81 N at ambient conditions using a dwell time of 15 s. The diagonals of the indentation were measured using a 3D laser scanning microscope (VK-9700K, Keyence). Refractive index (n) of samples before and after the DSC measurements (see below) were performed at 633 nm using a low range Precision Refractometer (Bausch & Lomb). 19. Wondraczek, L., Sen, S., Behrens, H. & Youngman, R. E. Structure-energy map of alkali borosilicate glasses: Effects of pressure and temperature. Phys. www.nature.com/scientificreports enthalpy landscape view of glasses40. At high temperatures (i.e., well above Tg), the system can flow freely among its configurational microstates, corresponding to the case of an ergodic, equilibrium liquid. As the system is cooled, the configurational transitions occur less frequently owing to the loss of thermal energy. At the glass transition, there is a continuous breakdown of ergodicity as the sys- tem gradually becomes trapped in a subset of the available config- urational phase space known as a ‘‘metabasin’’, i.e., a group of configurations that are mutually accessible at a given temperature and for a given observation time41. The relatively slow configura- tional transitions among different metabasins typically involve a concerted series of such transitions. During annealing of the borate glass in this work, there is more thermal energy available for the system to sample the phase space. However, since there is no decrease of N4 during annealing, the boron coordination state is thermome- chanically trapped in a basin in the energy landscape as a con- sequence of the isostatic compression although the macroscopic properties relax on the time scale of the annealing time. This kinetic effect is caused by a high activation barrier in the enthalpy landscape, preventing the boron coordination number from decreasing. Another possibility is the lack of a thermodynamic driving force for the coordination change. According to the random pair model of Gupta, the equilibrium N4 value of the studied composition is 49.2 at%42,43. Hence, the N4 of the compressed glass is closer to the ambi- ent equilibrium value than that of the as-prepared glass (see inset of Fig. 4b), i.e., there is no thermodynamic driving force to convert BIV into BIII units. determined from integration of these resonances and with consideration of a small determined from integration of these resonances and with consideration of a small correction in BIV intensity due to overlapping satellite transitions45. 23Na 3QMAS experiments were conducted also at 16.4 T (185.10 MHz resonance frequency) using a 3.2 mm MAS NMR probe and sample spinning of 20 kHz. The pulse sequence used two hard rf pulses (2.3 and 0.9 ms), followed by a z-filter echo using a delay of 10 ms and a soft reading pulse of 15 ms. 1. Dubrovinsky, L., Dubrovinskaia, N., Prakapenka, V. B. & Abakumov, A. M. Implementation of micro-ball nanodiamond anvils for high-pressure studies above 6 Mbar. Nat. Commun. 3, 1163 (2012). 2. Yang, W. G. www.nature.com/scientificreports et al. Coherent diffraction imaging of nanoscale strain evolution in a single crystal under high pressure. Nat. Commun. 4, 1680 (2013). 3. Mao, H. K. et al. Phonon density of states of iron up to 153 Gigapascals. Science 292, 914–916 (2001). 4. Mishima, O., Calvert, L. D. & Whalley, E. An apparently first-order transition between two amorphous phases of ice induced by pressure. Nature 314, 76–78 (1985). 5. Greaves, G. N. et al. Identifying vibrations that destabilize crystals and characterize the glassy state. Science 308, 1299–1302 (2005). g y 6. Elliot, S. R. Physics of Amorphous Materials (Wiley, New York, 1988 7. Yarger, J. L. et al. Al coordination changes in high-pressure aluminosilicate liquids. Science 270, 1964–1967 (1995). q 8. Guthrie, M. et al. The formation and structure of a dense octahedral glass. Phys. Rev. Lett. 93, 115502 (2004). 9. Meng, Y. et al. The formation of sp(3) bonding in compressed BN. Nat. Mater. 3, 111–114 (2004). 10. Monaco, A. et al. Effect of densification on the density of vibrational states of glasses. Phys. Rev. Lett. 97, 135501 (2006). g y 11. Bouhadja, M., Jakse, N. & Pasturel, A. Structural and dynamic properties of calcium aluminosilicate melts: A molecular dynamics study. J. Chem. Phys. 138, 224510 (2013). 12. Angell, C. A., Cheeseman, P. A. & Tamaddon, S. Pressure enhancement of ion mobilities in liquid silicates from computer simulations studies to 800 kbar. Science 218, 885–887 (1982). Discussion The irreversibility of the thermomechanically induced change in boron speciation upon annealing at 0.9Tg implies that the free energy barrier for forming a given structural state (i.e., boron speciation) can be overcome not only chemically, but also thermomechanically. This allows for tailoring of both microscopic and macroscopic properties, since the increased network connectivity that remains after anneal- ing should increase, e.g., chemical durability and thermal shock resistance. To further understand this effect, we consider the integration of peaks in Fig. 4a, with annealing duration. Inset: impact of isostatic pressure on N4 in absence of annealing (corresponding 11B spectra are shown in Supplementary Figure 2a). The error of N4 is 60.5 at% for the variable pressure series (inset) and 61 at% for the variable annealing time study. The latter has higher error due to changes in lineshape and thus more difficulty in fitting the 11B MAS NMR data. (c) Solid state 23Na MAS NMR spectra at 16.4 T of the samples compressed at 570 MPa and then annealed for various durations at 0.9Tg 5 688 K. SCIENTIFIC REPORTS | 4 : 3770 | DOI: 10.1038/srep03770 4 Author contributions Y.Z.Y. conceived the study. M.M.S., R.E.Y., S.S., M.P. and U.B. prepared the samples, performed the measurements, and contributed to analysis of the data. M.M.S. and Y.Z.Y. wrote the manuscript with inputs from R.E.Y., J.D., H.B. and J.C.M. All authors were involved in the discussions. 37. Deubener, J., Yue, Y. Z., Bornho¨ft, H. & Ya, M. Decoupling between birefringence decay, enthalpy relaxation and viscous flow in calcium boroalumosilicate glasses. Chem. Geol. 256, 298–304 (2008). 38. Reinsch, S, Mu¨ller, R., Deubener, J. & Behrens, H. Internal friction of hydrated soda-lime-silicate glasses. J. Chem. Phys. 139, 174506 (2013). Methods l Z., Wondraczek, L., Behrens, H. & Deubener, J. Glass transition in an isostatically compressed calcium metaphosphate glass. J. Chem. Phys. 126, 144902 (2007). 28. Yue, Y. Z., von der Ohe, R. & Jensen, S. L. Fictive temperature, cooling rate, and viscosity of glasses. J. Chem. Phys. 120, 8053–8059 (2004). Solid state NMR. 11B and 23Na MAS NMR experiments were conducted at 16.T using a commercial spectrometer and MAS NMR probes. Resonance frequencies for 11B and 23Na at this external magnetic field strength were 224.51 and 185.10 MHz, respectively. Samples were crushed using an agate mortar and pestle, packed into 3.2 mm zirconia rotors and spun at frequencies of nominally 20 kHZ. 11B and 23Na MAS NMR spectra were collected using short radio-frequency pulses (0.6 ms, equivalent to p/12 tip angles), relaxation delays of 2 seconds and signal averaging of 1000 to 2000 acquisitions. Data were frequency referenced to aqueous boric acid at 19.6 ppm and aqueous NaCl at 0 ppm for 11B and 23Na, respectively. 11B MAS NMR spectra were fit using DMfit44 to reproduce BIII and BIV lineshapes, and N4 values were 29. Yue, Y. Z. & Angell, C. A. Clarifying the glass-transition behaviour of water by comparison with hyperquenched inorganic glasses. Nature 427, 717–720 (2004). 30. Wolf, G. H. & McMillan, P. F. Pressure effects on silicate melt – Structure and properties. Rev. Mineral. 32, 505–561 (1995). 31. Wondraczek, L., Krolikowski, S. & Behrens, H. Kinetics of pressure relaxation in a compressed alkali borosilicate glass. J. Non-Crys. Solids 356, 1859–1862 (2010). 31. Wondraczek, L., Krolikowski, S. & Behrens, H. Kinetics of pressure relaxation in a compressed alkali borosilicate glass. J. Non-Crys. Solids 356, 1859–1862 (2010). 32. Allwardt, J. R. et al. Aluminum coordination and the densification of high- pressure aluminosilicate glasses. Am. Mineral. 90, 1218–1222 (2005). 32. Allwardt, J. R. et al. Aluminum coordination and the densification of high- pressure aluminosilicate glasses. Am. Mineral. 90, 1218–1222 (2005). SCIENTIFIC REPORTS | 4 : 3770 | DOI: 10.1038/srep03770 5 44. Massiot, D. et al. Modelling one- and two-dimensional solid-state NMR spectra. Magn. Reson. Chem. 40, 70–76 (2002). 33. Shannon, R. D. & Prewitt, C. T. Effective ionic radii in oxides and fluorides. Acta Crystallogr. B 25, 925–946 (1969). y g 34. Youngman, R. E. & Zwanziger, J. W. Network Modification in Potassium Borate Glasses: Structural Studies with NMR and Raman Spectroscopies. J. Phys. Chem. 100, 16720–16728 (1996). 45. Methods l Massiot, D., Bessada, C., Coutures, J. P. & Taulelle, F. A quantitative study of 27Al MAS NMR in crystalline YAG. J. Magn. Reson. 90, 231–242 (1990). 35. Ya, M., Deubener, J. & Yue, Y. Z. Enthalpy and anisotropy relaxation of glass fibers. J. Am. Ceram. Soc. 91, 745–752 (2008). 36. Sen, S., Topping, T., Yu, P. & Youngman, R. E. Atomic-scale understanding of structural relaxation in simple and complex borosilicate glasses. Phys. Rev. B 75, 094293 (2007). Additional information g y 39. Smedskjaer, M. M., Mauro, J. C. & Yue, Y. Z. Prediction of Glass Hardness Using Temperature-Dependent Constraint Theory. Phys. Rev. Lett. 105, 115503 (2010). 40. Stillinger, F. H. & Weber, T. A. Hidden structure in liquids. Phys. Rev. A 25, 978–989 (1982). 39. Smedskjaer, M. M., Mauro, J. C. & Yue, Y. Z. Prediction of Glass Hardness Using Temperature-Dependent Constraint Theory. Phys. Rev. Lett. 105, 115503 (2010). Supplementary information accompanies this paper at http://www.nature.com/ scientificreports 40. Stillinger, F. H. & Weber, T. A. Hidden structure in liquids. Phys. Rev. A 25, 978–989 (1982). Competing financial interests: The authors declare no competing financial interests. How to cite this article: Smedskjaer, M.M. et al. Irreversibility of Pressure Induced Boron Speciation Change in Glass. Sci. Rep. 4, 3770; DOI:10.1038/srep03770 (2014). 41. Gupta, P. K. & Mauro, J. C. The laboratory glass transition. J. Chem. Phys. 126, 224504 (2007). 42. Gupta, P. K. In Proceedings of the International Congress on Glass (New Delhi, India, 1986). This work is licensed under a Creative Commons Attribution 3.0 Unported license. To view a copy of this license, visit http://creativecommons.org/licenses/by/3.0 This work is licensed under a Creative Commons Attribution 3.0 Unported license. To view a copy of this license, visit http://creativecommons.org/licenses/by/3.0 43. Smedskjaer, M. M., Mauro, J. C., Sen, S. & Yue, Y. Z. Quantitative design of glassy materials using temperature-dependent constraint theory. Chem. Mater. 22, 5358–5365 (2010). SCIENTIFIC REPORTS | 4 : 3770 | DOI: 10.1038/srep03770 6
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Impact of meteorological conditions, canopy shading and leaf removal on yield, must quality, and norisoprenoid compounds content in Franciacorta sparkling wine
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OPEN ACCESS OPEN ACCESS EDITED BY Alessandra Ferrandino, University of Turin, Italy REVIEWED BY Silvia Guidoni, University of Turin, Italy Daniela Farinelli, University of Perugia, Italy Maurizio Petrozziello, Council for Agricultural and Economics Research (CREA), Italy *CORRESPONDENCE Isabella Ghiglieno isabella.ghiglieno@unibs.it †PRESENT ADDRESS Isabella Ghiglieno, Department of Civil, Environmental, Architectural Engineering and Mathematics, Agrofood Research Hub, University of Brescia, Brescia, Italy ‡These authors have contributed equally to this work and share first authorship RECEIVED 16 December 2022 ACCEPTED 17 April 2023 PUBLISHED 17 May 2023 CITATION Ghiglieno I, Carlin S, Cola G, Vrhovsek U, Valenti L, Garcia-Aloy M and Mattivi F (2023) Impact of meteorological conditions, canopy shading and leaf l i ld t lit d University of Turin, Italy REVIEWED BY Silvia Guidoni, University of Turin, Italy Daniela Farinelli, University of Perugia, Italy Maurizio Petrozziello, Council for Agricultural and Economics Research (CREA), Italy 1Department of Agricultural and Environmental Sciences - Production, Landscape, Agroenergy, University of Milan, Milan, Italy, 2Metabolomic Unit, Food Quality and Nutrition Department, Research and Innovation Center, Edmund Mach Foundation, S. Michele all’Adige, Italy Climate change is a major concern in agriculture; in grapevine production, climate change can affect yield and wine quality as they depend on the complex interactions between weather, plant material, and viticultural techniques. Wine characteristics are strongly influenced by microclimate of the canopy affecting primary and secondary metabolites of the grapevine. Air temperature and water availability can influence sugar and acid concentration in grapes and relative wines, and their content of volatile compounds such as norisoprenoids. This becomes relevant in sparkling wine production where grapes are generally harvested at a relatively low pH, high acidity, and low sugar content and where the norisoprenoids significantly contributes to the final aroma of the wine. The effect of climate change on grapevine and wine, therefore, calls for the implementation of on-field adaptation strategies. Among them canopy management through leaf removal and shading have been largely investigated in the wine growing sector. The present study, conducted over 4 years (2010-2013) aims at investigating how leaf removal and artificial shading strategies affect grape maturation, must quality and the production of norisoprenoids, analyzed using an untargeted approach, in sparkling wine. Specifically, this paper investigates the effect of meteorological conditions (i.e., water availability and temperatures) and the effect of leaf removal and shading on Vitis vinifera L. cv. COPYRIGHT © 2023 Ghiglieno, Carlin, Cola, Vrhovsek, Valenti, Garcia-Aloy and Mattivi. 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. TYPE Original Research PUBLISHED 17 May 2023 DOI 10.3389/fpls.2023.1125560 TYPE Original Research PUBLISHED 17 May 2023 DOI 10.3389/fpls.2023.1125560 1 Introduction lower soluble sugar content than those for table wine production (Alfonzo et al., 2020). The synthesis of these compounds tends to increase during the herbaceous phase of the berry, while their degradation is enhanced after veraison and during the final stages of ripening. Carotenoids are the precursors of the norisoprenoids, volatile compounds. Norisoprenoids can be generated by the direct degradation of carotenoids such as b-carotene and neoxanthin, and can be stored as glycoconjugates, which can then release their volatile aglycones during wine fermentation or ageing; hydrolysis is often slow and depends on various factors, such as storage duration, temperature, and pH (Winterhalter and Schreier, 1994; Deluc et al., 2009; Song et al., 2012). The olfactory perception thresholds of these compounds are very low and, therefore, they have a high sensorial impact on the wine aroma (Mendes-Pinto, 2009). The most important C13 norisoprenoids are: TDN, vitispirane, actinidols, b-damascenone, b-ionone, and E-1-(2,3,6- trimethylphenyl) buta-1,3diene (TBP-1). Climate change means any changes in weather patterns caused by natural events and human activities during a certain period (IPCC, 2007). The temperature increase that has characterized Europe and Italy since the end of the 1980s has relevant consequences on the quantity and quality of crops, representing a major concern in agriculture (Mariani et al., 2012; Suter et al., 2021). In grape-growing for wine production, climate change can significantly affect yield and, indirectly, wine quality (Fraga et al., 2012), as they depend on the complex interactions between weather variables, plant material, and viticultural techniques (Nesbitt et al., 2016; van Leeuwen et al., 2019; Mirás-Avalos and Araujo, 2021). Air temperature, water availability, and sunlight are the most relevant factors that, influencing grape and must quality, indirectly affect wine quality. Grapes and musts characteristics are, indeed, strongly influenced by climatic conditions as changes in the microclimate of the canopy have an impact on the primary and secondary metabolites of the grapevine. Although the concentration of norisoprenoids in grapes depends on many factors, such as variety and stage of ripeness, the environmental conditions, have a fundamental role in influencing their synthesis. This is confirmed by the behavior of norisoprenoid b-damascenone that decreases in white wines in conditions of exposure to light and higher temperature (Marais et al., 1992; Kwasniewski et al., 2010). OPEN ACCESS Chardonnay and Pinot noir, which are suitable to produce sparkling wine in the DOCG Franciacorta wine growing area (Lombardy, Italy). The effect of leaf removal and shading practices on norisoprenoids has been the focus of the study. No defoliation and artificial shading treatments play an important role in the preservation of the acidity in warm seasons and this suggests calibrating defoliation activities in relation to the meteorological trend without standardized procedures. This is particularly relevant in the case of sparkling wine, where the acidity is essential to determine wine quality. The CITATION Ghiglieno I, Carlin S, Cola G, Vrhovsek U, Valenti L, Garcia-Aloy M and Mattivi F (2023) Impact of meteorological conditions, canopy shading and leaf removal on yield, must quality, and norisoprenoid compounds content in Franciacorta sparkling wine. Front. Plant Sci. 14:1125560. doi: 10.3389/fpls.2023.1125560 01 Frontiers in Plant Science frontiersin.org Ghiglieno et al. 10.3389/fpls.2023.1125560 enhanced norisoprenoid aromas obtained with a total defoliation represent a further element to direct defoliation and shading strategies. The obtained results increase knowledge about the effect of different defoliation and artificial shading applications in relation to meteorological condition supporting the management decision-making in the Franciacorta wine growing area. Frontiers in Plant Science vine (Vitis vinifera L.), leaf removal, grapevine shading, must quality, yield, sparkling wine, aroma compounds, norisoprenoid frontiersin.org KEYWORDS Ghiglieno et al. 2.1 Experimental setting This study was carried out in the winegrowing area of Franciacorta DOCG, a famous Italian sparkling wine production area located in the Lombardy Region. The experiment was repeated for four consecutive years (from 2010 to 2013) in a vineyard (45.572850° Lat, 9.967622° Lon) belonging to Azienda Agricola Castello Bonomi Tenute in Franciacorta. This company is located in the southern part of the Franciacorta region (Supplementary Figure 1). The soil of experimental vineyard is grass-covered and characterized by a loamy texture, high total organic carbon content and CEC (Cation Exchange Capacity). The vineyard, with row orientation from north to south, was planted in 2004 with two international Vitis vinifera L. cultivars, Chardonnay (clone ENTAV-INRA® 96) and Pinot noir (clone 292), both grafted onto Kober 5BB rootstock. Both cultivars are cordon trained. The experimental plan included five different treatments: a control test without defoliation and shading (ND), a test where about six basal leaves, equal to about 35% of total leaf area, were removed, east and west side (TD), and three different systems adopting shading nets applied along the bunch zone; two of the shaded treatments were defoliated as for TD and covered with one layer of shading net (TD1) or two layers of shading net (TD2), while a third treatment was covered by only one layer of shading net, but not defoliated (ND1L). The present 4-year (from 2010 to 2013) study aims to investigate how leaf removal and artificial shading strategies affect grape maturation, must quality and the production of norisoprenoids in sparkling wine. Wine volatiles compounds were analyzed using an untargeted approach aimed at a holistic view rather than restricted only to a list of target compounds (Cozzolino, 2016). Specifically, the paper investigates the effect of meteorological conditions (i.e., water availability and temperatures) on grapevine production and must quality. Moreover, the practice of application of different canopy shading levels on Vitis vinifera L. cv. Chardonnay and Pinot noir is studied to understand their effect on grapevine production and must quality as well as the on the aroma of sparkling wine in the DOCG Franciacorta wine growing area (Lombardy, Italy). 2.1 Experimental setting Understanding the response of musts and wine quality to leaf removal and shading, in relation to the meteorological variability of each year, is useful to direct agronomical management in tackling the issue of climate change (e.g., early ripening, acidity degradation, wine aroma profile variation) that is affecting sparkling wine quality in particular. The leaf removal and shading practices on norisoprenoids compounds shall also be examined. The concentrations of these aromatic compounds tend to increase during wine aging, especially in sparkling wines, and can reach the level above the threshold of the characteristic aroma of aged wine which is sometimes detrimental for the wine quality. Since there aren’t, to our knowledge, studies that had been conducted on the modulation of norisoprenoids in relation to canopy management in finished sparkling wine, this work could be useful to understand the effect of these treatments in the final product. The treatments were replicated on both the Chardonnay and Pinot noir cultivars. All treatments were set in two replicates represented by two rows of the vineyard for each cultivar. Each treatment was extended for 25 vines for each of the two replicates. All treatments took place at about 20% veraison, corresponding to July 19th, July 14th, July 19th, July 29th, respectively, in 2010, 2011, 2012 and 2013. Both manual leaf removal and shading net application were carried out along the bunch zone; shading was realized through a UVstabilized polyethylene net of approximately 95 g m-2 (shading net OF50N supplied by Retes srl). The transmittance of global solar radiation of the single layer and double layer nets was preliminarily tested in order to evaluate the percentage of global solar radiation passing through the nets. A reduction by 50% and 70% was detected for single- and double- layer shading nets, respectively. 1 Introduction Water availability and water stress lead to different effects depending on the grapevine developmental stage, cultivar and wine target (Deloire et al., 2004; Santos et al., 2020; Mirás-Avalos and Araujo, 2021). During post-veraison excessive humidity tends to promote sugar dilution (Reynolds and Naylor, 1994), delaying the harvesting time (Tonietto, 1999) and avoiding an excessive accumulation of total soluble solids (Intrigliolo et al., 2016). In general, the water status correlates positively with berry size, total acidity, and malic acid concentration (Mirás-Avalos and Intrigliolo, 2017). The effect of climate change on grapes, musts and, consequently, on wine quality, therefore, calls for the implementation of on-field adaptation strategies. Among these, the management of the canopy through leaf removal and shading have been largely investigated in the wine growing sector (Downey et al., 2006; Caravia et al., 2016; Ghiglieno et al., 2020; Martı́nez- Lüscher et al., 2020). The influence of these agronomic practices on canopy microclimate has been widely studied (Crippen and Morrison, 1986; Jackson and Lombard, 1993; Downey et al., 2006; Ghiglieno et al., 2020). The direct solar radiation on grape caused by leaf removal and the consequent increase in berry temperature during maturation, influence berry ripening and metabolism, promoting sugar contents accumulation (Riou et al., 1994), reducing titratable acidity, and increasing malic acid degradation (Lakso and Kliewer, 1975; Conde et al., 2006; de Oliveira et al., 2019). Moreover, excessive sunlight inhibits the development of flavor and aroma components (Jones et al., 2005). Much has been written about how canopy management practices affect the content Many studies focused on the effect of temperature increases on grapevine and wine quality (Biasi et al., 2019; Santos et al., 2020). The mean air temperatures during the growing season are directly related to the length of the growing season for each variety (Jones et al., 2005); the different stages of development generally take place earlier and the time between veraison and ripening is shorter (Schultz, 2000; Jones et al., 2005). Excessively hot weather during the veraison–maturity period can significantly influence sugar accumulation (Greer, 2013), reducing the level of acidity in grapevines and, consequently, in wines (Pons et al., 2017). This becomes relevant in grapevines suitable to produce sparkling wines. In sparkling wines production grapevines are in fact generally harvested at a relatively low pH, higher titratable acidity, and 02 frontiersin.org Ghiglieno et al. 10.3389/fpls.2023.1125560 2 Materials and methods in norisoprenoids, especially TDN and vitispirane (Marais et al., 1992; Chen et al., 2017; Asproudi et al., 2018; Wang et al., 2018). Some authors (Kwasniewski et al., 2010) have indeed demonstrated that the timing of leaf removal can alter the carotenoid profile, as well as TDN and vitispirane precursors in mature Riesling grapes: if the leaves were removed one month after the berry set, they observed elevated total amounts in both must and wine. With regard to grapevine shading, although previous studies reported that artificial shading protects grapes from sunlight exposure but leads to an increase of air temperature in the fruit zone (Chorti et al, 2010), other researches carried out in Franciacorta wine growing area demonstrated that this agronomical practice also allows to decrease inner berry temperature (Ghiglieno et al., 2020). This effect of artificial on canopy microclimate makes this practice a good adaptation strategy to climate change. Many authors (Reynolds et al., 1986; Smart et al., 1990; Dokoozlian and Kliewer, 1996; Filippetti et al., 2015; Martin et al., 2016) have in fact demonstrated the effect of shading on delaying ripening and preserving acidity, in terms of both titratable acidity and malate concentration. The response of pH and potassium to artificial shading is less clear: some authors demonstrated a positive effect of shading on pH and potassium (Smart et al., 1985; Scafidi et al., 2013; Toda and Balda, 2014), while more recent studies reported that this treatment does not significantly affect these parameters (Filippetti et al., 2015). in norisoprenoids, especially TDN and vitispirane (Marais et al., 1992; Chen et al., 2017; Asproudi et al., 2018; Wang et al., 2018). Some authors (Kwasniewski et al., 2010) have indeed demonstrated that the timing of leaf removal can alter the carotenoid profile, as well as TDN and vitispirane precursors in mature Riesling grapes: if the leaves were removed one month after the berry set, they observed elevated total amounts in both must and wine. With regard to grapevine shading, although previous studies reported that artificial shading protects grapes from sunlight exposure but leads to an increase of air temperature in the fruit zone (Chorti et al, 2010), other researches carried out in Franciacorta wine growing area demonstrated that this agronomical practice also allows to decrease inner berry temperature (Ghiglieno et al., 2020). This effect of artificial on canopy microclimate makes this practice a good adaptation strategy to climate change. 2 Materials and methods Many authors (Reynolds et al., 1986; Smart et al., 1990; Dokoozlian and Kliewer, 1996; Filippetti et al., 2015; Martin et al., 2016) have in fact demonstrated the effect of shading on delaying ripening and preserving acidity, in terms of both titratable acidity and malate concentration. The response of pH and potassium to artificial shading is less clear: some authors demonstrated a positive effect of shading on pH and potassium (Smart et al., 1985; Scafidi et al., 2013; Toda and Balda, 2014), while more recent studies reported that this treatment does not significantly affect these parameters (Filippetti et al., 2015). Frontiers in Plant Science 2.2.1 Water stress * When the hourly air temperature is below LC: LHH=1, NHH=0, HHH=0, meaning that the whole hour was spent by the plant under stressing conditions caused by low temperatures. In order to provide a description of the relationship between plant growth and water resources, a single-layer reservoir model with a daily time step was created (Cola et al., 2014), considering a reference soil reservoir of 120 mm. The daily reference evapotranspiration (ET0) was calculated by means of the Hargreaves-Samani method and the daily maximum evapotranspiration of grapevine (ETM) was obtained by means of a dynamic crop multiplicative coefficient (Kc), as a function of the phenological stage of the plant, modelled on the base of air temperature-based course (Mariani et al., 2013). The daily real grapevine evapotranspiration (ETR) was obtained by means of the water limiting factor (WLFR) response curve, that relates the soil water content with the plant stress. When the soil water level is between the field capacity and the easily available water limit, the plant does not face stress, WLFR is equal to 1 and ETR=ETM. Otherwise, when the water content moves above the field capacity up to saturation or when it moves from the easily available water limit down to the wilting point, WLFR linearly decreases toward 0. As a consequence, the stress increases and ETR decreases. * When the hourly air temperature is above UC: LHH=0, NHH=0, HHH=1 meaning that the whole hour was spent by the plant under stressing conditions caused by high temperatures. * When the hourly air temperature sits between LOC and UOC: LHH=0, NHH=1, HHH=0 meaning that the whole hour was spent by the plant under restful conditions and the development was non limited. * Between LC and LOC the low temperature stress decreases linearly moving from LC to LOC, so that LHH moves from 1 to 0 and NHH from 0 to 1 (always with LHH+NHH=1 and HHH=0). * Between UOC and UC the high temperature stress increases linearly moving from UOC to UC so that NHH moves from 1 to 0 and HHH from 0 to 1 (always with NHH+HHH=1 and LHH=0). In this work, the seasonal water stress (WSTR) was obtained cumulating the daily values of 1-WLFR, so that a day without water stress weights 0 and a day with maximum water stress weights 1. 2.2.2 Thermal regime In this work, NHH, LHH and HHH were cumulated from fruit- set to harvest to describe the thermal course of the season. Phenological stages of each plot were determined on the base of weekly phenological monitoring. In order to describe the air thermal condition during fruit development and ripening, thermal resources and limitations were obtained on the basis of the air temperature, applying the Normal Heat Hours (NHH) response curve (Mariani et al., 2012; Mariani et al., 2013; Cola et al., 2020). This method tries to overcome the overestimation of high temperature in terms of plant development that characterizes the classic growing degree-days approach of the Winkler Index (Amerine and Winkler, 1944) in which a very warm day is translated into a high value of the index, meaning a strong positive contribution to plant growth when high air temperature values are detrimental for the plant development. Additionally, the stress caused by heat waves (33STR) was obtained by counting the number of days between fruit set and physiological maturity with a maximum temperature above 33°C (Cola et al., 2020). 2.2 Meteorological data and agrometeorological indices During the four years, weather conditions were monitored by means of the weather station of Rovato (BS) of the Agrometeorological Network of the Province of Brescia, which is located at a distance of 5 km from the experimental vineyard. To provide a more precise description of the thermal conditions in the experimental vineyard a four-channel data logger (Onset Frontiers in Plant Science 03 frontiersin.org Ghiglieno et al. 10.3389/fpls.2023.1125560 10.3389/fpls.2023.1125560 NHH indicates the total hourly thermal resources useful for berry maturation from fruit set to physiological maturity (Cola et al., 2017; Cola et al., 2020) and represent thermal resources. Low Heat Hours (LHH) represents the stress caused by hourly temperatures below the optimal level and High Heat Hours (HHH) is the stress caused by hourly temperatures above the optimal level. NHH indicates the total hourly thermal resources useful for berry maturation from fruit set to physiological maturity (Cola et al., 2017; Cola et al., 2020) and represent thermal resources. Low HOBO U12) equipped with an air temperature sensor (thermistor Onset HOBO S-THC), protected by a solar shield was placed in the vineyard. Precipitation data recorded by the Rovato weather station and temperature data from the vineyard thermometer were used to characterize the general meteorological features of the four years of investigation and to calculate thermal indices, the grapevine water balance and the derived water stress indices, as described further below. Heat Hours (LHH) represents the stress caused by hourly temperatures below the optimal level and High Heat Hours (HHH) is the stress caused by hourly temperatures above the optimal level. More in detail, air temperature is weighed on the base of four parameters, namely, low cardinal - LC, low optimal cardinal – LOC, upper optimal cardinal – UOC and upper cardinal UC, respectively: 2.2.1 Water stress Additionally, the cumulated evapotranspiration deficit (DET) was obtained, on the base of the daily difference [mm] between ETM and ETR throughout the season. The parametrization of the four parameters (LC=12°C, LOC=24°C, UOC=26°C, UC=33°C) proved to perform very well in describing the development of several cultivars (Cabernet- Sauvignon, Chardonnay, Barbera and Georgian cultivars Mtsvane Kakhuri, Rkatsiteli, Ojaleshi and Saperavi) (Mariani et al., 2013; Cola et al., 2014; Cola et al., 2017). Frontiers in Plant Science frontiersin.org 2.3 Yield components and the composition of must Indices were calculated considering meteorological conditions along the berry development period (i.e., from berry set stage to harvest) and specifically: from June 1st to August 26th for 2010; from May 20th to August 1st 2011; from May 29th to August 8th 2012; from June 6th to August 22nd 2013. With the aim to compare musts and wine composition at a standard potential alcohol degree, for both cultivars and all treatments, the harvesting time was established on the basis of the 04 frontiersin.org Ghiglieno et al. 10.3389/fpls.2023.1125560 strategy adopted by the wine company for the specific year and ranging from 10 to 10.7% of potential alcohol. each sample, 2 ml of wine with 50 ml of internal standard solution (2- octanol in ethanol at a concentration of 1 mg l-1) was mixed with 1 g NaCl in a 20 ml headspace vial. A Gerstel MultiPurpose Sampler autosampler (Gerstel GmbH & Co. KG Mülheim an der Ruhr Germany) with an agitator and SPME fiber 2 cm (50/30 DVB/ CAR/PDMS), from Supelco Merk KGaA (Darmstadt, Germany), was used to extract the volatiles from the sample vial headspace. The sample was pre-incubated for 5 min at 35°C. Adsorption lasted 20 min, at the same temperature, Then, desorption took place in the injector in splitless mode (4 min) at 250°C. The fiber was then reconditioned for 10 min at 250°C. The GC×GC system was the Agilent 7890 A (Agilent Technologies, Santa Clara, CA). Injections were performed in splitless mode. Equipped columns were the VF- Wax column (100% polyethylene glycol; 30 m × 0.25 mm × 0.25 mm, Agilent J&W Scientific Inc., Folsom, CA) as the 1st dimension and Rxi-17Sil MS 1.50 m × 0.15 mm × 0.15 mm, Restek Bellefonte, USA) as the 2nd dimension. The GC system was equipped with a secondary column oven and non-moving quadjet dual-stage thermal modulator. The injector/transfer line was maintained at 250°C. Oven temperature program conditions were as follows: initial temperature of 40°C for 4 min, programmed at 6°C min−1 at 250° C, where it remained for 5 min. The secondary oven was kept at 5°C above the primary oven throughout the chromatographic run. The modulator was offset by +15°C in relation to the secondary oven; the modulation time was 7 s and 1.4 s of hot pulse duration. Helium (99.9995% purity) was used as carrier gas at a constant flow of 1.2 ml min-1. 2.4 Wine production About 100 kg of grapes were harvested for each cultivar and treatment upon reaching the standard potential alcohol. Grapes were harvested in small boxes weighing maximum 15 kg, and stocked in a cold room at about 10°C. Microvinifications was then initiated according to a standardized protocol. The wine- making protocol was organized in different steps described in Supplementary Figure 2. For GC×GC-MS data, LECO ChromaTOF Version 4.22 software was used for all acquisition control, data processing and Fisher ratio calculations. Automated peak detection and spectral deconvolution with a baseline offset of 0.8 and signal-to-noise of 100 were used during data treatment. Before proceeding with the data analysis a quality control of the data sets, checking the distribution of the QC injections was carried out (Arapitsas et al., 2016). The identification of the wine volatile compounds was achieved by comparing the mass spectrometric information for each chromatographic peak with NIST 2.0, Wiley 8 and the FFNSC 2 mass spectral library (Chromaleont, Messina, Italy), with a library similarity match factor of 750 and comparing also the experimental linear temperature retention index (LTPRI) with retention indices reported in the literature for 1D-GC (VCF Volatile Compounds in Food 16.1 database). Retention data for a series of n-alkanes (C10– C30), under the same experimental conditions employed for chromatographic analysis of wine volatiles, were used for experimental LTPRI calculation. The content of norisoprenoids were expressed as peak areas. The wine so obtained was stocked in 15 l demijohns and periodically monitored for free sulfur content, using the WineScan™SO2 (FOSS) instrument, to avoid undesired malic fermentation. After about five months the wine was stabilized by adding 10 ml hl-1 silica sol and 1 ml hl-1 gelatin. The wine was then bottled using 0.75 l bottles (for a total of 18 bottles); nine bottles were then stocked and 0.3 g l-1 of yeast and 22 g l-1 of sugar were added to the other nine bottles to induce refermentation, thus allowing to produce a final sparkling wine. After this last step, all bottles were stocked and manually disgorged, after a manual remuage to remove the residue of yeasts without any further addition. Disgorgement was performed for all wine samples at the time of the volatile aroma compounds analysis in 2016 and 3 different bottles for each treatment were used as replicates. 2.3 Yield components and the composition of must The MS signal was obtained with a Pegasus IV time-of flight mass spectrometer (Leco Corporation, St. Joseph, MI) with electron ionization at 70 eV and the ion source temperature at 230°C, detector voltage of 1317 V, mass range of m/z 35–450 and acquisition rate of 200 spectra s−1. The evolution of ripening for both Chardonnay and Pinot noir was monitored weekly (maintaining an interval of 7-5 days between each sampling), in order to identify the relevant harvesting time for each treatment. Experimental harvesting was organized by selecting 5 grapevines from the two 25 vine replicates for each treatment, for a total of 10 vines per treatment (ND, TD, TD1, TD2 and ND1L). For each grapevine, the average bunch weight (ABW) was calculated counting the number of bunches and weighing the entire grapevine production. A sample of three bunches was collected from each grapevine to check must quality. These samples were then crushed and the total soluble solids concentration (TSS), pH, titratable acidity (TA) and malic acid (MA) concentration were analyzed in the resulting musts. These measurements were determined using a traditional handheld refractometer for soluble solids concentration, a Crison compact titrator analyser for both pH and TA, and the enzymatic method (Hyperlab wine analyser) to determine MA concentration. 2.5 Analysis of volatile aroma compounds by GC×GC-TOF-MS All the analyses were carried out following the method described in our previous works (Carlin et al., 2016; Carlin et al., 2022). In order to monitor instrument stability, pooled quality controls (QC) consisting of equal proportion of each sample were placed at the beginning of the run (n=5) and thereafter every 10th sample as a common practice in metabolomics studies (Arapitsas et al., 2016). For Frontiers in Plant Science frontiersin.org 3.1 Meteorological characterization Considering the limited number of biological replicates used for the untargeted volatile compounds analyses (i.e., 3 replicates for each cultivar, treatment, and year) a different approach was outlined. In this case, it was checked whether there were any statistically significant differences associated with each treatment compared to the untreated thesis (ND) by means of a linear model on log-scaled data using R software (R Core Team, 2022). The year 2012 was characterized by the highest water stress value over the four years (as shown by WSTR and DET values), mainly due to the limited precipitation during June, July and August that led to a long period of a limited water soil content. 3.1 Meteorological characterization Table 1 shows the monthly variation of temperature and precipitation by season, respectively, while the agrometeorological indices are shown in Table 2. The relationships between year and agrometeorological indices and AWB and must quality were also investigated through a principal component analysis (PCA) on autoscaled data (i.e., mean-centered and divided by the standard deviation of each variable), through R software (R Core Team, 2022). In the PCA analysis for each cultivar, five points for each year, corresponding to the average value of the single treatment in a specific year, were considered. This approach was used because one single value of agrometeorological indices was available for each year without having the possibility to differentiate between treatments. The year 2010 was characterized by optimal water conditions. The scarce precipitation of the winter period (Jan-Mar) was preceded by good precipitations in the 2009 Oct-Dec period and followed by abundant spring precipitations. From a thermal point of view, 2010 showed lower maximum temperatures during spring than 2011 and 2012, resulting in the highest NHH value in the series. However, the indices for this year were strongly affected by a longer interval between fruit set and harvest, than in the other three years. Low temperature stress (LHH) was the second highest over the four years and high temperature stress (HHH) was the highest. The 33°C limit was exceeded for 65 days during the season. Additional ANOVA and Siegel-Tukey’s post-hoc test were performed to determine the influence of the treatment on variables related to ABW and musts quality (TSS, TA, MA, pH). In this analysis, both the cultivars and the four years were considered separately. The decision to process the different years separately was determined by the meteorological differences in the individual years and the effect on the AWB and must quality revelead by the results obtained from the analysis of the relationship between year and these variables. The year 2011 was still characterized by very low water stress, thanks to the refilling of the water reservoir during winter and the good distribution of precipitation over the months. As compared to other years, low temperatures caused a high level of NHH (second highest), the highest value of low temperature stress (LHH) and the lowest value of high temperature stress (HHH), confirmed by only 2 days with maximum temperatures above 33°C. 2.6 Statistical analysis In the statistical analysis, Chardonnay and Pinot noir were kept separated in relation to the different composition of their grapes and 05 frontiersin.org Ghiglieno et al. 10.3389/fpls.2023.1125560 3 Results their different sensory characterization in wines (Herrero et al., 2016). The effect of the year was initially investigated for variables related to ABW and musts quality through an analysis of variance (ANOVA) and Siegel-Tukey’s post-hoc test using the SPSS software (Statistical Package for Social Science) (IBM Corp, 2021). Frontiers in Plant Science frontiersin.org 3.1 Meteorological characterization From a thermal point of view, the year 2012 featured the lowest levels in terms of NHH accumulation (though very close to 2013), Month Air Temperature [°C] Monthly Precipitation [mm] Minimum Maximum 2010 2011 2012 2013 2010 2011 2012 2013 2010 2011 2012 2013 Jan -1.3 (2.2) -0.6 (2.2) -1.4 (2.7) 0.4 (1.7) 4.9 (2.8) 6.1 (2.3) 8.8 (3.8) 7.3 (3.8) 32 29 26 59 Feb 0.9 (3.1) 1.7 (2.7) -2.8 (4.8) -0.1 (2.0) 8.9 (3.2) 11.4 (3.0) 7.3 (6.8) 7.6 (3.1) 93 62 12 61 Mar 3.7 (4.4) 5.2 (3.5) 6.8 (2.6) 3.8 (2.3) 13.5 (4.8) 14.7 (4.2) 19.3 (3.9) 11.3 (3.6) 49 46 11 137 Apr 8.1 (3.2) 10.1 (2.0) 7.9 (2.6) 9.4 (3.3) 19.9 (4.2) 23.0 (3.3) 17.8 (3.7) 18.5 (5.0) 71 13 146 96 May 12.2 (2.1) 12.9 (3.1) 12.1 (3.0) 10.9 (2.3) 23.1 (3.9) 26.5 (3.2) 24.5 (4.0) 22.1 (3.3) 165 44 128 180 Jun 16.8 (2.9) 16.0 (1.5) 17.4 (2.8) 15.5 (3.2) 28.7 (3.7) 26.9 (2.9) 30.0 (3.8) 28.7 (3.9) 94 122 33 53 Jul 19.2 (2.8) 16.7 (2.5) 18.6 (1.9) 19.2 (1.9) 32.0 (2.6) 28.7 (2.2) 32.2 (1.9) 32.1 (1.9) 91 79 39 50 Aug 16.7 (2.8) 18.2 (2.3) 19.7 (2.4) 17.5 (2.3) 29.2 (3.2) 31.0 (2.3) 33.5 (3.2) 30.9 (3.2) 154 93 35 109 Sep 13.1 (2.4) 16.4 (2.4) 14.4 (2.6) 14.3 (2.5) 24.8 (3.0) 28.3 (2.4) 25.9 (3.1) 26.1 (3.2) 105 143 125 23 Oct 8.1 (3.3) 8.5 (3.9) 10.5 (3.5) 11.8 (2.4) 17.5 (3.4) 20.2 (5.5) 19.9 (4.5) 19.0 (2.3) 128 60 146 91 Nov 6.0 (3.9) 4.6 (4.6) 6.8 (2.5) 5.6 (4.5) 12.6 (3.6) 13.9 (2.9) 14.3 (2.5) 13.4 (4.2) 178 63 140 111 Dec -1.7 (3.7) 1.6 (3.0) -0.9 (2.5) 1.4 (3.0) 5.3 (3.5) 9.4 (2.3) 7.2 (3.6) 10.9 (2.7) 151 32 68 62 06 10.3389/fpls.2023.1125560 Ghiglieno et al. TABLE 2 Environmental indices for the characterization of the four seasons. TABLE 2 Environmental indices for the characterization of the four seasons. WSTR DET NHH LHH HHH 33STR 2010 3.50 4.30 1276 412 324 65 2011 2.86 8.62 1151 468 140 2 2012 24.83 79.18 1020 288 315 93 2013 18.95 52.91 1075 315 321 99 and LHH and the highest level in HHH (thermal excess, with 93 days of maximum temperature above 33°C). the cv. Chardonnay, and from 26/08 to 6/09 for Pinot noir. 3.2.3 Must characterization Considering the thermal behavior in the four seasons in relative terms, in 2010 the percentages of NHH, LHH and HHH versus their total (NHH+LHH+HHH) were 63.85, 18.23 and 17.92 respectively. 2012 and 2013 showed a similar behavior with 62.85, 17.78, 19.38 and 62.81, 18.41, 18,77 respectively. The year 2011, with 65.41, 26.62, 7.97 showed a strongly different repartition, again emphasizing its unique environmental conditions. The results obtained in must characterization (Figure 2) underlined a similar response concerning TSS when comparing Chardonnay and Pinot noir, while some differences were observed in terms of acidity and pH. TSS was higher in 2012, while 2010 recorded the lowest value; in the case of Pinot noir no significant differences were recorded between 2012 and 2011. The year 2012 also showed a high TA value for both cultivars, as in 2011 for Chardonnay and in 2013 for Pinot noir. The year 2010 recorded the lowest TA value for Pinot noir, while no significant differences were recorded compared to the 2013 value for Chardonnay. 3.2.1 Average bunch weight The ABW varies over the years equally for both cultivars (Figure 1). The year 2012 showed a significantly lower value of ABW than that of the other years. The year 2013 showed an intermediate response, while 2011 and 2010 recorded higher values. Results related to MA were similar to TA: for Pinot noir, the year 2012 differed significantly from 2010, recording the highest and the lowest values, respectively; in the case of Chardonnay the highest value was recorded in 2011 and the lowest value in 2013. pH values were high in 2010 for both cultivars, as in 2012 for Chardonnay. Pinot noir showed no differences over 2011, 2012 and 2013 for this variable, while Chardonnay showed the lowest value in the case of 2013. 3.1 Meteorological characterization The delay in ripening was further confirmed by the lower average sugar content reached at harvesting time (i.e., 18°Brix). The years 2011 and 2012 had the earliest vintages, while year 2013 can be considered in between, having recorded harvesting times from 28/ 08 to 2/09 for Chardonnay and from 20/08 to 24/08 for Pinot noir. The year 2013 was very similar to 2012, with a certain amount of water stress (second highest in both indices), the second lowest in NHH and LHH, and the second highest in HHH and the first in the number of days with a maximum temperature above 33°C (99 days). 3.2.4 Years general responses concerning vectors related to average bunch weight (ABW) and musts quality variables (pH, TSS, TA, MA) confirms the findings previously reported in Sections 3.2.1 and 3.2.3. The biplots obtained from PCA analysis allowed to visualize the distributions of the data collected for Chardonnay and Pinot noir during the four-year 2010-2013 period (Figure 3). The pattern in the sample distribution highlighted a differentiation, especially between the years 2010, 2011 and 2012-2013. In both PCAs, the first two dimensions explained around 75% of variance. Vectors made it possible to display the inner relationship between years and variables (i.e., meteorological variables and ABW and must quality). Supporting the results described in the previous Section 3.1, the years 2012 and 2013 were associated, for both cultivar, to stress caused by water scarcity, higher evapotranspiration (DET) and temperatures (HHH and 33STR), while 2011 was associated with lower temperatures (LHH). The pattern in the distribution of years 3.2.2 Harvesting time The four years were characterized by different harvesting times due to varying ripening rates. The year 2010 was characterized by a delayed maturation. Harvesting times ranged from 24/08 to 2/09 for FIGURE 1 Boxplots showing average bunch weight (ABW in grams) over the 4 different vintages for Chardonnay (CH) and Pinot noir (PN): different letters indicate that values are significantly different at p < 0.05 obtained by one-way ANOVA and Siegel-Tukey’s post-hoc test. FIGURE 1 Boxplots showing average bunch weight (ABW in grams) over the 4 different vintages for Chardonnay (CH) and Pinot noir (PN): different letters indicate that values are significantly different at p < 0.05 obtained by one-way ANOVA and Siegel-Tukey’s post-hoc test. 07 07 Frontiers in Plant Science frontiersin.org Ghiglieno et al. 10.3389/fpls.2023.1125560 FIGURE 2 Boxplots showing data of total soluble solids concentration (TSS), titratable acidity (TA), malic acid (MA) and pH over the 4 different vintages for Chardonnay (CH) and Pinot noir (PN): different letters indicate that values are significantly different at p < 0.05 obtained by one-way ANOVA and Siegel-Tukey’s post-hoc test. Boxplots showing data of total soluble solids concentration (TSS), titratable acidity (TA), malic acid (MA) and pH over the 4 different vintages for Chardonnay (CH) and Pinot noir (PN): different letters indicate that values are significantly different at p < 0.05 obtained by one-way ANOVA and Siegel-Tukey’s post-hoc test. 3.3.1 Average bunch weight The results obtained for Chardonnay ABW by comparing treatments showed no homogeneous response over the years (Figure 4). In general, no significant differences were found between treatments. A significant difference emerged in 2011 between the less shaded (i.e., TD and TD1) and high shaded (i.e., ND1L, and TD2) treatments. Pinot noir recorded a generally FIGURE 3 Biplots showing the distribution of data along of the two first principal component dimensions for Chardonnay (CH) and Pinot noir (PN). Stress caused by heat waves (33STR), High Heat Hours (HHH), daily evapotranspiration deficit (DET), seasonal water stress (WSTR), Normal Heat Hours (NHH), Low Heat Hours (LHH), average bunch weight (ABW), total soluble solids concentration (TSS), titratable acidity (TA), malic acid (MA), pH are shown as vectors. Data from a single year is identified by different colored dots. Each dot represents the average value of the single treatment in a specific year. FIGURE 3 Biplots showing the distribution of data along of the two first principal component dimensions for Chardonnay (CH) and Pinot noir (PN). Stress caused by heat waves (33STR), High Heat Hours (HHH), daily evapotranspiration deficit (DET), seasonal water stress (WSTR), Normal Heat Hours (NHH), Low Heat Hours (LHH), average bunch weight (ABW), total soluble solids concentration (TSS), titratable acidity (TA), malic acid (MA), pH are shown as vectors. Data from a single year is identified by different colored dots. Each dot represents the average value of the single treatment in a specific year. 08 Frontiers in Plant Science frontiersin.org Ghiglieno et al. 10.3389/fpls.2023.1125560 this year, the defoliated treatment recorded a higher sugar concentration than ND1L. Regarding titratable acidity (TA) and malic acid concentration (MA), the treatments revealed similar trends in 2010 and 2011. Considering these two years, the totally defoliated (TD) and defoliated and shaded (TD1 and TD2) treatments recorded the lowest values in both titratable acidity and malic acid concentration, with the exception of the results obtained for TD1, which in 2010 were similar to those obtained for the not defoliated treatments (ND and ND1L). Significant differences were highlighted only in the case of titratable acidity in 2012 and in the case of malic acid concentration in 2013; in these cases, ND1L and TD recorded the highest values. pH didn’t show significant differences in 2010. As described in the case of Chardonnay, the trend observed for pH was not homogeneous over the years. 3.3.1 Average bunch weight negative effect of defoliation without shading (TD) on ABW in 2012 and 2013 (Figure 4). The years 2010 and 2011 showed a different response, reporting an intermediate value for TD. Other treatments related to defoliation and shading (TD1 and TD2) showed the opposite response in 2010 and 2011, resulting in a higher ABW in 2010 compared to no defoliated treatment (ND and ND1L) and a lower ABW in 2011 compared to ND and ND1L. 3.3.3 Effect on sparkling wine norisoprenoids As indicated in the Section 2.6 statistical analyses performed on norisoprenoids abundances were restricted to check whether, within a specific cultivar and year, there were statistically significant differences associated to each treatment compared to reference treatment, i.e. the not defoliated thesis (ND). In order to display the data, a matrix plot was performed (Figure 7) by indicating with colored cells the statistically significant comparisons, using a red color when the treatment mean value was statistically higher than the mean value of the reference treatment, or a blue color in the opposite case (grey cells indicate those cases of no statistical significance). 3.3.3.1 Chardonnay In the 2010 vintage, TDN, vitispirane, ethoxy actinidol 1 and the unknown norisoprenoid were present in greater quantities in the total defoliated treatment (TD), the unknown norisoprenoid was also greater in the TD2 treatment, while the lowest contents were observed in ND1L treatment. Vitispirane was high also in 3.3.2 Must characterization 3.3.2.1 Chardonnay Concerning sugar concentration (TSS), no significant differences were highlighted in 2013, while differences among treatments emerged in the other years (Figure 5). Specifically, the ND1L generally recorded high TSS values compared to other treatments. This can be observed by comparing ND1L and ND in 2010, ND1L and all other treatments in 2011 and comparing ND1L and TD2 in 2012. Titratable acidity (TA) and malic acid showed, in general, the same response (Figure 5). Not defoliated treatments (ND and ND1L) tended to preserve the acidity better than defoliated treatments (TD, TD1 and TD2). This can be seen in all the years considered, except in 2011, when no significant differences were found. The efficiency of the not defoliation and shading effect on acidity preservation is more evident in 2012 and 2013; in these years, the not defoliated and shaded treatment (ND1L) recorded the highest level of both titratable acidity and malic acid concentration. Significant differences can be observed in 2010, 2012 and 2013 for pH, even though the response was generally not uniform between years (Figure 5). In 2012 and 2013 the highest values recorded concerned TD2, while in 2010 the highest values can be associated to TD1. 3.3.2.2 Pinot noir Figure 6 shows the results obtained for Pinot noir musts characterization. Sugar concentration (TSS) showed no significant differences among treatments with the exception of year 2011. In FIGURE 4 Boxplots showing average bunch weight (ABW in grams) for each treatment considering 4 years, for Chardonnay (CH) and Pinot noir (PN): different letters indicate that values are significantly different at p < 0.05; NS indicates no significant differences among treatments at p < 0.05 obtained by one-way ANOVA and Siegel-Tukey’s post-hoc test. FIGURE 4 Boxplots showing average bunch weight (ABW in grams) for each treatment considering 4 years, for Chardonnay (CH) and Pinot noir (PN): different letters indicate that values are significantly different at p < 0.05; NS indicates no significant differences among treatments at p < 0.05 obtained by one-way ANOVA and Siegel-Tukey’s post-hoc test. 09 Frontiers in Plant Science frontiersin.org Ghiglieno et al. 10.3389/fpls.2023.1125560 FIGURE 5 Boxplots showing total soluble solids concentration (TSS), titratable acidity (TA), malic acid (MA) and pH for each treatment considering 4 years, 2010, 2011, 2012, 2013, for Chardonnay: different letters indicate that values are significantly different at p < 0.05; NS indicates no significant differences among treatments at p < 0.05 obtained by one-way ANOVA and Siegel Tukey’s post-hoc test. Each column is referred to one variable (each variable is reported in the upper part of the graph); each row is referred to one year (each year is reported in the left part of the graph). Boxplots showing total soluble solids concentration (TSS), titratable acidity (TA), malic acid (MA) and pH for each treatment considering 4 years, 2010, 2011, 2012, 2013, for Chardonnay: different letters indicate that values are significantly different at p < 0.05; NS indicates no significant differences among treatments at p < 0.05 obtained by one-way ANOVA and Siegel Tukey’s post-hoc test. Each column is referred to one variable (each variable is reported in the upper part of the graph); each row is referred to one year (each year is reported in the left part of the graph). defoliated and shaded (TD1 and TD2) treatments. In 2011, it was observed that all the norisoprenoids considered were higher in TD and some others, such as ethoxy actinidol 1 also increased in the TD1 and TD2 treatments. The ND1L treatment showed lower contents for ethoxy actinidol 1, vitispirane and TDN. 4.1 Effect of meteorological conditions The results obtained in this study highlight the influence of temperature and water availability on grapevine yield, musts quality and on the composition of aromas in wine. This confirms the results obtained by other authors that underlined the effect of meteorological conditions on grape and wine quality (Mariani et al., 2009). The average weight of the bunches was higher when water needs were satisfied; 2010 and 2011 were, indeed, characterised by lower water stress indices (WSTR and DET) and the highest values of ABW. The grapevine yield positive response to higher water availability was confirmed by other authors (Ramos 3.3.2.2 Pinot noir In 2012, a greater quantity was observed in TD for the unknown norisoprenoid, ethoxy actinidol 1 and vitispirane; ethoxy actinidol 1 was also more abundant in the TD1 treatment. In 2013, only vitispirane was higher in TD, and ethoxy actinidol 1 for TD1 treatment, while ethoxy actinidol 1 was present in less quantities in the ND1L treatment. Chardonnay wine seems to be highly influenced by the agronomic interventions on the canopy, indeed, it is clear that for the main norisoprenoids the total defoliated treatment (TD) was often higher followed by the artificial shading and defoliated treatments. The boxplots of norisoprenoids for each year and treatment are included in the supplementary material (Supplementary Figure 3). this treatment. In 2012 vitispirane was the highest in TD treatment while ethoxy actinidol 1 and the unknown norisoprenoid were the lowest in ND1L treatment. In the 2010 and 2013 vintages, safranal, ethoxy actinidol 1 and the unknown norisoprenoids were the highest in the TD1 treatment. In 2013 also the unknown norisoprenoid were highest in the TD treatment as well. The boxplots of norisoprenoids for each year and treatment are reported in the supplementary material (Supplementary Figure 4). Frontiers in Plant Science frontiersin.org 3.3.3.2 Pinot noir Conversely, Pinot Noir wine seems to be less influenced by the treatments (Figure 7), although showing similar trends to that seen for Chardonnay, in all the years surveyed ethoxy actinidol 1 was prevailing in the defoliated treatment. In 2010 all compounds except TPB-1 were prevailing in the TD2 treatment while in 2011 only TDN was highest in 10 frontiersin.org Ghiglieno et al. 10.3389/fpls.2023.1125560 FIGURE 6 Boxplots showing mean and unit variance of total soluble solids concentration (TSS), titratable acidity (TA), malic acid (MA) and pH for each treatment considering 4 years, 2010, 2011, 2012, 2013, for Pinot noir: different letters indicate that values are significantly different at p < 0.05; NS indicates no significant differences among treatments at p < 0.05 obtained by one-way ANOVA and Siegel-Tukey’s post-hoc test. Each column is referred to one variable (each variable is reported in the upper part of the graph); each row is referred to one year (each year is reported in the left part of the graph). Boxplots showing mean and unit variance of total soluble solids concentration (TSS), titratable acidity (TA), malic acid (MA) and pH for each treatment considering 4 years, 2010, 2011, 2012, 2013, for Pinot noir: different letters indicate that values are significantly different at p < 0.05; NS indicates no significant differences among treatments at p < 0.05 obtained by one-way ANOVA and Siegel-Tukey’s post-hoc test. Each column is referred to one variable (each variable is reported in the upper part of the graph); each row is referred to one year (each year is reported in the left part of the graph). and Mulligan, 2005; Ramos and Martı́nez-Casasnovas, 2006; Intrigliolo et al., 2016; Pérez-Á lvarez et al., 2021; Ohana-Levi et al., 2022). On the other hand, water scarcity in 2012 lead to a reduction of the bunch weight, causing in turn the typical phenomena of sugar and acidity concentration (Intrigliolo et al., 2016; Pérez-Á lvarez et al., 2021) and pH level increase (Reynolds and Naylor, 1994). values for meteorological indicators were reported, the reference sugar level was reached later (24 August - 6 September), leading to lower acidic levels, and higher pH levels. Moreover, the year 2010 reported the highest total amount of cumulated precipitation between berry set and harvest. 3.3.3.2 Pinot noir The low level of titratable acidity and the high value of pH in musts can therefore be related to the effect of dilution of acidic components (Keller, 2006) and the increase of K absorption (Marais et al., 1992) caused by water absorption. The negative effect of high temperatures on the must acidic content was evident in 2013, when a value of the stress caused by heat waves (33STR) was recorded. This was particularly evident in the case of malic acid in Chardonnay musts that showed low values during this vintage. Many previous studies reported the increase of malic acid degradation kinetics when high temperatures were recorded (Michelini et al., 2021). Previous studies reported how the ripening of grapes for sparkling wine production was positively influenced by a cool climate (Jones et al., 2014). The optimum temperature for the accumulation of malic acid during grape ripening was estimated to be around 20°C (Lakso and Kliewer, 1975), while the negative correlation between high temperatures and MA content after véraison was reported by many studies (Rienth et al., 2016; Blank et al., 2019). Preserving malic acid degradation made it possible to contrast the decrease of the acidic level in must (Volschenk et al., 2006), which is a desirable condition for sparkling wine production (Jones et al., 2014). In the present study, the positive effect of cooler temperatures on must quality was revealed in 2011, when high values of indicators of suboptimal temperature and optimal temperature were recorded. This led in 2011 to an early ripening (1 August – 12 August), while preserving the must sugar-acid balance, and a low pH. On the contrary in 2010, where similar Frontiers in Plant Science 4.2 The effect of leaf removal and artificial shading Leaf removal and artificial shading effects were differentiated by year and cultivar. In general, years characterized by satisfying water availability and cooler temperatures (i.e., 2010 and 2011) 11 frontiersin.org Ghiglieno et al. 10.3389/fpls.2023.1125560 FIGURE 7 Matrix plot colored cells showing the statistically significant comparison of some norisoprenoids using a red color when mean value of treatment was statistically higher than the mean value of the reference treatment (ND) or a blue color in the opposite case over the 4 years (2010, 2011, 2012, 2013) for Chardonnay and Pinot noir. NS means not significant, NA means not available data. GU Matrix plot colored cells showing the statistically significant comparison of some norisoprenoids using a red color when mean value of treatment was statistically higher than the mean value of the reference treatment (ND) or a blue color in the opposite case over the 4 years (2010, 2011, 2012, 2013) for Chardonnay and Pinot noir. NS means not significant, NA means not available data. two vintages of Pinot noir, and would suggest that it is the light that most influences the biosynthesis of this compound. TDN with a very low olfactory threshold (2 µg l-1), can sometimes represent a problem, especially in sparkling wines intended for a long period of aging, because it increases over time. It has been seen that the olfactory threshold of TDN in sparkling wine is slightly higher and goes from 2.1 µg l-1 in still Riesling to 4.0 µg l-1 in sparkling Riesling (Ziegler et al., 2019); however, this is a compound to pay attention to when deciding on agronomic practices. Several glycosylated precursors have also been reported to originate these compounds during fermentation and wine ageing, through acid-catalysed reactions, and these reactions are certainly promoted in a wine with a low pH, such as sparkling wine (Schneider et al., 2001; Janusz et al., 2003). emphasized the difference in ripening delay between the non- artificial shaded and the artificial shaded treatments. This is in agreement with previous papers that reported how shading makes it possible to slow down grape maturation (Caravia et al., 2016; Ghiglieno et al., 2020). The effect of no defoliation and artificial shading on acidity preservation was more evident in years characterized by high temperatures and high-water stress (i.e., 2012 and 2013). Frontiers in Plant Science frontiersin.org 4.2 The effect of leaf removal and artificial shading This can be related to the positive relationships between malic acid degradation and high temperature and water scarcity (Blank et al., 2019) that suggest the need for shading during warm and dry vintages. In the case of ABW and pH there was no unique response. The analysis of the main norisoprenoids showed how the canopy treatments greatly affect their quantity (boxplots in Supplementary Figures 3, 4). A general increasing effect of total defoliation can be highlighted, especially concerning Chardonnay. Even though Oliveira et al. (2004) reported that carotenoid contents were consistently higher in grapes in the shade than in those exposed to direct sunlight, the positive effect of exposure on norisoprenoids was underlined by many previous studies (Marais et al., 1991; Marais et al., 1992; Asproudi et al., 2016; Asproudi et al., 2020). The results obtained in the present study, in terms of TDN content in wines, showed that this aromatic compound, even if not always in a statistically significant way, was produced in larger quantities in TD while the shaded theses TD1 and TD2 contained less TDN; this behavior was clearly visible in Chardonnay and in The content in vitispirane was always much higher in TD theses and also in this case, although in Pinot noir there was no statistical proof, the defoliated and shaded theses presented a lower content of this compound. The olfactory descriptor of vitispirane is “camphor” or “eucalyptus”. Some studies (Silva Ferreira and Guedes de Pinho, 2004) showed that in aged wines the presence of vitispirane can reach the threshold of perception (800 µg l-1) and, consequently, that it participates in the aroma of wine. As with the TDN, pH and temperature are the two factors that have the greatest influence on its formation over time. (E)-1-(2,3,6-trimethylphenyl) buta-1,3-diene (TPB-1) was tendentially higher in the TD and less present in the shady theses 12 frontiersin.org Ghiglieno et al. 10.3389/fpls.2023.1125560 10.3389/fpls.2023.1125560 TD1-TD2; this compound, like TDN at high concentrations, could lead to an unpleasant chemical note at higher content (40 ng l-1 sensory threshold) (Janusz et al., 2003). The behavior of actinidols and their ethoxy forms was very similar to that seen previously with a higher content in TD, a little lower in the shaded theses and even lower in the not defoliated ones. For this compound the situation was very clear in both Chardonnay and Pinot Noir. 5 Conclusions The results obtained in the present study showed the importance of canopy management, in terms of defoliation and shading, on must and wine quality. This increases our knowledge about the effect of different defoliation and artificial shading application in relation to meteorological condition, supporting the management decision-making in the Franciacorta wine-growing area. The results obtained with no defoliation and artificial shading in the preservation of the acidic composition in warmer vintages suggest that defoliation activities should be calibrated in relation to the meteorological trends, without standardized procedures. This is particularly relevant in the case of sparkling wine production, where the acidic composition is essential to determine wine quality. The enhanced values of some norisoprenoids obtained with the total defoliation treatment represent a further element to direct defoliation and shading strategies. The very low sensory thresholds of some norisoprenoids place them among the important compounds for the final characteristics of the wine. Moreover, the concentrations of these aromatic compounds tend to increase during ageing, especially in sparkling wines, and can reach levels above the threshold, thus developing the characteristic aroma of aged wine, which is sometimes a detrimental wine quality. For example, TDN and TPB-1 found in wine, have a pleasant aroma Author contributions Another compound that has been increasingly found in TD treatments and less in the other not defoliated and shaded ones was safranal. This compound is responsible for the characteristic smell of saffron and can in some cases exceed the olfactory threshold and be considered too intense, leading to possible depreciation in sparkling wine. It is known that the principal monoterpene glycoside precursor of safranal in saffron is picrocrocin (Zougagh et al., 2006). We don’t know if picrocrocin is a precursor also in wine or if safranal is formed starting from some other precursor or by the rearrangement of some other molecules, but previous studies reported that the content in this compound tends to increase over time, especially at low pH and if the temperature during the storage of the wine is high. Very similar trends in both Chardonnay and Pinot noir, with high levels in the TD thesis and less content in other thesis, were also observed for an unidentified norisoprenoid. On the other hand, we did not observe any clear treatment effects for b- damascenone. IG, SC, GC, LV and FM contributed to the conception and design of the study. IG, SC, GC and UV performed the experiments and analyzed the data. IG, SC, GC and MG-A organized the databases. IG, SC and MG-A performed the statistical analysis. IG, SC and GC wrote the first draft of the manuscript. IG, SC and MG-A prepared figures and tables UV, LV and FM reviewed the drafts of the manuscript. All authors contributed to the article and approved the submitted version. Acknowledgments Our thanks go to Castello Bonomi Tenute in Franciacorta, which hosted the experimental tests and the ‘Consorzio per la tutela del Franciacorta’, which promoted and inspired a collaboration between the universities and the wine sector. Our heartful thanks also go to Pietro Franceschi for his helpful discussion. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Conflict of interest 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. 4.2 The effect of leaf removal and artificial shading Actinidols have an odor that has been described as camphoraceous or as woody and resinous, but their contribution to wine aroma is considered limited at best, as their concentrations are usually much lower than their detection threshold. Perhaps the corresponding ethyl ethers that have been found with the GC-O technique by Schneider et al. (2001), with fruity, citric and eucalyptus notes offer a greater contribution to the aroma. at low concentrations, but they can reduce the quality of the wine at high concentrations, and it is assumed that other compounds such as safranal, if present at high concentrations, may also be undesirable. 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The<i>Gaia</i>-ESO Survey: impact of extra mixing on C and N abundances of giant stars
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The Gaia-ESO Survey: impact of extra mixing on C and N abundances of giant stars N. Lagarde, C. Reylé, A. C. Robin, G. Tautvaišienė, A. Drazdauskas, Š. Mikolaitis, R. Minkevičiūtė, E. Stonkutė, Y. Chorniy, V. Bagdonas, et al. The Gaia-ESO Survey: impact of extra mixing on C and N abundances of giant stars N. Lagarde, C. Reylé, A. C. Robin, G. Tautvaišienė, A. Drazdauskas, Š. Mikolaitis, R. Minkevičiūtė, E. Stonkutė, Y. Chorniy, V. Bagdonas, et al. To cite this version: N. Lagarde, C. Reylé, A. C. Robin, G. Tautvaišienė, A. Drazdauskas, et al.. The Gaia-ESO Survey: impact of extra mixing on C and N abundances of giant stars. Astronomy and Astrophysics - A&A, 2019, 621, pp.A24. ￿10.1051/0004-6361/201732433￿. ￿hal-02066786￿ 1. Introduction stars (e.g. Gilroy 1989; Gilroy & Brown 1991; Gratton et al. 2000; Luck 1994; Tautvaišien˙e et al. 2000, 2001, 2005; Smiljanic et al. 2009; Mikolaitis et al. 2010, 2012). Over the last decade, the understanding of our Galaxy has dramatically increased thanks to the development of large spectroscopic surveys that provide fundamental prop- erties of a large number of stars in different regions of our Galaxy (e.g. RAVE: Steinmetz et al. 2006; SEGUE: Yanny et al. 2009; Gaia-ESO: Gilmore et al. 2012; APOGEE: Blanton et al. 2017; LAMOST: Cui et al. 2012; GALAH: De Silva et al. 2015). Some of them provide abundances deduced from high-resolution spectra, allowing the determina- tion of light chemical elements such as carbon, nitrogen, or lithium. Different transport processes have been discussed in the lit- erature to explain the abundance anomalies in giants. Paramet- ric computations have been proposed to better understand the behaviour of chemical abundances at the stellar surface in low- and intermediate-mass stars. After showing that the hot bot- tom burning (HBB) process, which was previously proposed by Cameron & Fowler (1971) to allow Li production in AGB stars, can explain the oxygen isotopic ratios in AGB stars with ini- tial stellar mass between 4.5 and 7.0 M, Boothroyd et al. (1995) introduced the notion of cool bottom processing (i.e. ad hoc transport material from the cool bottom of stellar convective envelope to deeper and hotter radiative regions where nuclear reactions occur) to explain surface abundances of giant stars with masses lower than 2.0 M⊙(see also Wasserburg et al. 1995; Boothroyd & Sackmann 1999; Sackmann & Boothroyd 1999). Denissenkov & Weiss (1996) suggested, as did Wasserburg et al. (1995), the presence of non-standard mixing of unknown phys- ical origin, between the hydrogen burning and the base of convective envelope after the bump to explain abundances anomalies in red giant stars. To understand variations in The carbon and nitrogen abundances, and also the carbon isotopic ratio, are key chemical tracers used to constrain the stellar evolution of giant stars. Indeed, low- mass stars experience the well-known first dredge-up at the bottom of the red giant branch (RGB), implying changes of the surface abundances of C and N (Iben 1967). Open Access article, published by EDP Sciences, under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT Overall, the current model including thermohaline mixing is able to reproduce very well the C and N metallicity range investigated by the Gaia-ESO survey data. he current model including thermohaline mixing is able to reproduce very well the C and N abundances over the wh gated by the Gaia-ESO survey data. Key words. stars: abundances – stars: evolution – Galaxy: stellar content – Galaxy: abundances Received 7 December 2017 / Accepted 5 June 2018 Received 7 December 2017 / Accepted 5 June 2018 ABSTRACT Context. The Gaia-ESO Public Spectroscopic Survey using FLAMES at the VLT has obtained high-resolution UVES spectra for a large number of giant stars, allowing a determination of the abundances of the key chemical elements carbon and nitrogen at their surface. The surface abundances of these chemical species are known to change in stars during their evolution on the red giant branch (RGB) after the first dredge-up episode, as a result of the extra mixing phenomena. ffects of thermohaline mixing on C and N abundances using the first comparison between the Gaia-ESO survey [C/N] ions of the observed fields using a model of stellar population synthesis. s. We investigate the effects of thermohaline mixing on C and N abundances using the first comparison between the G rminations with simulations of the observed fields using a model of stellar population synthesis. s g g g p y [ / ] determinations with simulations of the observed fields using a model of stellar population synthesis. Methods. We explore the effects of thermohaline mixing on the chemical properties of giants through stellar evolutionary models computed with the stellar evolution code STAREVOL. We include these stellar evolution models in the Besançon Galaxy model to simulate the [C/N] distributions determined from the UVES spectra of the Gaia-ESO survey and to compare them with the observations. Methods. We explore the effects of thermohaline mixing on the chemical properties of giants through stellar evolutionary models computed with the stellar evolution code STAREVOL. We include these stellar evolution models in the Besançon Galaxy model to simulate the [C/N] distributions determined from the UVES spectra of the Gaia-ESO survey and to compare them with the observations. Results. Theoretical predictions including the effect of thermohaline mixing are in good agreement with the observations. However, the field stars in the Gaia-ESO survey with C and N abundance measurements have a metallicity close to solar, where the efficiency of thermohaline mixing is not very large. The C and N abundances derived by the Gaia-ESO survey in open and globular clusters clearly show the impact of thermohaline mixing at low metallicity, which explains the [C/N] value observed in lower mass and older giant stars. Using independent observations of carbon isotopic ratio in clump field stars and open clusters, we also confirm that thermohaline mixing should be taken into account to explain the behaviour of 12C/13C as a function of stellar age. g Conclusions. HAL Id: hal-02066786 https://hal.science/hal-02066786v1 Submitted on 11 Nov 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. A&A 621, A24 (2019) https://doi.org/10.1051/0004-6361/201732433 c⃝ESO 2018 Astronomy & Astrophysics Astronomy & Astrophysics Astronomy & Astrophysics The Gaia-ESO Survey: impact of extra mixing on C and N abundances of giant stars N. Lagarde1, C. Reylé1, A. C. Robin1, G. Tautvaišien˙e2, A. Drazdauskas2, Š. Mikolaitis2, R. Minkeviˇci¯ut˙e2, E. Stonkut˙e2, Y. Chorniy2, V. Bagdonas2, A. Miglio3, G. Nasello1, G. Gilmore4, S. Randich5, T. Bensby6, A. Bragaglia7, E. Flaccomio8, P. Francois9, A. J. Korn10, E. Pancino5,11, R. Smiljanic12, A. Bayo13, G. Carraro14, M. T. Costado15, F. Jiménez-Esteban16,17, P. Jofré18, S. L. Martell19, T. Masseron20,21, L. Monaco22, L. Morbidelli23, L. Sbordone24, S. G. Sousa25, and S. Zaggia26 (Affiliations can be found after the references) Received 7 December 2017 / Accepted 5 June 2018 N. Lagarde1, C. Reylé1, A. C. Robin1, G. Tautvaišien˙e2, A. Drazdauskas2, Š. Mikolaitis2, R. Minkeviˇci¯ut˙e2, E. Stonkut˙e2, Y. Chorniy2, V. Bagdonas2, A. Miglio3, G. Nasello1, G. Gilmore4, S. Randich5, T. Bensby6, A. Bragaglia7, E. Flaccomio8, P. Francois9, A. J. Korn10, E. Pancino5,11, R. Smiljanic12, A. Bayo13, G. Carraro14, M. T. Costado15, F. Jiménez-Esteban16,17, P. Jofré18, S. L. Martell19, T. Masseron20,21, L. Monaco22, L. Morbidelli23, L. Sbordone24, S. G. Sousa25, and S. Zaggia26 (Affiliations can be found after the references) Received 7 December 2017 / Accepted 5 June 2018 N. Lagarde1, C. Reylé1, A. C. Robin1, G. Tautvaišien˙e2, A. Drazdauskas2, Š. Mikolaitis2, R. Minkeviˇci¯ut˙e2, E. Stonkut˙e2, Y. Chorniy2, V. Bagdonas2, A. Miglio3, G. Nasello1, G. Gilmore4, S. Randich5, T. Bensby6, A. Bragaglia7, E. Flaccomio8, P. Francois9, A. J. Korn10, E. Pancino5,11, R. Smiljanic12, A. Bayo13, G. Carraro14, M. T. Costado15, F. Jiménez-Esteban16,17, P. Jofré18, S. L. Martell19, T. Masseron20,21, L. Monaco22, L. Morbidelli23, L. Sbordone24, S. G. Sousa25, and S. Zaggia26 (Affiliations can be found after the references) Received 7 December 2017 / Accepted 5 June 2018 2. Data set and simulations (2017), improvements have been extended to all populations other than the thin disc, imply- ing the computation of stellar evolution models with different α-enhancements ([α/Fe] = 0.15 and 0.30), following the obser- vational [α/Fe] versus [Fe/H] trend observed by Data Release 12 of APOGEE (Majewski et al. 2017). Namely, for [Fe/H] < 0.1, To exploit all the potential of the spectroscopic data, Lagarde et al. (2017; hereafter L17) improved the Besançon Galaxy model (hereafter BGM) including the stellar evolution models that provide surface chemical and seismic properties of stars during their life. In addition to global properties, the BGM is now able to explore the effects of extra mixing on the sur- face abundances of different chemical species. The BGM allows us to compute the stellar component of our Galaxy taking into account errors and the selection function of the observations, drawing a consistent picture of the Galaxy with the formation and evolution scenarios of the Milky Way, stellar formation and evolution theory, models of stellar atmospheres, and dynamical constraints (Robin et al. 2003; Czekaj et al. 2014). This popula- tion synthesis model is a powerful tool that can improve current stellar evolution models and the physics of different transport processes occurring in stellar interiors using a comparison with observations of field stars at different stellar masses, metallic- ities, ages, evolutionary stages, or at different locations in the Galaxy. [α/Fe] =  0.014 + 0.0140675 × [Fe/H] + 0.101262 × [Fe/H]2 for the thin disc stars, 0.320 −exp(1.19375 × [Fe/H] −1.6) for the thick disc stars, 0.3 for halo stars. (1) [α/Fe] =  0.014 + 0.0140675 × [Fe/H] + 0.101262 × [Fe/H]2 for the thin disc stars, 0.320 −exp(1.19375 × [Fe/H] −1.6) for the thick disc stars, 0.3 for halo stars. (1) for halo stars. for halo stars. (1) For [Fe/H] > 0.1, [α/Fe] is assumed solar. To these relations an intrinsic Gaussian dispersion of 0.02 dex is added. y In this paper, we study the effects of thermohaline mixing with metallicity and mass of giant stars, using the C and N abun- dances derived for the giants in the Gaia-ESO survey. To this end, we perform simulations using the BGM with and without the effects of thermohaline instability. The simulations and data used for this study are described in Sect. 2, while the theoretical effects of thermohaline instability with stellar mass and metal- licity are discussed in Sect. 2. Data set and simulations All data used in this paper are included in the second, fourth, and fifth internal GES data releases (iDR2, iDR4, and iDR5) to have a sample that is as large as possible. The main atmospheric parameters of the stars were determined as described by Smiljanic et al. (2014), the carbon and nitrogen abundances were determined as described by Tautvaišien˙e et al. (2015). We separated the stars into two groups: Thermohaline instability driven by 3He-burning through the pp-chain has been proposed to govern the photo- spheric compositions of bright low-mass red giant stars (e.g. Charbonnel & Zahn 2007; Charbonnel & Lagarde 2010). This double diffusive instability, induced by a mean molecular weight inversion due to the 3He(3He,2p)4He reaction in the thin radiative layer between the convective envelope and the hydrogen-burning shell (Eggleton et al. 2008; Lattanzio et al. 2015), is a physical mechanism that best reproduces the observational abundances (e.g. of C and N) in giant stars (e.g. Charbonnel & Lagarde 2010; Angelou et al. 2011, 2012; Henkel et al. 2017). A few recent papers have suggested that magnetic fields might play a role in stellar mixing, alone (Busso et al. 2007) or in combination with thermohaline mixing (Denissenkov & Merryfield 2011; Palmerini et al. 2011). Since thermohaline also provides a physical solution of the 3He problem, well-known in Galactic chemical evolution models (Lagarde et al. 2011, 2012), we focus on this mechanism in this paper. p g p – 324 giant field stars with 173 stars located in the Galactic bulge; – Giants belonging to open and globular clusters (see Table 1). The simulations were made using the revised version of BGM (Paper I of this series, Lagarde et al. 2017), where a new grid of stellar evolution models computed with the code STAREVOL (e.g. Lagarde et al. 2012; Amard et al. 2016) has been imple- mented. This new grid provides the global properties (e.g. sur- face gravity, effective temperature) and chemical abundances (for 54 stable and unstable species). These models also take into account the effects of thermohaline instability during the red giant branch (e.g. Charbonnel & Lagarde 2010). As discussed in Paper I, thermohaline instability changes the photospheric composition of low-mass brighter giant stars, with a decrease in carbon and an increase in nitrogen. In addition to the sim- ulations discussed in Lagarde et al. 2. Data set and simulations surface abundances of giant stars, they proposed a deep diffusive mixing (e.g. Denissenkov & Weiss 1996; Weiss et al. 1996; Denissenkov et al. 1998). Nevertheless, these two proposi- tions of extra mixing are then not related to any physi- cal mechanism to explain changes in surface abundances, and depend on free parameters. On the other hand, rota- tion has been investigated as a possible source of mixing in RGB stars by several authors (Sweigart & Mengel 1979; Charbonnel 1995; Denissenkov & Tout 2000; Palacios et al. 2006; Chanamé et al. 2005), showing that the total trans- port coefficient of rotation at this phase is too low to imply abundance variation on the first ascent giant branch as requested by observations of RGB stars brighter than the RGB bump. surface abundances of giant stars, they proposed a deep diffusive mixing (e.g. Denissenkov & Weiss 1996; Weiss et al. 1996; Denissenkov et al. 1998). Nevertheless, these two proposi- tions of extra mixing are then not related to any physi- cal mechanism to explain changes in surface abundances, and depend on free parameters. On the other hand, rota- tion has been investigated as a possible source of mixing in RGB stars by several authors (Sweigart & Mengel 1979; Charbonnel 1995; Denissenkov & Tout 2000; Palacios et al. 2006; Chanamé et al. 2005), showing that the total trans- port coefficient of rotation at this phase is too low to imply abundance variation on the first ascent giant branch as requested by observations of RGB stars brighter than the RGB bump. The Gaia-ESO survey (Gilmore et al. 2012; Randich et al. 2013, hereafter GES) uses the Fibre Large Array Multi Element Spec- trograph (FLAMES) multifibre facility (Pasquini et al. 2002) of the Very Large Telescope (VLT) to obtain a better understand- ing of the kinematic and chemical evolution of our Galaxy. Giraffe, the medium-resolution spectrograph (R ∼20000), and UVES (Dekker et al. 2000), the high-resolution spectrograph (R ∼47000), are used to observe up to 105 stars in the Milky Way. y For our study, we used the observations of giant stars made with UVES, and the carbon and nitrogen abundances derived from their spectra. These giant stars lie in different Galactic regions (see Fig. 1). 1. Introduction After this episode, numerous spectroscopic observa- tions show that an extra mixing occurs after the bump lumi- nosity on the red giant branch changing the abundances of elements lighter than oxygen at the surface of bright red giant A24, page 1 of 11 A&A 621, A24 (2019) 2. Data set and simulations 3. We start with field stars observed with UVES in Sect. 4, and then enlarge our study to the open and globular clusters observed by the Gaia-ESO survey and com- piled from the literature (see Sect. 4.2). We draw our conclusions in Sect. 5. As discussed in Czekaj et al. (2014), the BGM also simulates the Poisson noise in the Monte Carlo generation of the simulated stars. We performed simulations in every GES field referred to in the iDR5 using this new version of the BGM. As shown in Fig. 2, all stars in the sample have 0.5 < J −K < 1.0 and 5.0 < J < 14, so we restricted our simulations to these colour and magnitude ranges. We did two sets of simulations, with and without the effects of thermohaline instability. The selection bias introduced by the additional requirement of measurable carbon and nitrogen abundances cannot be taken into account in our sample, which is why simulations produce more stars than are present in the A24, page 2 of 11 N. Lagarde et al.: The Gaia-ESO survey: impact of extra mixing on C and N abundances of giant stars Fig. 1. All-sky view centred on the Milky Way Plane in an Aitoffprojection from Mellinger (2009) with a Galactic coordinate grid. Giants observed by the Gaia-ESO survey for which C and N abundances are determined are colour-coded: clusters members (red) and field stars (yellow). We also represent nine open clusters (Collinder 261, Melotte 66, NGC 6253, NGC 3960, NGC 2324, NGC 2477, NGC 2506, IC4651, NGC 6134) for which 12C/13C is derived (cyan diamond, see Sect. 4.3). Fig. 1. All-sky view centred on the Milky Way Plane in an Aitoffprojection from Mellinger (2009) with a Galactic coordinate grid. Giants observed by the Gaia-ESO survey for which C and N abundances are determined are colour-coded: clusters members (red) and field stars (yellow). We also represent nine open clusters (Collinder 261, Melotte 66, NGC 6253, NGC 3960, NGC 2324, NGC 2477, NGC 2506, IC4651, NGC 6134) for which 12C/13C is derived (cyan diamond, see Sect. 4.3). sample. This difference does not affect the conclusions of this paper. predict that thermohaline instability is not efficient enough to explain surface chemical abundances (Wachlin et al. 2014) with the thermohaline fingers becoming more like blobs. Future hydrodynamical simulations closer to the conditions met in stel- lar interiors (Prat et al. 3.1. Physics Thermohaline mixing, as discussed in Paper I, is a double diffusive process conducted in RGB stars by an inversion of mean molecular weight induced by 3He(3He,2p)4He reaction (Charbonnel & Zahn 2007; Siess 2009; Charbonnel & Lagarde 2010; Henkel et al. 2017) and a temperature gradient. For this study we use the prescriptions advocated by Charbonnel & Zahn (2007) given by Ulrich (1972) with an aspect ratio of instabil- ity fingers α ∼6 as referred by Krishnamurti (2003). The ther- mohaline diffusion coefficient used in stellar evolution models includes the correction for non-perfect gas and is given by 2. Data set and simulations 2015) or including the effects of other hydrodynamical processes as recently by Sengupta & Garaud (2018) would shed light on this discrepancy. Hence, we choose to use the prescriptions described above to compare theoreti- cal predictions with current observations at different masses and metallicities. 1 [X/Y] = A(X)−A(X)⊙−A(Y) + A(Y)⊙, with A(X) = log(N(X)/ N(H)) + 12. 3.2. Impact along the evolution This double diffusive instability develops starting from the lumi- nosity of the bump during the RGB (e.g. Charbonnel & Lagarde 2010, and Paper I). Indeed, the steady increase in the stellar luminosity along the RGB momentarily stops when the hydro- gen burning shell (HBS) crosses the molecular weight barrier left behind by the first dredge-up. At that moment the mean molecular weight of the HBS becomes smaller, which implies a decrease in the total stellar luminosity. This is called a bump in the luminosity function. When the region of nuclear energy pro- duction has passed this discontinuity, the mean molecular weight slightly increases and the stellar luminosity increases again. This variation in the luminosity causes an accumulation of stars in the colour-magnitude diagram leading to a bump in the lumi- nosity distribution (e.g. Iben 1967, 1968; Fusi Pecci et al. 1990; Charbonnel 1994; Christensen-Dalsgaard 2015). The impact of thermohaline instability on the theoretical [C/N]1 distributions at different evolutionary states are shown in Fig. 3. With stel- lar models, giant stars are divided into three groups: (1) low- RGB stars: stars ascending the red giant branch before the Dt = Ct K ϕ δ  −∇µ (∇ad −∇) for ∇µ < 0, (2) (2) where K is the thermal diffusivity; ϕ = (∂ln ρ/∂ln µ)P,T; δ = −(∂ln ρ/∂ln ν)P,µ; ∇= (∂ln T/∂ln P); ∇µ and ∇ad are respec- tively the molecular weight gradient and the adiabatic gradient; and with the non-dimensional coefficient Ct = 8 3π2α2· (3) Ct = 8 3π2α2· (3) The value of α is still discussed in the literature by hydrodynamic simulations in 2D or 3D (Denissenkov et al. 2009; Denissenkov 2010; Denissenkov & Merryfield 2011; Rosenblum et al. 2011; Traxler et al. 2011; Brown et al. 2013; Garaud & Brummell 2015). Although these simulations are still far from stellar conditions and do not take into account the coupling of this instability with other hydrodynamic processes occurring in red giant stars (e.g. rotation, magnetic field), they 1 [X/Y] = A(X)−A(X)⊙−A(Y) + A(Y)⊙, with A(X) = log(N(X)/ N(H)) + 12. A24, page 3 of 11 A&A 621, A24 (2019) nces for the chemical properties of globular and open clusters used in the comparison with model predictions. Table 1. References for the chemical properties of globular and open clusters used in the comparison with model predictions. Cluster MTO Age Ref. Mass & Age Ref. 3.3. Impact as a function of stellar mass RGB bump2. These stars have not yet undergone thermoha- line mixing; (2) upper-RGB: RGB stars brighter than the RGB- bump (with logg ≲2.2); (3) clump stars selected according to their asymptotic period spacing of g-modes ∆Πℓ=1. As dis- cussed by Charbonnel & Lagarde (2010) and Paper I, thermoha- line instability occurring at the RGB-bump luminosity changes the surface abundances of C and N for giant stars brighter than the RGB-bump and along the red giant branch, resulting in a decrease in [C/N] (see middle panel of Fig. 3). While thermo- haline mixing is no longer happening in red clump stars, they have the lowest [C/N] (as shown in Fig. 3) because they have undergone a full RGB phase of extra mixing. RGB bump2. These stars have not yet undergone thermoha- line mixing; (2) upper-RGB: RGB stars brighter than the RGB- bump (with logg ≲2.2); (3) clump stars selected according to their asymptotic period spacing of g-modes ∆Πℓ=1. As dis- cussed by Charbonnel & Lagarde (2010) and Paper I, thermoha- line instability occurring at the RGB-bump luminosity changes the surface abundances of C and N for giant stars brighter than the RGB-bump and along the red giant branch, resulting in a decrease in [C/N] (see middle panel of Fig. 3). While thermo- haline mixing is no longer happening in red clump stars, they have the lowest [C/N] (as shown in Fig. 3) because they have undergone a full RGB phase of extra mixing. As discussed and explained by Charbonnel & Lagarde (2010; see Sect. 3.1.2), the global efficiency of thermohaline mixing increases when considering less massive stars at a given metal- licity or more metal-poor stars at a given stellar mass. This results from the combination of several factors like the thermo- haline diffusion timescale compared to the secular timescale, the compactness of the hydrogen burning shell and of the thermoha- line unstable region, and the amount of 3He available to power the thermohaline instability. Figure 4 compares the effect of thermohaline mixing on the surface abundances of red clump stars with (blue) and without (grey) extra mixing, for 1 M⊙(upper panel) and 2 M⊙ (lower panel). On the main sequence, low-mass stars (LMS, M ≲1.7 M⊙) burn hydrogen mainly through pp-chains rather than CNO cycle as in intermediate-mass stars (IMS, 1.7 ≲ M ≲ 2.2 M⊙). 2 Since the gravity (and thus luminosity) of the RGB-bump changes with the metallicity of stars, we establish a simple empirical relation based on stellar evolution models (without the effects of rotation on the evolutionary path) allowing the distinction of low-RGB and upper-RGB stars: log gRGBbump = 0.32·[Fe/H]+2.44. 3.2. Impact along the evolution 2012) with the corresponding ages and metallicities. For globular clusters see the text; for clusters already published, we use the turn-offmass and age indicated in the corresponding article. Notes. For GES clusters, the approximate turn-offmasses were evaluated using the theoretical PARSEC isochrones (Bressan et al. 2012) with the corresponding ages and metallicities. For globular clusters see the text; for clusters already published, we use the turn-offmass and age indicated in the corresponding article. 3.2. Impact along the evolution [C/N] Symbols Tr 20 1.9 1.4 Carraro et al. (2010), Donati et al. (2014) GES Black plus sign NGC 104 0.89 12 Charbonnel & Chantereau (2016) GES Dark cyan circle according to VandenBerg et al. (2013) and Parada et al. (2016) NGC 1851 0.87 11 Charbonnel & Chantereau (2016) GES Magenta square according to VandenBerg et al. (2013) NGC 5927 0.94 11 Charbonnel & Chantereau (2016) GES Blue diamond according to VandenBerg et al. (2013) NGC 6705 3.30 0.3 Santos et al. (2005) GES Yellow square Cantat-Gaudin et al. (2014) NGC 2243 1.19 4.5 WEBDA database GES Red square Br 81 2.06 1.0 Sagar & Griffiths (1998) GES Blue open diamond NGC 6005 2.05 1.2 Piatti et al. (1998) GES Black down triangle NGC 6802 1.90 0.9 Tang et al. (2017) GES Purple circle Tr 23 2.05 0.8 Overbeek et al. (2017) GES Green square Br 31 1.52 2.9 Cignoni et al. (2011) GES Yellow open square Br 36 1.39 7 Donati et al. (2012) GES Magenta plus sign Melotte 71 3.65 0.2 WEBDA database GES Pink open circle NGC 6067 4.75 0.1 WEBDA database GES Magenta circle according to Alonso-Santiago et al. (2017) NGC 6253 1.32 3-5 WEBDA database GES Purple plus sign M67 1.6 2.6 WEBDA database GES Green circle NGC 6259 3.73 0.2 WEBDA database GES Dark blue circle Dias et al. (2002) Rup 134 2.18 1.0 Carraro et al. (2006) GES Red circle NGC 2324 2.7 0.44 Tautvaišien˙e et al. (2016) Red cross NGC 3960 2.2 0.9 Tautvaišien˙e et al. (2016) Blue plus sign NGC 6253 1.4 0.6 Mikolaitis et al. (2012) Black open circle NGC 2477 2.3 0.82 Tautvaišien˙e et al. (2016) Magenta up triangle Melotte 66 1.2 4 Drazdauskas et al. (2016) Black cross Collinder 261 1.1 6.0 Mikolaitis et al. (2012) Yellow circle Drazdauskas et al. (2016) NGC 6134 2.34 0.7 Mikolaitis et al. (2010) Blue open square NGC 2506 1.69 1.7 Mikolaitis et al. (2011a) Green up triangle IC 4651 1.69 1.7 Mikolaitis et al. (2011b) Red down triangle Notes. For GES clusters, the approximate turn-offmasses were evaluated using the theoretical PARSEC isochrones (Bressan et al. 2012) with the corresponding ages and metallicities. For globular clusters see the text; for clusters already published, we use the turn-offmass and age indicated in the corresponding article. Notes. For GES clusters, the approximate turn-offmasses were evaluated using the theoretical PARSEC isochrones (Bressan et al. N. Lagarde et al.: The Gaia-ESO survey: impact of extra mixing on C and N abundances of giant stars Fig. 2. Colour-magnitude diagram for stars observed by the GES survey (grey dots), and for which C and N abundances have been determined (magenta dots). The colour and magnitude values are from the 2MASS Catalog. Low-RGB ¡1:6 ¡1:4 ¡1:2 ¡1:0 ¡0:8 ¡0:6 ¡0:4 ¡0:2 0 [C=N] 0 2 4 6 8 N?;norm Up-RGB ¡1:6 ¡1:4 ¡1:2 ¡1:0 ¡0:8 ¡0:6 ¡0:4 ¡0:2 0 [C=N] 0 2 4 6 8 N?;norm Low-RGB ¡1:6 ¡1:4 ¡1:2 ¡1:0 ¡0:8 ¡0:6 ¡0:4 ¡0:2 0 [C=N] 0 2 4 6 8 N?;norm Up-RGB ¡1:6 ¡1:4 ¡1:2 ¡1:0 ¡0:8 ¡0:6 ¡0:4 ¡0:2 0 [C=N] 0 2 4 6 8 N?;norm Clump/early-AGB ¡1:6 ¡1:4 ¡1:2 ¡1:0 ¡0:8 ¡0:6 ¡0:4 ¡0:2 0 [C=N] 0 2 4 6 8 N?;norm Fig. 3. [C/N] distributions normalised to the histogram area of the synthetic population simulated with the BGM with the effects of ther- mohaline instability (blue solid histograms) and without (grey shaded histograms) during the red giant branch. Giant stars are divided into three groups: low-RGB stars (before the RGB-bump luminosity, top panel), upper-RGB stars (after the RGB-bump luminosity, middle panel), and clump/early-AGB stars (bottom panel). Low-RGB ¡1:6 ¡1:4 ¡1:2 ¡1:0 ¡0:8 ¡0:6 ¡0:4 ¡0:2 0 [C=N] 0 2 4 6 8 N?;norm Up-RGB ¡1:6 ¡1:4 ¡1:2 ¡1:0 ¡0:8 ¡0:6 ¡0:4 ¡0:2 0 [C=N] 0 2 4 6 8 N?;norm Up-RGB Fig. 2. Colour-magnitude diagram for stars observed by the GES survey (grey dots), and for which C and N abundances have been determined (magenta dots). The colour and magnitude values are from the 2MASS Catalog. [C/N] distribution is larger for 1.0 M⊙stars than for 2.0 M⊙ stars. Clump/early-AGB ¡1:6 ¡1:4 ¡1:2 ¡1:0 ¡0:8 ¡0:6 ¡0:4 ¡0:2 0 [C=N] 0 2 4 6 8 N?;norm Clump/early-AGB On the other hand, the RGB-bump occurs on the evolution of low- and intermediate-mass stars only, and depends on the metal- licity of these stars. More massive stars (HMS, M ≳2.2 M⊙) ignite central helium burning in a non-degenerate core at rel- atively low luminosity on the RGB, well before the hydrogen burning shell reaches the mean molecular weight discontinuity caused by the first dredge-up. Consequently, these objects do not go through the bump on their short ascent of the RGB, and thus thermohaline instability does not develop in this kind of star. [C=N] Fig. 3. 3.4. Impact as a function of metallicity In low-mass, low-metallicity giants the thermohaline unstable region is more compact and has a steeper temperature gradient, resulting in a higher diffusion coefficient and then a more effi- cient transport process (see Fig. 6 of Lagarde et al. 2011). p p g g The effect of thermohaline mixing at two metallicities on the [C/N] value at the surface of clump stars simulated by the BGM is shown on Fig. 5 (top and middle panels). The mass distribu- tions for each metallicity range are also shown in Fig. 5 (bot- tom panel). Although thermohaline mixing has a larger impact on [C/N] when the metallicity decreases, the figure also clearly shows a non-negligible impact at solar metallicity. panel, respectively). Low-RGB, up-RGB, and clump stars are included in this figure (as described above). This figure clearly shows a stronger impact of thermohaline instability on the sur- face abundances of giants (here, [C/N]) with [Fe/H] < −0.5. Lower metallicity populations are composed essentially of low- mass stars, implying thick disc and halo as key populations to test the efficiency of this extra mixing (see Figs. 5 and 6). g g p y Because of their large mass and metallicity ranges, field stars provide key data to constrain the efficiency of thermohaline instability occurring in giant stars. In the next section, we study the effects of thermohaline instability with mass and metallicity, comparing the population synthesis and the GES observations. In the same figure, the [C/N] value derived from the observed field stars are also shown (red dots). Since UVES is centred on solar neighbourhood MSTO stars (plus bulge, see Fig. 1), where approximately solar metallicity is expected, the metallicity range of observations is around the solar metallicity (Stonkut˙e et al. (2016), −0.5 ≲[Fe/H] ≲0.5). Figure 7 presents a com- parison between the [C/N] distribution predicted by the BGM with and without the effects of themohaline instability and the observed distribution of [C/N] in the GES field stars sample. Since the [C/N] range in the simulation including the effects of thermohaline instability or following the standard theory are the same, Fig. 6 does not allow us to discriminate between both N. Lagarde et al.: The Gaia-ESO survey: impact of extra mixing on C and N abundances of giant stars [C/N] distributions normalised to the histogram area of the synthetic population simulated with the BGM with the effects of ther- mohaline instability (blue solid histograms) and without (grey shaded histograms) during the red giant branch. Giant stars are divided into three groups: low-RGB stars (before the RGB-bump luminosity, top panel), upper-RGB stars (after the RGB-bump luminosity, middle panel), and clump/early-AGB stars (bottom panel). 3.3. Impact as a function of stellar mass Consequently, a large production of 3He occurs in LMS, favouring the development of thermohaline instability during the red giant branch. Then the impact on A24, page 4 of 11 N. Lagarde et al.: The Gaia-ESO survey: impact of extra mixing on C and N abundances of giant stars 4.1. C and N abundances in field stars 4.1. C and N abundances in field stars Figure 6 displays the surface [C/N] of giant stars simulated with the BGM (grey dots) as a function of stellar metallicity, with and without the effects of thermohaline mixing (right and left A24, page 5 of 11 A24, page 5 of 11 A&A 621, A24 (2019) M = 1.0 M⊙ ¡1:2 ¡1:0 ¡0:8 ¡0:6 ¡0:4 ¡0:2 [C=N] 0 5 10 15 N?;norm M = 2.0 M⊙ ¡1:2 ¡1:0 ¡0:8 ¡0:6 ¡0:4 ¡0:2 [C=N] 0 5 10 15 N?;norm ¡1:5 ¡1:0 ¡0:5 0 0:5 [Fe=H] 0 200 400 600 800 N? M » 1:0M¯ M » 2:0M¯ Fig. 4. [C/N] distributions for two stellar masses ranges, 0.95 ≤ M/M⊙≤1.05 and 1.95 ≤M/M⊙≤2.05 (top and bottom panels, respectively), for clump stars simulated with the BGM with the effects of thermohaline instability (blue solid histograms) and without (grey shaded histograms). Bottom panel: mass distributions for clump stars simulated with the BGM at both mass ranges. [Fe/H] ∼0 ¡1:2 ¡1:0 ¡0:8 ¡0:6 ¡0:4 ¡0:2 0 [C=N] 0 5 10 15 N?;norm [Fe/H] ∼−0.8 ¡1:2 ¡1:0 ¡0:8 ¡0:6 ¡0:4 ¡0:2 0 [C=N] 0 5 10 15 N?;norm 1:0 1:5 2:0 2:5 3:0 3:5 4:0 4:5 5:0 5:5 M=M¯ 0 1000 2000 3000 N? [Fe=H] » ¡0:8 [Fe=H] » 0 Fig. 5. [C/N] distributions at two metallicities (top and middle panels, respectively) for clump stars simulated with the BGM with the effects of thermohaline instability (blue solid histograms) and without (grey shaded histograms). Bottom panel: mass distributions for clump stars simulated with the BGM at −0.85 ≤[Fe/H] ≤−0.75 and −0.05 ≤ [Fe/H] ≤0.05. M = 1.0 M⊙ ¡1:2 ¡1:0 ¡0:8 ¡0:6 ¡0:4 ¡0:2 [C=N] 0 5 10 15 N?;norm [Fe/H] ∼0 ¡1:2 ¡1:0 ¡0:8 ¡0:6 ¡0:4 ¡0:2 0 [C=N] 0 5 10 15 N?;norm M = 1.0 M⊙ M = 2.0 M⊙ ¡1:2 ¡1:0 ¡0:8 ¡0:6 ¡0:4 ¡0:2 [C=N] 0 5 10 15 N?;norm [Fe/H] ∼−0.8 ¡1:2 ¡1:0 ¡0:8 ¡0:6 ¡0:4 ¡0:2 0 [C=N] 0 5 10 15 N?;norm M = 2.0 M⊙ ¡1:5 ¡1:0 ¡0:5 0 0:5 [Fe=H] 0 200 400 600 800 N? M » 1:0M¯ M » 2:0M¯ 1:0 1:5 2:0 2:5 3:0 3:5 4:0 4:5 5:0 5:5 M=M¯ 0 1000 2000 3000 N? [Fe=H] » ¡0:8 [Fe=H] » 0 Fig. 5. 4.1. C and N abundances in field stars [C/N] distributions at two metallicities (top and middle panels, respectively) for clump stars simulated with the BGM with the effects of thermohaline instability (blue solid histograms) and without (grey shaded histograms). Bottom panel: mass distributions for clump stars simulated with the BGM at −0.85 ≤[Fe/H] ≤−0.75 and −0.05 ≤ [Fe/H] ≤0.05. Fig. 4. [C/N] distributions for two stellar masses ranges, 0.95 ≤ M/M⊙≤1.05 and 1.95 ≤M/M⊙≤2.05 (top and bottom panels, respectively), for clump stars simulated with the BGM with the effects of thermohaline instability (blue solid histograms) and without (grey shaded histograms). Bottom panel: mass distributions for clump stars simulated with the BGM at both mass ranges. prescriptions directly. However, Fig. 7 allows a quantitative com- parison and shows the necessity of extra mixing to reproduce the observations even at higher metallicities. In this case, infor- mation on stellar masses and evolutionary states of field stars are required to add more constraints on the efficiency of extra mixing with stellar mass. This is now possible with asteroseis- mology, which can be combined with astrometry from Gaia (Gaia Collaboration 2016), and will be discussed in a forthcom- ing paper. 4.2. C and N abundances in clusters [C/N] as a function of [Fe/H] for synthetic populations computed with the BGM with the effects of thermohaline instability (right panel) and without (left panel). The [C/N] values for our sample of UVES giant field stars are also shown (red dots). Field stars NGC 104 ¡0:8 ¡0:6 ¡0:4 ¡0:2 0 [C=N] 0 1 2 3 4 N?;norm ¡0:8 ¡0:6 ¡0:4 ¡0:2 0 [C=N] 0 1 2 3 4 N?;norm ¡0:9 ¡0:8 ¡0:7 ¡0:6 ¡0:5 ¡0:4 ¡0:3 ¡0:2 [C=N] 0 1 2 3 4 5 6 7 8 N?;norm Fig. 7. Left and middle panels: [C/N] distributions for the synthetic populations in Fig. 6 with the effects of thermohaline instability (middle panel) and without (left panel). [C/N] for our sample of UVES giant field stars are also shown (red histogram). Right panel: [C/N] distributions for a synthetic populations computed with the BGM for the globular cluster NGC 104 with the effect of thermohaline instability (blue histogram) and without (black histogram). The observed [C/N] derived by GES survey is shown (red histogram). NGC 104 ¡0:9 ¡0:8 ¡0:7 ¡0:6 ¡0:5 ¡0:4 ¡0:3 ¡0:2 [C=N] 0 1 2 3 4 5 6 7 8 N?;norm eld stars ¡0:8 ¡0:6 ¡0:4 ¡0:2 0 [C=N] 0 1 2 3 4 N?;norm Fie ¡0:8 ¡0:6 ¡0:4 ¡0:2 0 [C=N] 0 1 2 3 4 N?;norm Fig. 7. Left and middle panels: [C/N] distributions for the synthetic populations in Fig. 6 with the effects of thermohaline instability (middle panel) and without (left panel). [C/N] for our sample of UVES giant field stars are also shown (red histogram). Right panel: [C/N] distributions for a synthetic populations computed with the BGM for the globular cluster NGC 104 with the effect of thermohaline instability (blue histogram) and without (black histogram). The observed [C/N] derived by GES survey is shown (red histogram). Fig. 8. [C/N] as a function of [Fe/H] for synthetic populations computed with the BGM with the effects of thermohaline instability (right panel) and without (left panel). [C/N] for our sample of UVES giant stars members of open and globular clusters are also shown (each symbol represents a cluster, see Table 1). A typical error bar is indicated. Fig. 8. [C/N] as a function of [Fe/H] for synthetic populations computed with the BGM with the effects of thermohaline instability (right panel) and without (left panel). 4.2. C and N abundances in clusters The GES has observed many different clusters in different regions of our Galaxy (see red circles in Fig. 1), providing the homogeneous observational data needed to constrain stellar and Galactic evolution. We investigate the [C/N] value derived in giant members of those open and globular clusters. We do not want to study each cluster in detail; instead, clusters are used here as tracers of extra mixing. Since stars belonging to a cluster were formed together, we can assume that they have the same age, distance, and metallicity, resulting in stronger constraints of thermohaline efficiency. In addition, the right panel of Fig. 7 shows the simula- tion performed specifically for NGC 104 (with and without extra mixing), focussing on cluster region in the sky and the specific metallicity range. The mean [C/N] and the standard deviation predicted by the simulations including extra mix- ing (⟨[C/N]⟩= −0.48 ± 0.19) are in better agreement with observations in NGC 104 (⟨[C/N]⟩ = −0.53 ± 0.20), than for simulations following the standard stellar evolution model (⟨[C/N]⟩= −0.28 ± 0.05). With this very promising result, we focus on clusters observed by the GES survey in the next section. Figures 8–10 show the [C/N] value derived by GES for evolved stars belonging to different globular and open clus- ters as a function of metallicity, turn-offmass, and age. We also add clusters for which [C/N] and 12C/13C determinations are available from the literature (not from GES): Collinder 261, Melotte 66, NGC 6253, NGC 3960, NGC 2324, NGC 2477, NGC 2506, IC 4651, NGC 6134 (Mikolaitis et al. 2012, 2011a,b, 2010; Drazdauskas et al. 2016; Tautvaišien˙e et al. 2016). These A24, page 6 of 11 N. Lagarde et al.: The Gaia-ESO survey: impact of extra mixing on C and N abundances of giant stars N. Lagarde et al.: The Gaia-ESO survey: impact of extra mixing on C and N abundances of giant stars Fig. 6. [C/N] as a function of [Fe/H] for synthetic populations computed with the BGM with the effects of thermohaline instability (right panel) and without (left panel). The [C/N] values for our sample of UVES giant field stars are also shown (red dots). N. Lagarde et al.: The Gaia-ESO survey: impact of extra mixing on C and N abundances of giant sta Fig. 6. 4.2. C and N abundances in clusters [C/N] for our sample of UVES giant stars members of open and globular clusters are also shown (each symbol represents a cluster, see Table 1). A typical error bar is indicated. A24, page 7 of 11 A&A 621, A24 (2019) Fig. 9. [C/N] as a function of stellar mass for synthetic populations computed with the BGM with the effects of thermohaline instability (right panel) and without (left panel). [C/N] for our sample of UVES giant stars members of open and globular clusters are also shown (each symbol represents a cluster, see Table 1). A typical error bar is indicated. Fig. 9. [C/N] as a function of stellar mass for synthetic populations computed with the BGM with the effects of thermohaline instability (right panel) and without (left panel). [C/N] for our sample of UVES giant stars members of open and globular clusters are also shown (each symbol represents a cluster, see Table 1). A typical error bar is indicated. Fig. 10. [C/N] as a function of stellar ages for synthetic populations computed with the BGM with the effects of thermohaline instability (right panel) and without (left panel). [C/N] for our sample of UVES giant stars members of open and globular clusters are also shown (each symbol represents a cluster, see Table 1). Fig. 10. [C/N] as a function of stellar ages for synthetic populations computed with the BGM with the effects of thermohaline instability (right panel) and without (left panel). [C/N] for our sample of UVES giant stars members of open and globular clusters are also shown (each symbol represents a cluster, see Table 1). As discussed by Charbonnel & Lagarde (2010) and described in Sect. 3, three mass ranges can be evoked to quantify the efficiency of thermohaline instability: clusters were acquired by two complementary programmes, and were analysed in a homogeneous way by the same group that produces C and N determinations in GES, result- ing in a robust comparison with our models. Individual stars are attributed the turn-offmass and age of their host clus- ters (e.g. VandenBerg et al. 2013, Chantereau’s priv. comm. using stellar models discussed in Charbonnel & Chantereau 2016, and Drazdauskas et al., in prep.). Synthetic popula- tions with and without the effects of thermohaline instability are shown (right and left panels, respectively) for low-RGB, upper-RGB, and clump stars (as defined in Sect. 4). 4.2. C and N abundances in clusters Simu- lations and observations in each cluster are a mix of giant stars at different evolutionary states (i.e. at different luminosi- ties or gravities on the RGB), implying a wide [C/N] range for the synthetic populations and for the determination in each cluster. clusters were acquired by two complementary programmes, and were analysed in a homogeneous way by the same group that produces C and N determinations in GES, result- ing in a robust comparison with our models. Individual stars are attributed the turn-offmass and age of their host clus- ters (e.g. VandenBerg et al. 2013, Chantereau’s priv. comm. using stellar models discussed in Charbonnel & Chantereau 2016, and Drazdauskas et al., in prep.). Synthetic popula- tions with and without the effects of thermohaline instability are shown (right and left panels, respectively) for low-RGB, upper-RGB, and clump stars (as defined in Sect. 4). Simu- lations and observations in each cluster are a mix of giant stars at different evolutionary states (i.e. at different luminosi- ties or gravities on the RGB), implying a wide [C/N] range for the synthetic populations and for the determination in each cluster. – For low-mass and low-metallicity stars, thermohaline insta- bility is the most efficient transport process that can change the C and N surface abundances, which is why the simula- tions presented here (which take into account thermohaline mixing only) reproduce very well the observed [C/N] in this mass range (see Fig. 9) including a very good fit for stars older than ∼1 Gyr (see Fig. 10). – For intermediate-mass stars (1.7 ≲M ≲2.2 M⊙), our simu- lations present a slightly higher [C/N] than observations (see right panel of Fig. 9). Charbonnel & Lagarde (2010) show that in addition to thermohaline mixing, rotation-induced mixing plays an equivalent role in changing the surface abun- dances of the stars in this mass range, resulting in a slightly A24, page 8 of 11 N. Lagarde et al.: The Gaia-ESO survey: impact of extra mixing on C and N abundances of giant stars lower [C/N] at the surface of intermediate-mass stars (see Fig. 17 of Charbonnel & Lagarde 2010). lower [C/N] at the surface of intermediate-mass stars (see Fig. 17 of Charbonnel & Lagarde 2010). ¡0:8 ¡0:7 ¡0:6 ¡0:5 ¡0:4 ¡0:3 ¡0:2 ¡0:1 0 [C=N] 0 10 20 30 40 50 60 70 12C=13C Fig. 11. 4.2. C and N abundances in clusters 12C/13C as a function of [C/N] for synthetic populations com- puted with the BGM with the effects of thermohaline instability (grey dots) and without (green dots) for upper-RGB and clump stars. A sam- ple of clump field stars are represented by black dots, and nine clus- ters (Collinder 261, Melotte 66, NGC 6253, NGC 3960, NGC 2324, NGC 2477, NGC 2506, IC4651, NGC 6134) using different colours and symbols (see Table 1). ¡0:8 ¡0:7 ¡0:6 ¡0:5 ¡0:4 ¡0:3 ¡0:2 ¡0:1 0 [C=N] 0 10 20 30 40 50 60 70 12C=13C – For high-mass stars, thermohaline mixing plays no role because these stars do not go through the RGB-bump on their short first ascent of the red giant branch, and thus thermohaline instability does not occur (see Sect. 3). This explains why our simulations do not reproduce the spread of [C/N] observed in clusters more massive than 2.2 M⊙(see Fig. 9) and younger than 0.5 Gyr (see Fig. 10). As known for a long time (e.g. Meynet & Maeder 2002; Palacios et al. 2006; Charbonnel & Lagarde 2010), rotation-induced mix- ing has an impact on the internal chemical structure of main sequence stars, although its signatures are revealed later, at the beginning of the RGB. This results in a decrease in the surface abundances of C while N increases. We plan to focus on the effects of rotation on stellar ages and chemical prop- erties in a forthcoming paper. g p p We note that using the Data Release 12 of the APOGEE sur- vey, Masseron & Gilmore (2015) showed that extra mixing has occurred in thin disc stars, but indicated that thick disc stars do not show any evidence of this extra mixing process. They pro- posed that the thick disc stars could be formed with a differ- ent initial abundances than thin disc stars. We also note that our simulations reproduce very well observations in clusters having metallicity corresponding to the thick disc or halo population assuming the same initial abundances for all populations. How- ever, our comparison is based on clusters members, and com- plementary to this study it is crucial to consider a larger sample of thick disc field stars such as those observed by the APOGEE survey. 4.3. 12C/13C in field stars and clusters The GES cannot derive the carbon isotopic ratio due to the wavelength regions observed with no good 13CN features, thus in this part we use data from other studies to investigate the importance of 12C/13C to constrain extra mixing on the red giant branch. We compared data from the Gaia-ESO survey with predic- tions computed using the Besançon Galaxy model in which we included stellar evolution models taking into account (and not) the effects of thermohaline instability (Paper I). To date, this mixing is the only physical process proposed in the literature to explain the photospheric composition of evolved red giant stars. We focus in the first part of this paper on field stars because of their wide-coverage properties (e.g. mass, metallicity, and ages) to deduce an observational trend between [C/N] and stellar mass, metallicity, or age. Due to the lack of C and N determinations in field stars at low metallicity, we cannot investigate further the observational constraints in the metallicity domain where ther- mohaline instability is more efficient. Nevertheless, the theoret- ical distribution of [C/N] predicted by the BGM including the effects of thermohaline instability is in better agreement than the distribution predicted by the standard model at metallicity close to solar. Figures 11 and 12 present the carbon isotopic ratio as a func- tion of C/N and as function of stellar masses and ages, for syn- thetic populations computed with the BGM taking into account the effects of thermohaline mixing (grey dots) or not (green dots). The carbon isotopic ratio decreases abruptly when the thermohaline mixing develops in RGB stars, as already shown by Charbonnel & Lagarde (2010). This is in agreement with the observed abundance ratios found in field stars and open clusters, shown with symbols in the figures (Mikolaitis et al. 2012; Drazdauskas et al. 2016; Tautvaišien˙e et al. 2016). Even though the range of [C/N] values agrees with both models in the observed range, the low values of 12C/13C cannot be reproduced without extra mixing process. As discussed below in the [C/N] case, thermohaline mixing explains very well the low-mass (and older) giants stars, but not the higher mass stars. This proves that 12C/13C is a more powerful parameter for constraining extra mixing on the RGB than [C/N], including for solar metallicity stars. We also investigate the [C/N] derived in giant members of open and globular clusters by the Gaia-ESO survey and litera- ture. 4.2. C and N abundances in clusters In addition, the chemical evolution model and the popu- lation synthesis model should be combined to study the effects of different initial abundances for the different populations before drawing any conclusions. Fig. 11. 12C/13C as a function of [C/N] for synthetic populations com- puted with the BGM with the effects of thermohaline instability (grey dots) and without (green dots) for upper-RGB and clump stars. A sam- ple of clump field stars are represented by black dots, and nine clus- ters (Collinder 261, Melotte 66, NGC 6253, NGC 3960, NGC 2324, NGC 2477, NGC 2506, IC4651, NGC 6134) using different colours and symbols (see Table 1). the C and N abundances derived by the Gaia-ESO survey in field stars and in different clusters, both globular and open. We conclude from this work that it is crucial to take into account thermohaline mixing to understand the C and N observed at the surface of low-mass stars, and in the determination of the stellar mass and age from their chemical properties (Martig et al. 2015; Ness et al. 2016). 4.3. 12C/13C in field stars and clusters This comparison shows a very good agreement with stellar evolution models including thermohaline mixing over the whole scrutinized metallicity range, explaining the [C/N] observed in lower mass and older giant stars. This confirms that thermoha- line instability is crucial to understand the chemical properties of giant stars. The next step is to use a larger sample of field stars to strengthen this encouraging result. 5. Conclusions l’Univers (INSU). G.T., A.D., Š.M., R.M., E.S., Y.Ch., and V.B acknowledge support from the Research Council of Lithuania (MIP-082/2015). R.S. acknowl- edges support from the Polish Ministry of Science and Higher Education. F.J.E. acknowledges financial support from ASTERICS project (ID:653477, H2020- EU.1.4.1.1. - Developing new world-class research infrastructures). T.B. was funded by the project grant “The New Milky Way?” from the Knut and Alice Wallenberg Foundation. A.J.K acknowledges support from the Swedish National Space Board (SNSB). T.M. acknowledges support provided by the Spanish Ministry of Economy and Competitiveness (MINECO) under grant AYA-2017- 88254-P. Additionally, and independently of spectroscopy, asteroseis- mology paves the way to a better understanding of stellar interiors, providing valuable and independent constraints on cur- rent stellar evolution models and on the physics of different transport processes. 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These data have been obtained from the Gaia-ESO Survey Data Archive, prepared and hosted by the Wide Field Astronomy Unit, Institute for Astronomy, University of Edinburgh, which is funded by the UK Science and Technology Facilities Council. This work was partly supported by the European Union FP7 programme through ERC grant number 320360 and by the Lever- hulme Trust through grant RPG-2012-541. We acknowledge the support from INAF and Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR) in the form of the grant “Premiale VLT 2012”. The results presented here bene- fit from discussions held during the Gaia-ESO workshops and conferences sup- ported by the ESF (European Science Foundation) through the GREAT Research Network Programme. N.L., C.R., A.R., and G.N. acknowledge financial support from the “Programme National de Physique Stellaire” (PNPS) and the “Pro- gramme National de cosmologie et Galaxie” (PNCG) of CNRS/INSU, France. 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These data have been obtained from the Gaia-ESO Survey Data Archive, prepared and hosted by the Wide Field Astronomy Unit, Institute for Astronomy, University of Edinburgh, which is funded by the UK Science and Technology Facilities Council. This work was partly supported by the European Union FP7 programme through ERC grant number 320360 and by the Lever- hulme Trust through grant RPG-2012-541. We acknowledge the support from INAF and Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR) in the form of the grant “Premiale VLT 2012”. The results presented here bene- fit from discussions held during the Gaia-ESO workshops and conferences sup- ported by the ESF (European Science Foundation) through the GREAT Research Network Programme. N.L., C.R., A.R., and G.N. acknowledge financial support from the “Programme National de Physique Stellaire” (PNPS) and the “Pro- gramme National de cosmologie et Galaxie” (PNCG) of CNRS/INSU, France. N.L. acknowledges financial support from the CNES. Simulations have been executed on computers from the Utinam Institute of the Université de Franche- Comté, supported by the Région de Franche-Comté and Institut des Sciences de 5. Conclusions In this paper, we present the first comparison between synthetic populations computed with the Besançon Galaxy model and On the other hand, we show that the observed behaviour of 12C/13C with stellar ages is clearly reproduced by models which A24, page 9 of 11 A24, page 9 of 11 A&A 621, A24 (2019) 1:0 1:5 2:0 2:5 3:0 3:5 4:0 4:5 5:0 5:5 M=M¯ 0 5 10 15 20 25 30 35 40 12C=13C 0:1 0:2 0:5 1 2 5 10 Age [Gyr] 0 5 10 15 20 25 30 35 40 12C=13C Fig. 12. 12C/13C as a function of stellar mass and ages for synthetic populations computed with the BGM with the effects of thermohaline instability (grey dots) and without (green dots). A sample of clump field stars are represented by black dots in the right panel, and nine clusters (Collinder 261, Melotte 66, NGC 6253, NGC 3960, NGC 2324, NGC 2477, NGC 2506, IC4651, NGC 6134) using different colours and symbols (see Table 1). 1:0 1:5 2:0 2:5 3:0 3:5 4:0 4:5 5:0 5:5 M=M¯ 0 5 10 15 20 25 30 35 40 12C=13C 0:1 0:2 0:5 1 2 5 10 Age [Gyr] 0 5 10 15 20 25 30 35 40 12C=13C Fig. 12. 12C/13C as a function of stellar mass and ages for synthetic populations computed with the BGM with the effects of thermohaline instability (grey dots) and without (green dots). A sample of clump field stars are represented by black dots in the right panel, and nine clusters (Collinder 261, Melotte 66, NGC 6253, NGC 3960, NGC 2324, NGC 2477, NGC 2506, IC4651, NGC 6134) using different colours and symbols (see Table 1). include the effect of thermohaline mixing. 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English
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The effects of virtual reality game training on trunk to pelvis coupling in a child with cerebral palsy
Journal of neuroengineering and rehabilitation
2,013
cc-by
4,692
Barton et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:15 http://www.jneuroengrehab.com/content/10/1/15 Barton et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:15 http://www.jneuroengrehab.com/content/10/1/15 Barton et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:15 http://www.jneuroengrehab.com/content/10/1/15 JNER JOURNAL OF NEUROENGINEERING AND REHABILITATION RESEARCH Open Access © 2013 Barton 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: Good control of trunk and pelvic movements is necessary for well controlled leg movements required to perform activities of daily living. The nature of movement coupling between the trunk and pelvis varies and depends on the type of activity. Children with cerebral palsy often have reduced ability to modulate coupling between the trunk and pelvis but movement patterns of the pelvis can be improved by training. The aim of this study was to examine how pelvis to trunk coupling changed while playing a computer game driven by pelvic rotations. Methods: One boy with cerebral palsy diplegia played the Goblin Post Office game on the CAREN virtual rehabilitation system for six weeks. He navigated a flying dragon in a virtual cave towards randomly appearing targets by rotating the pelvis around a vertical axis. Motion of the pelvis and trunk was captured in real-time by a Vicon 612 optoelectronic system tracking two clusters of three markers attached to the sacrum and thoracic spine. Results: Convex hull areas calculated from angle-angle plots of pelvic and trunk rotations showed that coupling increased over game training (F1,11 = 7.482, p = 0.019). Reaching to targets far from the midline required tighter coupling than reaching near targets (F1,12 = 10.619, p = 0.007). Conclusions: Increasing coupling appears to be an initial compensation mechanism using the better controlled trunk to drive rotation of the pelvis. Co-contractions causing increased coupling are expected to reduce over longer exposure to training. The control scheme of the training game can be set to facilitate de-coupling of pelvic movements from the trunk. Using large ranges of pelvic rotation required more coupling suggesting that training of selective pelvic movements is likely to be more effective close to a neutral pelvic posture. Keywords: Virtual rehabilitation, Cerebral palsy, Core control, Pelvis and trunk coupling The effects of virtual reality game training on trunk to pelvis coupling in a child with cerebral palsy Gabor J Barton1*, Malcolm B Hawken1, Richard J Foster1, Gill Holmes2 and Penny B Butler3 * Correspondence: g.j.barton@ljmu.ac.uk 1Research Institute for Sport and Exercise Sciences, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF, UK Full list of author information is available at the end of the article Background [6,7], reduced strength and impaired proprioception around the core (pelvis and trunk) were found to be associated with increased risk of injuries, specifically around the knee. This suggests that good control of the movement of the core is a prerequisite for well con- trolled use of the legs. There is evidence to support the hypothesis that the pel- vis and trunk play an active role in gait, as opposed to the traditional view [1] which regards the core of the body as a “passenger unit” carried by the lower extrem- ities, which are termed the “locomotor unit”. Responses triggered by balance perturbations involve neck, trunk and thigh muscles which contract simultaneously, even before the activation of muscles around the ankle joint in unimpaired subjects [2-5]. In prospective studies Good control of the interaction between the trunk and pelvis is required to carry out activities of daily living. During level walking the nature of this interaction depends on walking speed [8-12]. At slow speeds the pelvis and trunk are coupled in the transverse plane and move in-phase, but as walking speed increases there is a transition from in-phase to anti-phase coupling [9,10]. Retraction of the trunk, together with protraction of the * Correspondence: g.j.barton@ljmu.ac.uk 1Research Institute for Sport and Exercise Sciences, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF, UK Full list of author information is available at the end of the article Barton et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:15 http://www.jneuroengrehab.com/content/10/1/15 Barton et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:15 http://www.jneuroengrehab.com/content/10/1/15 Page 2 of 6 Page 2 of 6 pelvis on the swing leg side, increases step length and therefore improves the efficiency of gait. Ethical approval was obtained from the NHS National Re- search Ethics Service (07/H1014/83) and local ethics com- mittees (Royal Liverpool Children’s NHS Trust and Liverpool John Moores University), confirming that the research carried out was in compliance with the Helsinki Declaration. The subject’s parent provided written consent for the study. Ethical approval was obtained from the NHS National Re- search Ethics Service (07/H1014/83) and local ethics com- mittees (Royal Liverpool Children’s NHS Trust and Liverpool John Moores University), confirming that the research carried out was in compliance with the Helsinki Declaration. The subject’s parent provided written consent for the study. One of the primary problems in cerebral palsy is reduced selective control of movement [13]. Background Altered se- lective motor control of the pelvis and trunk hinders efficient gait and thereby activities of daily living. Improved ability to reduce in-phase coupling between the trunk and pelvis is expected to improve gait at nor- mal walking speeds characterised by anti-phase coupling. Activities of daily living are also expected to benefit from the reduced in-phase coupling. The subject’s task in the game was to use the horn on a dragon’s head to burst balloons containing letters. After one balloon target was either hit or missed, the next target appeared at some distance in front of the dragon in one of four unpredictable locations (Figure 1). Forward speed of the dragon through the virtual cave was controlled by the game software, the subject con- trolled left- and rightward movement (speed) of the dragon by rotating the pelvis about a vertical axis in a high kneeling posture. This posture was used to simplify control requirements by eliminating the complexity of ankle motion. High kneeling ensured that the pelvis and trunk remained in the same relative posture as standing, thus giving a more realistic training opportunity. Motion of the dragon and location of the targets were confined to a single horizontal plane. Rotations of the pelvis and trunk about a vertical axis were captured by tracking the 3D orientation of two triangular clusters of three retro- Due to their central location in the body’s linked chain of segments the trunk and pelvis are mechanically restricted to a greater degree than the extremities and the head. In spite of this constraint, movement of the trunk and pelvis can be altered through training [14,15]. There are numerous reports of successfully using cus- tom made computer games to both test and train move- ment control of the upper or lower extremities [16-19]. Applications of serious gaming focused on the pelvis and trunk, however, are scarce. Our Goblin Post Office (GPO) training game [20-25] is derived from the con- ceptual framework of Targeted Training [26] in that it allows sequential training of body segments from upper to lower, thereby increasing successively the complexity of the motor task. We report here some findings (from a larger training study) in which we observed changes in coupling between the pelvis and trunk when the game was controlled by movement of the pelvis. Figure 1 The Goblin Post Office game. Background The dragon flies forwards through the cave at a speed set by the game software, and individual targets appear at some distance ahead unpredictably in one of the four horizontal locations shown. The subject directs the dragon towards the target by left- and rightward pelvic rotation, registered by a triangular array of reflective markers. A second set of markers registers trunk motion, enabling evaluation of trunk to pelvis coupling. Methods b One boy with cerebral palsy spastic diplegia (age: 10 years; height: 1.34 m; mass: 36 kg) trained for 6 weeks, twice a week for 30 minutes (13 sessions in total) on our custom-made GPO computer game, developed in the CAREN system (Motek Medical, Amsterdam, The Netherlands). The child had no history of surgical inter- vention, and was receiving no conventional physiotherapy or core specific rehabilitation which might have influenced his responses to game training. His Gross Motor Function Classification System (GMFCS [27]) score of 1 indicates that he walks indoors and outdoors without limitations and can run and jump but speed, balance and co- ordination may be reduced. He attends mainstream edu- cation and does not have learning difficulties. On physical examination he had a 10° equinus contracture in the right ankle and bilaterally tight hamstrings (popliteal angles of 60°). The hips were offset into internal rotation bilaterally together with external tibial torsion. Muscle strength was reduced in right dorsi/plantarflexors, right hip extensors and bilaterally in hip abductors and foot in/evertors. There were positive signs of spasticity bilaterally in the plantar- flexors (Tardieu 5 on right and 2 on left side), hamstrings (Tardieu 2) and quadriceps (positive Duncan Ely signs). Figure 1 The Goblin Post Office game. The dragon flies forwards through the cave at a speed set by the game software, and individual targets appear at some distance ahead unpredictably in one of the four horizontal locations shown. The subject directs the dragon towards the target by left- and rightward pelvic rotation, registered by a triangular array of reflective markers. A second set of markers registers trunk motion, enabling evaluation of trunk to pelvis coupling. Page 3 of 6 Barton et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:15 http://www.jneuroengrehab.com/content/10/1/15 reduced Areas of the convex hull indicating a more syn- chronised rotation of the trunk and pelvis (Figure 3). reflective markers placed over the thoracic spine and on the sacrum. Neither rotation of the pelvis about the left/ right axis (tilt), nor trunk rotations about either axis, had any effect on the motion of the dragon. At the be- ginning of the first training session the control scheme of the game was explained to the participant and he played the training level of the game which responded to rotations of the pelvis but the dragon was not moving forwards. Methods b Values of Area were not normally distributed, but a natural log transform corrected the distribution (logArea). The mean, maximum, minimum and standard deviation of logArea over all targets (trials) were calcu- lated for each Session. Regression of mean logArea against Session showed that mean logArea reduced sig- nificantly over the training sessions (F1,11 = 7.482, p = To quantify coupling between the trunk and pelvis, angle-angle plots of trunk and pelvic rotation, normal- ised to their respective ranges of motion over all ses- sions, were generated for sections of gameplay between consecutive targets. The area of the convex hull contain- ing all data points of the angle-angle plot was calculated using the CONVHULL function in MATLAB (The Mathworks, Natick MA, USA). This area was used to quantify the level of coupling between the trunk and pel- vis (Figure 2). Low values of Area indicate tight in-phase or anti-phase coupling and high values indicate a lack of association between rotation of the trunk and of the pelvis. -1 -0.5 0 0.5 1 -1 -0.5 0 0.5 1 Left Pelvic rotation Right Left Trunk rotation Right First assessment -1 -0.5 0 0.5 1 -1 -0.5 0 0.5 1 Left Pelvic rotation Right Left Trunk rotation Right Last assessment a b Figure 3 Examples of angle-angle plots (black line) and convex hulls (bold grey line) representing coupling between the trunk and pelvis a) in the first assessment and b) in the last (13th) assessment of the six weeks training. -1 -0.5 0 0.5 1 -1 -0.5 0 0.5 1 Left Pelvic rotation Right Left Trunk rotation Right First assessment a a Discussion Over the period of six weeks while targeting movement training to the pelvis in a patient with cerebral palsy, a gradual development of increased coupling of the pelvis to the trunk was found. This strategy improved perform- ance during gameplay which we have reported elsewhere [22] but improved selective movement control of pelvic rotation was not achieved. Previous findings indicate better movement control of the trunk in comparison to the pelvis in children with cerebral palsy during com- puter based game-play [25]. The tighter coupling of the pelvis to the trunk appears to be a compensatory mech- anism which enables the child to improve control of the pelvis indirectly by locking it to the better controlled trunk. Development of strategies compensating for impaired movement can be prevented by constraint induced movement therapy which physically inhibits the com- pensation thereby forcing use of the targeted movement [29]. Such a constraint preventing the use of trunk rota- tion while driving the GPO game with pelvic rotation can be provided by the game software and may be a means to facilitate use of the pelvis independent of the trunk. The GPO game can be set to either give a visual warning in case of trunk rotation or to be driven by the difference between pelvic and trunk rotation thereby preventing coupled motion. Initial tests however showed that typically developing children found it more difficult to play the game with such control schemes so they were not examined in the present study. Co-contraction, that is simultaneous activation of agonist and antagonist muscles, is a common motor control strategy used to improve stability and accuracy -2.8 -2.4 -2.0 -1.6 -1.2 -0.8 -0.4 0.0 1 2 3 4 5 6 7 8 9 10 11 12 13 logArea Session mean+2SD mean mean-2SD Figure 4 The reducing means of convex hull areas indicate increased coupling between the trunk and pelvis over the 13 game sessions. An increased level of coupling was found when using a large range of pelvic rotation needed to reach targets far from the midline. The child was able to move the pelvis with less in-phase coupling when small ranges were required (indicated by higher logArea) but pelvic movement was aided by in-phase coupled trunk rotation when reaching targets further away from the midline (confirmed by lower logArea). Results A l Angle-angle plots from the first session showed a low level of in-phase coupling between the pelvis and trunk, indicated by higher Areas of the convex hull. In the final (13th session) at the end of the sixth week the angle- angle plots showed tighter in-phase coupling with Trunk rotation Right -1 -0.5 0 0.5 1 -1 -0.5 0 0.5 1 Left Pelvic rotation Right L ft Trunk rotation Ri ht Last assessment b -1 -0.5 0 0.5 1 -1 -0.5 0 0.5 1 Left Pelvic rotation Right Left Trunk rotation Right Figure 2 Angle-angle plot (black line) visualising the interaction between rotation of the trunk and pelvis. The area of the convex hull surrounding the angle-angle plot (bold grey line) was used to quantify coupling. Four quadrants of the chart represent the possible combinations of trunk and pelvic rotation. -1 -0.5 0 0.5 1 -1 -0.5 0 0.5 1 Left Pelvic rotation Right Left Trunk rotation Right Last assessment Left Trunk rotation Right Left Figure 2 Angle-angle plot (black line) visualising the interaction between rotation of the trunk and pelvis. The area of the convex hull surrounding the angle-angle plot (bold grey line) was used to quantify coupling. Four quadrants of the chart represent the possible combinations of trunk and pelvic rotation. Figure 2 Angle-angle plot (black line) visualising the interaction between rotation of the trunk and pelvis. The area of the convex hull surrounding the angle-angle plot (bold grey line) was used to quantify coupling. Four quadrants of the chart represent the possible combinations of trunk and pelvic rotation. Figure 3 Examples of angle-angle plots (black line) and convex hulls (bold grey line) representing coupling between the trunk and pelvis a) in the first assessment and b) in the last (13th) assessment of the six weeks training. Figure 3 Examples of angle-angle plots (black line) and convex hulls (bold grey line) representing coupling between the trunk and pelvis a) in the first assessment and b) in the last (13th) assessment of the six weeks training. Barton et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:15 http://www.jneuroengrehab.com/content/10/1/15 Barton et al. Results A l Journal of NeuroEngineering and Rehabilitation 2013, 10:15 http://www.jneuroengrehab.com/content/10/1/15 Page 4 of 6 -1.8 -1.5 -1.2 -0.9 -0.6 1 2 3 4 5 6 7 8 9 10 11 12 13 logArea Session Near Far Figure 5 The convex hull areas indicate increased coupling (smaller logArea) between the trunk and pelvis when navigating to Far targets as compared to Near targets. 0.019), Figure 4. This, however, concealed more complex behaviour. The maximum logArea did not change over the training sessions, but the mean and minimum logArea reduced significantly as training progressed. As the maximum and minimum values are not unbiased measures of data spread values of Mean + 2 × STD and Mean - 2 × STD of logArea were used instead in regres- sions against Session. The difference in slopes seen with maximum and minimum values was again present (Figure 4: regressions: Mean + 2 × STD F1,11 = 0.539, p = 0.478, Mean - 2 × STD F1,11 = 15.526, p = 0.002). Due to their spatial distribution, reaching half of the balloon targets required less pelvic rotation (Near tar- gets) than reaching the other half (Far targets), Figure 1. Separating the means of logArea for Near and Far tar- gets for all the Sessions, a paired t-test showed that trunk to pelvis in-phase coupling is stronger for Far tar- gets than for Near targets (Figure 5), t12 = 3.259, p = 0.007. Figure 5 The convex hull areas indicate increased coupling (smaller logArea) between the trunk and pelvis when navigating to Far targets as compared to Near targets. when performing an unpractised task [28]. Levels of co- contraction decrease over time with practice. Playing the training game twice a week for 30 minutes over 6 weeks appears to have resulted in the interlocking of the trunk and pelvis, representing the initial response to the new game task. Intensifying exposure to the game by means of increased frequency or duration of play, or both, may lead to reduced coupling between the trunk and pelvis as selective control improves. Conclusions d 8. Bruijn S, Lamoth C, Kingma I, Meijer O, van Dieën J: Coordination between pelvis, thorax and leg movements in gait. Gait Posture 2006, 24(S2):S10–S11. Our case study of a child with cerebral palsy diplegia demonstrated that control was translated from the trunk to the pelvis through tighter coupling over six weeks training consisting of thirteen game sessions. Increased exposure to the game is expected to lead to reduction of coupling due to reduced co-contraction in muscles link- ing the trunk and pelvis as selective control improves. The Goblin Post Office game has means to facilitate se- lective control of the pelvis and this might reduce the time needed to achieve improved selective movements. Pelvic rotation is helped more by trunk rotation at the ends of the pelvic rotation range due to mechanical con- straints and so training of selective pelvic control is likely to be more productive near to the neutral orientation. 9. Bruijn SM, Meijer OG, van Dieën JH, Kingma I, Lamoth CJC: Coordination of leg swing, thorax rotations, and pelvis rotations during gait: The organisation of total body angular momentum. Gait Posture 2008, 27:455–462. 10. Lamoth CJC, Beek PJ, Meijer OG: Pelvis-thorax coordination in the transverse plane during gait. Gait Posture 2002, 16:101–114. 11. Lamoth CJC, Daffertshofer A, Meijer OG, Beek PJ: How do persons with chronic low back pain speed up and slow down? Trunk-pelvis coordination and lumbar erector spinae activity during gait. Gait Posture 2006, 23:230–239. 12. Lamoth CJC, Meijer OG, Daffertshofer A, Wuisman PIJM, Beek PJ: Effects of chronic low back pain on trunk coordination and back muscle activity during walking: changes in motor control. Eur Spine 2006, 15:23–40. 13. Gage JR, Novacheck TF: An update on the treatment of gait problems in cerebral palsy. J Pediatr Orthop B 2001, 10:265–274. 14. Aruin AS: Knee position feedback: Its effect on management of pelvic instability in a stroke patient. Disabil Rehabil 2000, 22:690–692. 15. Janssen LJF, Verhoeff LL, Horlings CGC, Allum JHJ: Directional effects of biofeedback on trunk sway during gait tasks in healthy young subjects. Gait Posture 2009, 29:575–581. Received: 16 March 2012 Accepted: 5 February 2013 Published: 7 February 2013 23. Foster RJ, Hawken MB, Barton GJ: Movement co-ordination of the pelvis in a virtual game environment. Gait Posture 2008, 28S:S10–S11. 24. Barton GJ: Virtual rehabilitation in cerebral palsy. J Sports Sci 2009, 27S1:S3. Discussion Over the duration of six weeks training the level of coupling increased but the difference of coupling over small and large ranges of pel- vic rotations remained (Figure 5). Figure 4 The reducing means of convex hull areas indicate increased coupling between the trunk and pelvis over the 13 game sessions. Functional measures of the patient showed mixed results when comparing performance before and after game training. On one hand the Segmental Assessment Page 5 of 6 Page 5 of 6 Barton et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:15 http://www.jneuroengrehab.com/content/10/1/15 Barton et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:15 http://www.jneuroengrehab.com/content/10/1/15 of Trunk Control scores increased (SATCo Static:2 →4 Active:1 →1 Reactive:NA →2 [30]), the child’s parents commented on improved sitting posture and the swim- ming coach reported “improvements” in swimming. On the other hand an objective evaluation of the patient’s deviation from normality using the Gait Deviation Index [31] showed negligible changes in gait (75.6 →75.0). 4. Allum JH, Honegger F, Acuña H: Differential control of leg and trunk muscle activity by vestibulo-spinal and proprioceptive signals during human balance corrections. Acta Otolaryngol 1995, 115:124–129. 5. Forssberg H, Hirschfeld H: Postural adjustments in sitting humans following external perturbations: muscle activity and kinematics. Exp Brain Res 1994, 97:515–527. 6. Leetun DT, Ireland ML, Willson JD, Ballantyne BT, Davis IM: Core stability measures as risk factors for lower extremity injury in athletes. Med Sci Sports Exerc 2004, 36:926–934. 7. Zazulak BT, Hewett TE, Reeves NP, Goldberg B, Cholewicki J: The effects of core proprioception on knee injury: a prospective biomechanical- epidemiological study. Am J Sports Med 2007, 35:368–373. Authors’ contribution GJB i d h d GJB conceived the study and drafted the manuscript. MBH participated in the design of the study and performed the statistical analysis. RJF carried out the data collection and analysis and helped to draft the manuscript. GJB, MBH and RJF developed the Goblin Post Office game with contribution from GH and PBB on the conceptual and practical aspects of targeted training. All authors read and approved the final manuscript. 18. Holden MK: Virtual environments for motor rehabilitation: review. Cyberpsychol Behav 2005, 8:187–211. discussion 212–189. 19. You SH, Jang SH, Kim YH, Kwon YH, Barrow I, Hallett M: Cortical reorganization induced by virtual reality therapy in a child with hemiparetic cerebral palsy. Dev Med Child Neurol 2005, 47(9):628–635. 20. Hawkins PJR, Hawken MB, Barton GJ: Effect of game speed and surface perturbations on postural control in a virtual environment, Proceedings of the 7th ICDVRAT with ArtAbilitation: 8–11 September 2008; Maia & Porto. Reading, United Kingdom: ICDVRAT; http://www. icdvrat.reading.ac.uk/2008/index2008.htm. Competing interests Th f h h The first two authors have received financial support from Motek Medical (Amsterdam, The Netherlands) to attend user group meetings and scientific conferences. Their host university would receive a proportion of the income Motek Medical generates by sale of the Goblin Post Office game. 16. Bryanton C, Bosse J, Brien M, McLean J, McCormick A, Sveistrup H: Feasibility, motivation, and selective motor control: Virtual reality compared to conventional home exercise in children with cerebral palsy. Cyberpsychol Behav 2006, 9:123–128. 17. Deutsch JE, Latonio J, Burdea GC, Boian R: Post-stroke rehabilitation with the Rutgers ankle system: A case study. Presence-Teleop Virt 2001, 10:416–430. Acknowledgements This project was funded by The WellChild Trust and Alder Hey Children’s NHS Foundation Trust. Author details 1R h I i 21. Barton GJ: Virtual rehabilitation – a focus on movement function. Biomech Hung 2009, 2/2:7–14. 1Research Institute for Sport and Exercise Sciences, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF, UK. 2North West Movement Analysis Centre, Alder Hey Children’s NHS Foundation Trust, Eaton Road, Liverpool L12 2AP, UK. 3The Movement Centre, Robert Jones and Agnes Hunt Hospital, Oswestry SY10 7AG, UK. 22. Barton GJ, Hawken MB, Foster RJ, Holmes G, Butler PB: Playing the goblin post office game improves movement control of the core: a case study, Proceedings of the international conference on virtual rehabilitation; 27–29 June 2011; Zurich. http://dx.doi.org/10.1109/ ICVR.2011.5971811. Received: 16 March 2012 Accepted: 5 February 2013 Published: 7 February 2013 References 25. Barton GJ, Hawken MB, Butler P, Holmes G, Foster RJ: Movement control of the trunk and pelvis in cerebral palsy diplegia. Gait Posture 2009, 30(S1):147–148. 1. Perry J: Gait analysis: normal and pathological function. Thorofare, NJ: SLACK- Inc.; 1992. 2. Allum JH, Bloem BR, Carpenter MG, Hulliger M, Hadders-Algra M: Proprioceptive control of posture: a review of new concepts. Gait Posture 1998, 8:214–242. 26. Butler PB, Major RE: The learning of motor control. Biomechanical considerations. Physiother 1992, 78:1–6. 3. Keshner EA, Woollacott MH, Debu B: Neck, trunk and limb muscle responses during postural perturbations in humans. Exp Brain Res 1988, 71:455–466. 27. Palisano R, Rosenbaum P, Walter S, Russell D, Wood E, Galuppi B: Development and reliability of a system to classify gross motor function in children with cerebral palsy. Dev Med Child Neurol 1997, 39(4):214–223. Page 6 of 6 Page 6 of 6 Barton et al. Journal of NeuroEngineering and Rehabilitation 2013, 10:15 http://www.jneuroengrehab.com/content/10/1/15 28. Gribble PL, Mullin LI, Cothros N, Mattar A: Role of cocontraction in arm movement accuracy. J Neurophysiol 2003, 89:2396–2405. 29. Gordon AM: To constrain or not to constrain, and other stories of intensive upper extremity training for children with unilateral cerebral palsy. Dev Med Child Neurol 2001, 53:56–61. 30. Butler P, Saavedra S, Sofranac M, Jarvis S, Woollacott M: Refinement, reliability and validity of the segmental assessment of trunk control (SATCo). Pediatr Phys Ther 2010, 22(3):246–257. 31. Schwartz MH, Rozumalski A: The gait deviation index: a new comprehensive index of gait pathology. Gait Posture 2008, 28(3):351–357. doi:10.1186/1743-0003-10-15 Cite this article as: Barton et al.: The effects of virtual reality game training on trunk to pelvis coupling in a child with cerebral palsy. Journal of NeuroEngineering and Rehabilitation 2013 10:15. 28. Gribble PL, Mullin LI, Cothros N, Mattar A: Role of cocontraction in arm movement accuracy. J Neurophysiol 2003, 89:2396–2405. 29. Gordon AM: To constrain or not to constrain, and other stories of intensive upper extremity training for children with unilateral cerebral palsy. Dev Med Child Neurol 2001, 53:56–61. 30. Butler P, Saavedra S, Sofranac M, Jarvis S, Woollacott M: Refinement, reliability and validity of the segmental assessment of trunk control (SATCo). Pediatr Phys Ther 2010, 22(3):246–257. 31. Schwartz MH, Rozumalski A: The gait deviation index: a new comprehensive index of gait pathology. Gait Posture 2008, 28(3):351–357. References doi:10.1186/1743-0003-10-15 Cite this article as: Barton et al.: The effects of virtual reality game training on trunk to pelvis coupling in a child with cerebral palsy. Journal of NeuroEngineering and Rehabilitation 2013 10:15. doi:10.1186/1743-0003-10-15 Cite this article as: Barton et al.: The effects of virtual reality game training on trunk to pelvis coupling in a child with cerebral palsy. Journal of NeuroEngineering and Rehabilitation 2013 10:15. 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:
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https://www.qeios.com/read/FFHNHO/pdf
English
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Adenocarcinoma of the anal canal
Definitions
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cc-by
115
Qeios · Definition, February 10, 2020 Open Peer Review on Qeios Adenocarcinoma of the anal canal INSERM Qeios ID: FFHNHO · https://doi.org/10.32388/FFHNHO Source INSERM. (1999). Orphanet: an online rare disease and orphan drug data base. Adenocarcinoma of the anal canal. ORPHA:424016 Adenocarcinoma of the anal canal is a very rare tumor of the intestine, originating from the epithelium of the anal canal (including the mucosal surface, anal glands, and lining of fistulous tracts), macroscopically appearing as a nodular, often ulcerated, invasive mass located in the anal canal. Patients often present with rectal bleeding, as well as difficulty and pain during defecation. Inguinal lymphadenopathy, if present, usually indicates metastatic spread. Qeios ID: FFHNHO · https://doi.org/10.32388/FFHNHO 1/1
https://openalex.org/W4391548062
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English
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Baggage Cart with Weighing Mechanism for Hotels and Airlines
Journal of engineering research and sciences
2,024
cc-by-sa
2,417
Baggage Cart with Weighing Mechanism for Hotels and Airlines Vishal Verma*,1 , Kuldeep Kumar2 , Rashmi Aggarwal1 , Tanvi Verma1 1 Chitkara College of Hospitality Management, Chitkara University, Rajpura, 140401, Punjab India 2 Chitkara Business School, Chitkara University, Rajpura, 140401, Punjab India *Corresponding author: Vishal Verma, vishal.verma@chitkara.edu.in, +91 9041078993 ABSTRACT: In this article, it has been proposed a functional design of Baggage Cart with Weighing Mechanism for Hospitality Industry based on empirical observations. This design is expected to promote and become one of the high demand products which can later be used especially by hotels and respective airlines. It has been often observed that travelers are always concerned about the maximum weight of their baggage allowed in order to board a flight. Because as per norms of airlines one needs to carry a specific amount of baggage in the flight, in case of extra weighed baggage carried by a guest, he/she is charged extra amount as per KG by the respective airline. Keeping this point in mind, travelers are always conscious about the weight of their baggage before boarding a flight to avoid the last-minute hassle of paying huge extra money, and sometimes they need to drop the necessary items out from the baggage to adjust the weight of the baggage. In hotels, a guest request to weigh his/ her luggage is dealt with in the following ways: The front office associate/porter first needs to fetch the baggage from the guest room and bring it up to the bell desk or need to drag it up till time office / receiving area where it is weighed on a heavy-duty weighing scale. It is the traditional way of measuring baggage. The traditional way is time-consuming, uncomfortable, and less suitable to support the need of the traveler. Hence, to minimize the time and efforts, we wanted to invent a mechanism that will help and reduce the burden of managing the separate weighing machine, and therefore we came up with a baggage cart with a weighing mechanism in it. KEYWORDS: Baggage Cart, Weighing Mechanism, Customer Satisfaction, Service Quality weighing his baggage. Moreover, Airports’ key customers are passengers and their initial impressions of airport facilities and services can significantly impact their perceptions and evaluations of the airport. Therefore, it is vital for the airports to provide safe, comfortable and convenient services in a cost-effective manner [2]. Transactions involving goods and services are not the only aspects of business. Baggage Cart with Weighing Mechanism for Hotels and Airlines Its capacity to last is significantly reliant on a variety of humanitarian aspects related to the management and upkeep of commercial relationships with clients, suppliers, and staff, particularly when conducting business internationally [3]. Keeping these in mind, Quality of Service as apparent by customers is a comparison amongst performance and expectations [4]. As per [5], The overall observation of service quality is determined by the modification between customers’ expectations and their actual experiences. When a Special Issue on Computing, Engineering and Sciences Received: 04 October, 2023, Revised: 06 January, 2024, Accepted: 06 January, 2024, Online: 30 January, 2024 Received: 04 October, 2023, Revised: 06 January, 2024, Accepted: 06 January, 2024, Online: 30 January, 2024 Received: 04 October, 2023, Revised: 06 January, 2024, Accepted: 06 January, 2024, Online: 30 January, 2024 DOI: https://doi.org/10.55708/js0301002 1. Introduction Passenger Satisfaction is a crucial metric in order to evaluate airport performance. International airports in various regions or countries usually do not compete with each other. Passengers generally do not have the choice to choose between airports, irrespective of the quality and price of airport services. However, Passenger’s demand for airport services is likely to be fairly inflexible [1]. To minimize time, effort, and ease out the task, the design provides a mechanism to weigh the baggage as per the guest’s request. While working in the hotel industry, it was a routine task to deal with guests and their needs and out of all to weigh their baggage was one of them. One genuine problem which is paid less attention and provided with an ordinary, less effective, or non- hospitable solution is the handling of a traveler’s need of Journal of Engineering Research and Sciences, 3(1): 06-10, 2024 6 V. Verma et al., Baggage Cart with Weighing Mechanism customer’s experience matches their expectation, service quality is perceived as upright. In other words, Quality of the service is measured by comparing customer expectations with their experiences and this comparison is based on the performance of service delivery. The goal of providing better service to clients is to meet their needs in addition to making sure they are completely satisfied with the service they are utilizing [6]. In the hospitality industry, it is a routine task to weigh the baggage of guests. The proposed design provides a mechanism to weigh the baggage as per the guest's request with minimal time, effort. Present solutions are not convenient and need the efforts of the porter to fetch the baggage to and fro between the guests' rooms and hotel utilities which is a time-consuming process. weighing scale kept in their bathroom. However, this system is not suitable as it is meant for weighing human body weight. In case one holds a bag and stands on the machine, it gives total weight which is not an appropriate method. it’s not even feasible to keep a large piece of baggage on the surface of a weighing scale. 2. Technical Background Figure 2: Traditional way of Measuring Baggage through a weighing device with a lifting hook (top view) At present, the hospitality industry is using the following mechanisms to weigh the baggage. Based on empirical observations and as shown in Figure 1 below, Earlier In a traditional way, a porter needs to fetch the bag from the guest room which is taken to the time office / receiving area where it is weighed on a heavy-duty weighing scale. The moment it’s being measured, it is properly tagged and delivered to the respective guest room. The process needs to be executed immediately on the guest’s demand as the guest has to check out and take a flight or train. Even a reasonable delay in the process upsets the guests and creates a bad impression of the hotel. Figure 3: Traditional way of Measuring Baggage through a weighing device with a lifting hook (front view) Figure 3: Traditional way of Measuring Baggage through a weighing device with a lifting hook (front view) Figure 1: Traditional way of Measuring Baggage through the help of Porter Figure 4: Traditional way of Measuring Baggage through the weighing scale kept in their bathroom Figure 4: Traditional way of Measuring Baggage through the weighing scale kept in their bathroom Figure 1: Traditional way of Measuring Baggage through the help of Porter 1. Introduction Figure 2: Traditional way of Measuring Baggage through a weighing device with a lifting hook (top view) Figure 3: Traditional way of Measuring Baggage through a weighing device with a lifting hook (front view) Figure 2: Traditional way of Measuring Baggage through a weighing device with a lifting hook (top view) 3. Related Work In another mechanism, as per empirical observations and as shown in Figures 2 & 3 below, some hotels use a weighing device with a lifting hook. The porter has to hold the device and lift the bag hooked to the device to weigh it. Sometimes the process may result in a muscular sprain in the porter’s hand or back or lead to dropping the bag, which could again lead to a bad impression of the hotel. 4. Hardware Implementation As per empirical observations, the surface of the baggage cart has a mechanism to measure the weight. Upon placing a bag on the cart's surface, it will display the weight on the digital display mounted on the cart. The baggage cart will appear like a regular baggage cart when the digital meter is turned off. KULDEEP KUMAR has a decade of experience in the hospitality industry, working with top brands such as Marriott International, The Oberoi Hotels and Resorts, Kempinski Hotels, and Wyndham Hotels. For the last five years, he has been sharing his industry insights with students at the prestigious Chitkara University, while also pursuing research and innovation V. Verma et al., Baggage Cart with Weighing Mechanism by hospitality professionals because of lesser effort, easy handling, and being time effective. moving towards their flight one needs to finish check-in formalities including the weighing of their bags. Using this mechanism would enable them to get the results faster than the existing ones. (https://creativecommons.org/licenses/by-sa/4.0/). VISHAL VERMA is an Experienced hotel professional with 8 years of hotel experience with 5-star hotel brands, like Oberoi Hotels & Resorts, Leela Kempinski, Carlson Rezidor, Wyndham, Post that it’s been 8 years since he’s working with Chitkara University as an Assistant Professor. He has publications in various reputable journals with exceptional orientation of research. His area of interest is Revenue Management and research skills in Tourism, Travel & Hospitality with expertise in Room Division, Sales and Marketing as well. The authors declare no conflict of interest. Figure 5: Traditional way of Measuring Baggage through the weighing scale References [1] R. Doganis, The Airline Business, Routledge, 2005, doi:10.4324/9780203596807. [2] R. Rendeiro Martín-Cejas, “Tourism service quality begins at the airport,” Tourism Management, vol. 27, no. 5, pp. 874–877, 2006, doi:10.1016/j.tourman.2005.05.005. [3] B.K. Das, S. Sharma, “Influence of Relationship & Behavioural Aspects on Customer Retention & Loyalty in B2B Flexible Packaging Industry in Bangladesh,” International Journal of Applied Business and Economic Research, vol. 15, no. 22, pp. 71–81, 2017. Figure 5: Traditional way of Measuring Baggage through the weighing scale [4] A. Parasuraman, V.A. Zeithaml, L.L. Berry, “A Conceptual Model of Service Quality and Its Implications for Future Research,” Journal of Marketing, vol. 49, no. 4, pp. 41–50, 1985, doi:10.1177/002224298504900403. 3.2. Advantage of the proposed system in comparison with prior art 3.2. Advantage of the proposed system in comparison with prior art • Time Effective: It saves time as it is always available with Front Office / Bell Desk. [5] C. Gronroos, Service management and marketing: managing the moments of truth in service competition, Lexington Books, 1990. [6] J. Sharma, J. Singh, A. Singh, “Impact of E-Banking Service Quality on Customer Satisfaction,” International Journal of Recent Technology and Engineering (IJRTE), vol. 8, no. 5, pp. 2296–2300, 2020, doi:10.35940/ijrte.E5841.018520. • Easy Handling: It is always handy and in use by Bell Desk Staff. • Reduced Efforts: No more to-and-fro efforts, as a handy baggage cart with a weighing mechanism, is available all the time. Copyright: This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-SA) license Copyright: This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-SA) license 3.3. Proposed Solution The mechanism was designed to minimize the time and effort required in the traditional weighing method through a separate weighing machine. The proposed design allows installing the weighing mechanism on the baggage cart. The proposed design for Baggage Cart with weighing mechanism for hotels and airlines are shown in figure 6. (https://creativecommons.org/licenses/by-sa/4.0/). 3.1. Prior Art 3.1. Prior Art As per empirical observations, figure 5 represents the traditional way of measuring the baggage. It would be easy and convenient for the front office associate/porter to use efficient ways to reduce their efforts and save time. As per empirical observations, the use of the proposed mechanism in baggage cart is not restricted or limited to just hotels only, even the respective airlines can use it during a passenger's arrival inside the airport, before In a few hotels, as per empirical observations and as shown in Figure 4 below, the guest is prompted to measure his / her baggage on his own by using the Journal of Engineering Research and Sciences, 3(1): 06-10, 2024 7 V. Verma et al., Baggage Cart with Weighing Mechanism V. Verma et al., Baggage Cart with Weighing Mechanism enrs.com Journal of Engineering Research and Sciences, 3(1): 06-10, 2024 8 Figure 6: Proposed design for Baggage Cart with Weighing Mechanism for Hotels and Airlines Figure 6: Proposed design for Baggage Cart with Weighing Mechanism for Hotels and Airlines Figure 6: Proposed design for Baggage Cart with Weighing Mechanism for Hotels and Airlines Journal of Engineering Research and Sciences, 3(1): 06-10, 2024 Journal of Engineering Research and Sciences, 3(1): 06-10, 2024 8 5. Conclusions The product is designed to eliminate the existing flaws used by respective hotels and airlines of guests for weighing their luggage, especially at the eleventh hour of checking out of the hotel. As per the enclosed pictures from 1-4, traditionally these are the methods of fetching baggage and measuring them too which are not at all convenient and time-effective. The most significant advantage of the baggage cart with a weighing mechanism is that it can be used by both hotels and airlines for their purposes. This product is expected to find high acceptance RASHMI AGGARWAL is a Professor and Dean Commerce at Chitkara Business School, Chitkara University, Punjab. She is PhD, M. Phil, M. Com and UGC-NET qualified, with a vast experience of 23 years in teaching and administration at various positions. Her areas of specialization are Accounting, Finance and Management. She has publications in many reputable journals with excellent research orientation. She has been awarded with the excellence Award for Most Collaborating Employee. She also got the letters of appreciation for strenuous and meticulous efforts by her in guiding and mentoring the students. She is an effective communicator along with the exceptional Journal of Engineering Research and Sciences, 3(1): 06-10, 2024 9 V. Verma et al., Baggage Cart with Weighing Mechanism ability of relationship management. She is always committed to creating an inclusive learning environment for the students where they can explore their potential. TANVI VERMA holds a doctorate from University Business School, Panjab University, in Finance and Strategic Management. She possesses a decade of experience in the fields of Accountancy, Performance Management, Financial Reporting, Strategic Management, and subjects related to the Association of Chartered Certified Accountants (ACCA). At present, she holds the esteemed position of Assistant Professor at Chitkara University in Punjab. She has a strong focus on research and a solid list of papers to her name. Her work has been published in a multitude of prestigious journals. In several of the national and international conferences where she has presented, Ms. Verma has been honored with the Best Paper Award. Journal of Engineering Research and Sciences, 3(1): 06-10, 2024 10 www.jenrs.com
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https://leicester.figshare.com/articles/journal_contribution/Quantifying_Uncertainty_in_Satellite-Retrieved_Land_Surface_Temperature_from_Cloud_Detection_Errors/10226807/1/files/18448589.pdf
English
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Quantifying Uncertainty in Satellite-Retrieved Land Surface Temperature from Cloud Detection Errors
Remote sensing
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Received: 9 March 2018; Accepted: 6 April 2018; Published: 17 April 2018 Abstract: Clouds remain one of the largest sources of uncertainty in remote sensing of surface temperature in the infrared, but this uncertainty has not generally been quantified. We present a new approach to do so, applied here to the Advanced Along-Track Scanning Radiometer (AATSR). We use an ensemble of cloud masks based on independent methodologies to investigate the magnitude of cloud detection uncertainties in area-average Land Surface Temperature (LST) retrieval. We find that at a grid resolution of 625 km2 (commensurate with a 0.25◦grid size at the tropics), cloud detection uncertainties are positively correlated with cloud-cover fraction in the cell and are larger during the day than at night. Daytime cloud detection uncertainties range between 2.5 K for clear-sky fractions of 10–20% and 1.03 K for clear-sky fractions of 90–100%. Corresponding night-time uncertainties are 1.6 K and 0.38 K, respectively. Cloud detection uncertainty shows a weaker positive correlation with the number of biomes present within a grid cell, used as a measure of heterogeneity in the background against which the cloud detection must operate (e.g., surface temperature, emissivity and reflectance). Uncertainty due to cloud detection errors is strongly dependent on the dominant land cover classification. We find cloud detection uncertainties of a magnitude of 1.95 K over permanent snow and ice, 1.2 K over open forest, 0.9–1 K over bare soils and 0.09 K over mosaic cropland, for a standardised clear-sky fraction of 74.2%. As the uncertainties arising from cloud detection errors are of a significant magnitude for many surface types and spatially heterogeneous where land classification varies rapidly, LST data producers are encouraged to quantify cloud-related uncertainties in gridded products. Keywords: uncertainties; land surface temperature; cloud detection errors Quantifying Uncertainty in Satellite-Retrieved Land Surface Temperature from Cloud Detection Errors s Meteo o og ca st tute ( M ), 00560 e s k , a d * Correspondence: c.e.bulgin@reading.ac.uk (C.E.B.); c.j.merchant@reading.ac.uk (C.J.M.); djg20@leicester.ac.uk (D.G.); thomas.popp@dlr.de (T.P.); Larisa.Sogacheva@fmi.fi(L.S.); Tel.: +44-118-378-6732 (C.E.B.) remote sensing remote sensing remote sensing www.mdpi.com/journal/remotesensing remote sensing Article Quantifying Uncertainty in Satellite-Retrieved Land Surface Temperature from Cloud Detection Errors Claire E. Bulgin 1,2,* ID , Christopher J. Merchant 1,2,* ID , Darren Ghent 2,3,* ID , Lars Klüser 4,5, Thomas Popp 4,* ID , Caroline Poulsen 6 ID and Larisa Sogacheva 7,* ID 1 Department of Meteorology, University of Reading, Reading, RG6 6AL, UK 2 National Centre for Earth Observation, Leicester, UK 3 Space Research Centre, University of Leicester, Leicester, LE1 7RH, UK 4 Deutsches Zentrum für Luft- und Raumfahrt (DLR), 82234 Oberpfaffenhofen, Germany 5 Now at: Adesso Insurance Solutions GmbH, 81541 Munich, Germany; lars.klueser@imail.de 6 Rutherford Appleton Laboratory (RAL), Harwell Campus, Didcot, OX11 0QX, UK; caroline.poulsen@stfc.ac.uk 7 Finnish Meteorological Institute (FMI), F1-00560 Helsinki, Finland * Correspondence: c.e.bulgin@reading.ac.uk (C.E.B.); c.j.merchant@reading.ac.uk (C.J.M.); djg20@leicester.ac.uk (D.G.); thomas.popp@dlr.de (T.P.); Larisa.Sogacheva@fmi.fi(L.S.); Tel.: +44-118-378-6732 (C.E.B.) Received: 9 March 2018; Accepted: 6 April 2018; Published: 17 April 2018   Ab t t Cl d i f th l t f t i t i t i f f remote sensing Article Quantifying Uncertainty in Satellite-Retrieved Land Surface Temperature from Cloud Detection Errors Claire E. Bulgin 1,2,* ID , Christopher J. Merchant 1,2,* ID , Darren Ghent 2,3,* ID , Lars Klüser 4,5, Thomas Popp 4,* ID , Caroline Poulsen 6 ID and Larisa Sogacheva 7,* ID 1 Department of Meteorology, University of Reading, Reading, RG6 6AL, UK 2 National Centre for Earth Observation, Leicester, UK 3 Space Research Centre, University of Leicester, Leicester, LE1 7RH, UK 4 Deutsches Zentrum für Luft- und Raumfahrt (DLR), 82234 Oberpfaffenhofen, Germany 5 Now at: Adesso Insurance Solutions GmbH, 81541 Munich, Germany; lars.klueser@imail.de 6 Rutherford Appleton Laboratory (RAL), Harwell Campus, Didcot, OX11 0QX, UK; caroline.poulsen@stfc.ac.uk 7 Finnish Meteorological Institute (FMI), F1-00560 Helsinki, Finland * Correspondence: c.e.bulgin@reading.ac.uk (C.E.B.); c.j.merchant@reading.ac.uk (C.J.M.); djg20@leicester.ac.uk (D.G.); thomas.popp@dlr.de (T.P.); Larisa.Sogacheva@fmi.fi(L.S.); Tel.: +44-118-378-6732 (C.E.B.) Received: 9 March 2018; Accepted: 6 April 2018; Published: 17 April 2018   Ab t t Cl d i f th l t f t i t i t i f f 1. Introduction All geophysical variables are retrieved with a level of uncertainty, which is a function of both the data used and the retrieval methods. Taking account of uncertainty is fundamental to the appropriate scientific application of these data, and uncertainty information should be provided in the most complete state possible by data producers, preferably for each datum where uncertainty varies Remote Sens. 2018, 10, 616; doi:10.3390/rs10040616 www.mdpi.com/journal/remotesensing 2 of 20 Remote Sens. 2018, 10, 616 sufficiently to justify this [1]. Within the surface temperature remote sensing community, there have been recent efforts to quantify measurement uncertainties on a per-pixel basis. This has been achieved either by developing a model of how uncertainties vary under different retrieval conditions [2,3] or by estimating uncertainties within the retrieval process itself [4,5]. In the latter case, the uncertainties can be validated in addition to the measurement data, using independent reference datasets [6]. A method for estimating per-pixel uncertainty in sea surface temperature retrieval was developed within the context of the European Space Agency (ESA) Climate Change Initiative (CCI) programme [4]. It has since been adopted within the lake surface temperature community via the European Commission EU Surface Temperature of All Corners of Earth (EUSTACE) project, and within the land surface temperature (LST) community via the ESA Data User Element (DUE) GlobTemperature project [5,7]. These data producers consider the propagation of uncertainties arising from random and systematic errors from Level 1b data into Level 2 and Level 3 products. The investment of these different data producers in using the same approach to characterise uncertainty in surface temperature retrievals is an important step in providing comparable products across different domains (land, ocean, lake, ice). The uncertainties currently characterised by these data producers include both random and systematic components. Sources of uncorrelated (random) errors in land surface temperature data include instrument noise and sampling uncertainties. Systematic uncertainties arise due to retrieving surface temperature through the atmosphere, sub-pixel variability in emissivity and from calibration and instrument characteristics that change over time. Surface temperature retrievals are predominantly made using measurements in the infrared region of the electromagnetic spectrum and are therefore affected by the presence of clouds. Sampling uncertainties due to the presence of cloud limiting the observations available in gridded data have been considered in sea surface temperature products [8], but uncertainties due to imperfect cloud screening impacting the retrieved surface temperature have not previously been quantified. 1. Introduction Cloud detection is an essential pre-processing step in the retrieval of Earth surface temperatures, as cloud will affect the observed brightness temperatures. Clouds are the source of two different types of uncertainty in surface temperature retrieval: (1) uncertainty due to ‘missed clouds’ (errors of omission), which typically result in a cold bias in the retrieved surface temperature; and (2) uncertainty due to ‘missed measurements’ (errors of commission), resulting from clear-sky pixels falsely classified as cloud. At Level 2, (2) simply leads to omission of valid data from a given dataset, but at Level 3, this has an impact on the averaged surface temperatures within a given gridded domain, not explicitly accounted for within the sampling uncertainty. Where clouds are missed (1), uncertainties may also include errors arising from the application of a clear-sky infrared LST retrieval algorithm to cloud-affected observations. Cloud detection algorithms predominantly work on the concept that clouds are ‘bright’ in the visible part of the spectrum and ‘cold’ in the infrared part of the spectrum. Cloud screening techniques range from threshold-based testing [9–11], to neural networks [12–15], probabilistic approaches [16,17] and full Bayesian probability calculations [18]. Problems typically occur where the clouds are of a similar temperature to the underlying surface (e.g., low-level fog), along clear-cloud transition zones and over bright and/or cold surfaces [19–22]. At night, visual detection efficiency is reduced, amplifying the challenge of detecting low-level warm clouds. Detection difficulties can also be magnified over land due to rapid spatial variations in land surface emissivity and surface reflectance, making retrievals more difficult over pixels containing multiple land surface types and varying surface topography [17,23,24]. Cloud detection errors contribute to structural uncertainty in surface temperature retrieval, an uncertainty arising due to the methodological approaches adopted in the retrieval (e.g., which type of cloud detection algorithm is used) [25]. Quantifying structural uncertainties in remotely-sensed LST due to cloud detection failures is challenging without a ‘truth’ mask against which to make comparisons, and will of course be dependent on the cloud masking technique chosen in the creation 3 of 20 Remote Sens. 2018, 10, 616 of any given data product. There is an awareness within the remote sensing community that cloud mask failures do impact retrieval uncertainty [2,26], but the magnitude of cloud detection uncertainties in LST retrieval have yet to be fully quantified. 2. Cloud Clearing Inter-Comparison The cloud clearing inter-comparison within the ESA DUE GlobTemperature project was designed to assess a range of different cloud detection techniques for their suitability in land surface temperature (LST) retrieval from the Advanced Along-Track Scanning Radiometer (AATSR). AATSR, the third in a series of such instruments (following ATSR-1 and ATSR-2), flew aboard Envisat and was the precursor to the now operational Sea and Land Surface Radiometer (SLSTR) aboard Sentinel-3. This instrument series was designed specifically for the retrieval of surface temperature and is therefore the optimal choice for the production of a long-term LST climate data record (CDR) [27]. AASTR is a dual-view radiometer, making observations at 1-km resolution at the sub-satellite point in the nadir view (with viewing angles of 0–22◦) and a second observation in the forward view (with viewing angles of 52–56◦). AATSR has seven channels covering the visible and infrared spectrum, centred at 0.55, 0.66, 0.87, 1.6, 3.7, 10.8 and 12 µm. It flew between 2002 and 2012 in Sun-synchronous orbit with an Equator overpass time of 10.30 a.m. For LST retrieval, only observations in the nadir view are used. The cloud-clearing inter-comparison exercise had three phases designed to give participants an opportunity to develop their algorithms prior to the final comparison: training, testing and submission. At each stage, data for ten AATSR sub-scenes were provided, with a size of 512 × 512 pixels. These scenes were chosen to cover a wide range of conditions, specifically focussing on those where cloud detection is known to be more challenging. Within each set of ten scenes, as many as possible of the following conditions were represented: cumulus, stratus and cirrus cloud types; desert, vegetated, forest, ice and coast land surface types; a range of atmospheric total column water vapour (TCWV) and scenes where some dust or smoke aerosol was present. The training phase was designed for algorithm development, and the test dataset was available to ensure that developers had not tuned their algorithm too closely to a particular scene selection. At these stages, a ‘truth’ cloud mask was created manually by expert inspection and provided to algorithm developers to assess their cloud mask performance. The analysis in this paper uses the final submission dataset where participants provided their data ‘blind’ without reference to a truth cloud mask (although this was available at the point of analysing the ensemble). 1. Introduction In this paper, we use data from multiple cloud masks collated within a cloud detection inter-comparison exercise for the Advanced Along-Track Scanning Radiometer (AATSR), within the European Space Agency (ESA) Data User Element (DUE) GlobTemperature project. Collating a range of leading cloud masks over a consistent set of images enables us to evaluate structural uncertainties due to clouds using an ensemble approach. Multiple independent datasets help to resolve the state-space of the structural uncertainty [25] and provide an estimate of its magnitude. The paper proceeds as follows: we describe the cloud clearing inter-comparison (Section 2), our approach to deriving LST uncertainties within the retrieval process (Section 3) and our understanding of cloud masking uncertainties using the ensemble data (Section 4). We present the discussion and conclusions in Section 5. 5 of 20 Figure 1. The 10.8-µm brightness temperatures for the 10 submission scenes in the cloud clearing inter-comparison (Table 1) at native resolution (∼1 km). Individual plot headings give the scene number, date and orbit start time. Figure 1. The 10.8-µm brightness temperatures for the 10 submission scenes in the cloud clearing inter-comparison (Table 1) at native resolution (∼1 km). Individual plot headings give the scene number, date and orbit start time. The data provided to algorithm developers with each scene were designed to be comprehensive enough to implement any type of cloud detection scheme. Observations were provided for collocated views (forward and nadir) in all channels, in addition to the land-sea mask, and confidence flags from the L1b data. Numerical weather prediction (NWP) data were provided for those algorithms dependent on radiative transfer simulation, including skin temperature and TCWV from ERA-Interim data [28], and surface emissivity and reflectance from the UVIREMIS and BRDF atlases [29,30]. These are used as inputs into the RTTOV 11 fast radiative transfer model [31] to simulate top of atmosphere reflectance and brightness temperatures. We also supplied land surface cover data, which is a variant of the GlobCover dataset [5] and all-sky LST (i.e., with no cloud detection pre-processing). y p p g Expert cloud masks were generated for each of the 30 scenes across the three phases of the inter-comparison exercise. These were constructed using a bespoke tool developed for this purpose by the Institute for Environmental Analytics at the University of Reading [32]. It enables the user to view a given scene from a number of different instrument channels concurrently and to easily construct channel inter-comparisons (such as differences, RGB images and local standard deviations). The majority of cloud in a given scene can generally be masked using thresholds on the channel data specific to the image (set using sliders), and in challenging regions, the mask can be edited by hand. This allows the user to refine the masking in areas such as cloud edges and sunglint where thresholds appropriate for the majority of the image may not apply locally. Figure 2 shows this manual cloud mask for each of the scenes in the submission dataset for land only (ocean and inland water bodies are masked out and shown in blue). The classification tool allowed pixels to be classified as ‘clear’, ‘probably clear’, ‘probably cloud’ and ‘cloud’. 2. Cloud Clearing Inter-Comparison A full description of the submission scenes is provided in Table 1. Figure 1 shows the 10.8-µm brightness temperatures for each of the ten scenes at native resolution (∼1 km). Particular features to note are that Scene 3 contains a fire with some smoke aerosol in the top right quadrant of the image, but the spatial extent of high aerosol loading is limited. Scene 5 has a significant dust aerosol loading across the bottom-right of the image, which will affect the LST where a retrieval is made. Scene 7 is a composite of ice over land and sea-ice with some cloud cover over the left side of the peninsula ridge and towards the top of the image. LST is only retrieved over the Antarctic Peninsula and not over the sea-ice region (GT_ATS_2P v1.0 dataset). Remote Sens. 2018, 10, 616 4 of 20 Table 1. Key characteristics of the ten submission scenes used within the cloud clearing inter-comparison. Scene Location Date Orbit No. Start Time Lines Time of Day Land Cover Cloud Type Aerosol Elevation 1 China 21/05/2007 27304 14:34:28 3,600–4,111 Night Cropland/Shrubland/Forest Stratus, Cumulus None 0–4000 m 2 Russia 22/05/2007 27314 07:20:27 13,789–14,300 Day Forest/Snow/Ice Stratus/Cumulus None 0–300 m 3 Florida 23/05/2007 27333 15:11:50 17,450–17,961 Day Forest/Vegetation Cumulus/Cirrus Smoke 0–250 m 4 UK 23/08/2007 28647 10:18:40 14,675–15,186 Day Grassland/Shrubland/Cropland Stratus/Cirrus None 0–600 m 5 Algeria 11/06/2010 43290 09:26:40 18,140–18,651 Day Bare Soil Cirrostratus/Altostratus Dust 300–600 m 6 Ukraine 05/08/2010 44083 19:01:32 6,250–6,761 Night Vegetation/Cropland/River Cumulostratus None 0–400 m 7 Antarctica 08/08/2010 44121 10:44:17 29,320–29,831 Night Snow/Ice Stratus None 0–2250 m 8 Mauritania 08/80/2010 44128 22:28:29 3,050–3,561 Night Bare Soil Stratus None 100–700 m 9 Canada 07/06/2011 48474 17:22:31 14,500–15,011 Day Evergreen Forest Stratus/Cirrus None 300–800 m 10 Uruguay 11/11/2009 40250 00:26:21 37,570–38,081 Night Vegetation/Cropland/Shrubland Stratus None 0–600 m 5 of 20 Remote Sens. 2018, 10, 616 5 of 20 ‘Clear’ and ‘cloud’ were the most commonly-used categories, with ‘probably clear’ and ‘probably cloud’ used infrequently, typically in the proximity of aerosol. For analysis purposes, the few ‘probably clear’ pixels are interpreted as clear-sky and ‘probably cloud’ pixels interpreted as cloud. The dust aerosol in Scene 5 is set as clear-sky by the manual mask for the purpose of considering cloud masking uncertainties, but aerosol with a high optical depth will also affect the retrieved LST. Remote Sens. 2018, 10, 616 6 of 20 6 of 20 6 of 20 Figure 2. Manual cloud mask for the 10 submission scenes in the cloud clearing inter-comparison (Table 1) at native resolution (∼1 km). Individual plot headings give the scene number, date and orbit start time. Ocean and inland water bodies are plotted in blue. Figure 2. Manual cloud mask for the 10 submission scenes in the cloud clearing inter-comparison (Table 1) at native resolution (∼1 km). Individual plot headings give the scene number, date and orbit start time. Ocean and inland water bodies are plotted in blue. There were eight participants in the cloud clearing inter-comparison from across Europe, including the AATSR operational cloud mask from the Synthesis of ATSR Data Into Sea-Surface Temperature (SADIST) [9], the AATSR Dual View V2 (ADV V2) [11,33], the Avhrr Processing scheme Over Land, cLouds and Ocean- Next Generation (APOLLO_NG) [16], Optimal Retrieval of Aerosol and Cloud (ORAC) [10], Generalised Bayesian Cloud Screening (Bayes Min and Bayes Max) [17,18], Community Cloud Retrieval for Climate (CC4CL) [14,15] and the University of Leicester Version 3 (UoL_V3) [17,34] algorithms. This selection of algorithms represents a wide range of cloud detection techniques, including threshold-based testing, neural networks, probabilistic approaches and full Bayesian calculations. With the exception of SADIST, the algorithms have all been developed within the following European Space Agency-funded projects: GlobTemperature Data User Element, Long-Term Land Surface Temperature Validation, Aerosol Climate Change Initiative (Aerosol_cci) and Cloud CCI; and have a wider use within the Earth Observation community beyond the extensive datasets produced within these projects. Full details on each of the contributing algorithms are provided in Table 2. Subsequent to the time of submission to the cloud clearing inter-comparison exercise, the ORAC algorithm has been replaced by the CC4CL algorithm within Aerosol_cci and is not therefore included in the ensemble analyses presented in this paper. 7 of 20 7 of 20 Remote Sens. 2018, 10, 616 Table 2. Key characteristics of the cloud detection algorithms submitted to the GlobTemperature Cloud Clearing Inter-Comparison exercise. Expansion of algorithm acronyms are as follows: Synthesis of ATSR Data Into Sea-Surface Temperature (SADIST); AATSR Dual View V2 (ADV 2); the Avhrr Processing scheme Over Land, cLouds and Ocean-Next Generation (APOLLO_NG); Optimal Retrieval of Aerosol and Cloud (ORAC); Bayesian Cloud Detection (Bayes Min and Bayes Max); Community Cloud Retrieval for Climate (CC4CL); University of Leicester Version 3 (UoL_V3). 3. Uncertainties in LST Retrieval LST from AATSR is retrieved using the 10.8 and 12 µm channels in a nadir-only split window algorithm [5,35]. LST from AATSR is retrieved using the 10.8 and 12 µm channels in a nadir-only split window algorithm [5,35]. LST = a f,i,pw + bf,i(T11 −T12)p(θ) + (bf,i + c f,i)T12 (1) (1) where a, b and c are retrieval coefficients, dependent on biome (i), fractional vegetation cover (f), precipitable water (pw) and satellite viewing angle (p(θ)). T11 and T12 denote the 10.8 and 12 µm brightness temperatures, respectively. The retrieval coefficients vary as a function of time of day (day or night), and the dependent parameters vary on differing length scales: the biome or land cover classification is fixed for a given location throughout time, whilst the precipitable water and fractional vegetation cover change seasonally. The land cover classification is based on the GlobCover dataset [36], regridded at 1/120◦resolution. The bare soil category is sub-divided into six different classes, giving a total of twenty-seven distinct land cover classifications. Fractional vegetation cover is calculated from the Copernicus Global Land Services dataset [37] at ten-day temporal resolution, whilst the precipitable water is derived from the six-hourly ERA-Interim dataset [28]. Uncertainties in the LST retrieval arise from instrument noise, instrument calibration, specification of surface parameters (e.g., surface emissivity, land cover classification) and because the retrieval is made through the Earth’s atmosphere. Radiative transfer is simulated in the retrieval process using a fast forward model (RTTOV 11) constrained by Numerical Weather Prediction (NWP) data. Uncertainties are characterised using an approach consistent with the production of sea surface temperature data within the ESA CCI project [4,8], where an uncertainty budget is constructed from a number of distinct components: uncorrelated, locally correlated and systematic uncertainties [5]. Random errors are those that have no correlation between observations and include instrument noise and sub-pixel variability in surface emissivity not captured by the auxiliary data. Locally systematic uncertainties arise from errors in the surface emissivity for different land cover classifications (captured implicitly in the fractional vegetation cover), errors in the atmospheric state (via the precipitable water and radiative transfer parametrisations), errors in the selection of retrieval coefficients, errors in geolocation and uncertainty in the knowledge of the underlying biome. Within the retrieval, these locally systematic uncertainties are considered as two separate components relating to the surface (sfc) and atmosphere (atm). 6 of 20 Name Project Type Institution Day/Twilight (µm) Night (µm) Views RT 1 Current Application References SADIST Operational Threshold RAL a 10.8, 12 3.7, 10.8, 12 Nadir, Forward No ATSR Series [9] ADV V2 Aerosol_cci Threshold FMI b 0.6, 0.8, 10.8, 12 NA Nadir, Forward No AATSR [11,33] APOLLO_NG Aerosol_cci Probabilistic DLR c 0.6, 0.8, 10.8, 12 NA Nadir Only No AVHRR, AATSR [16] ORAC Aerosol_cci Threshold RAL a 0.5, 0.6, 0.8, 1.6, 10.8, 12 NA Nadir, Forward No ATSR Series [10] Bayes Min GlobTemperature Bayesian UoR d 10.8, 12 10.8, 12 Nadir Only Yes ATSR Series, AVHRR, SEVIRI, GOES [17,18] Bayes Max GlobTemperature Bayesian UoR d 0.6, 1.6, 10.8, 12 3.7, 10.8, 12 Nadir Only Yes ATSR Series, AVHRR, SEVIRI, GOES [17,18] CC4CL Cloud CCI Neural Network RAL a 0.6, 0.8, 1.6, 10.8, 12 3.7, 10.8, 12 Nadir Only No AATSR [14,15] UoL_V3 GlobTemperature Probabilistic UoL e 3.7, 10.8, 12 3.7, 10.8, 12 Nadir Only Yes ATSR Series [17,34] a Rutherford Appleton Laboratory; b Finnish Meteorological Institute; c Deutsches Zentrum für Luft- und Raumfahrt; d University of Reading; e University of Leicester; 1 Radiative Transfer. Remote Sens. 2018, 10, 616 8 of 20 3. Uncertainties in LST Retrieval Finally, the large-scale systematic uncertainty arises from calibration errors and the bias in the satellite surface temperatures relative to other sources of temperature data, once all known residual biases are corrected for [38]. This is currently set to a fixed value of 0.03 K [5]. Although the magnitude of individual uncertainty components can be algorithm dependent, the total uncertainty budget tends to be similar across different LST retrieval schemes. Within this paper, we consider uncertainties in the LST data when gridded at a 25 × 25 pixel resolution corresponding to a surface area of 625 km2. This is commensurate with a gridded resolution of 0.25◦at the tropics, a typical grid size for global surface temperature products. At Level 3, an additional source of uncertainty is introduced via the gridding process as LST can only be retrieved for clear-sky pixels. The sampling uncertainty (σl3 samp) is calculated as follows: σl3 samp = s ncld × σ2 LST,clr nland −1 (2) (2) where ncld is the number of cloudy pixels in the grid cell, nland is the number of land pixels in the grid cell and σLST is the standard deviation of the LST across the clear-sky pixels in the grid cell. Where there is only a single clear-sky pixel in the grid cell (and the LST standard deviation cannot be calculated), the variance is set to the maximum value across the scene. The sampling uncertainty is uncorrelated between Level 3 grid cells and is added to the upscaled Level 2 random uncertainty, where σl2 ran and σl3 ran denote the Level 2 and Level 3 random uncertainty components, respectively. Remote Sens. 2018, 10, 616 9 of 20 σl3 ran = v u u t∑σ2(l2) ran,clr n2 land + σ2samp (3) (3) The subscript ‘clr’ denotes clear sky pixels over land within the 625 km2 grid cell. The Level 2 atmospheric uncertainty component (σl2 atm) is upscaled to Level 3 (σl3 atm) as follows, assuming that atmospheric state effects are correlated over length scales of 25 km: The subscript ‘clr’ denotes clear sky pixels over land within the 625 km2 grid cell. 3. Uncertainties in LST Retrieval Individual plot headings give the scene number, date and orbit start time. We calculate a cloud masking uncertainty for these ten scenes using the ensemble of contributing cloud masks. Seven masks are applicable during the day and only five at night (as ADV and APOLLO_NG were designed for daytime classification only). We apply each cloud mask to the unfiltered LST field in turn and calculate an average LST in each 625-km2 grid cell. We then calculate the LST difference between applying the manual mask (MM) in each grid cell and applying each of the contributed masks (CM) in turn (LSTCM −LSTMM). The cloud masking structural uncertainty is then the standard deviation of the ensemble of LST differences. We classify any grid cells with 600 or more pixels with an ocean biome (out of 625 pixels in a given grid cell) as water and do not consider them in this analysis. For any land grid cells, the lower threshold on the number of clear-sky pixels required to calculate the mean LST is one, as this methodology is applied in the generation of operational products. Figure 4 shows the cloud masking uncertainties for each of the ten scenes. The magnitude of the cloud masking uncertainty is 0 K where all masks (including the manual mask) agree that there is clear-sky, and grid cells where all masks agree that it is cloudy are shown in white. Cloud masking uncertainties over largely clear regions range between 0 and 0.5 K. Small variations in the pixels flagged as cloud within a given grid cell will cause some uncertainty in the retrieved LST, as this is not homogeneous at 1-km resolution. Cloud masking uncertainty typically increases around the edges of cloud features and some land features where one or more masks incorrectly interpret them as cloud, being of the order of 1.5–3 K in magnitude. There is also some evidence of increased cloud detection uncertainty over broken cloud fields (Scenes 3 and 9). In some regions, the cloud masking uncertainty increases significantly to >10 K, and this uncertainty has higher spatial variability than the LST retrieval uncertainty. Figure 5 shows the retrieval (Equation (7)) and cloud masking uncertainties added in quadrature. The spatial variability from the cloud masking uncertainty dominates, with minimum total uncertainties typically of the order 1.5 K in clear-sky regions. 3. Uncertainties in LST Retrieval The Level 2 atmospheric uncertainty component (σl2 atm) is upscaled to Level 3 (σl3 atm) as follows, assuming that atmospheric state effects are correlated over length scales of 25 km: σl3 atm = v u u t∑σ2(l2) atm,clr nland (4) (4) For the surface component, it is assumed that the uncertainties are correlated with a length scale of 5 km, consistent with the resolution of the auxiliary surface datasets, but are uncorrelated over larger spatial scales. The AATSR data are at a native resolution of 1 km, so to calculate the Level 3 surface uncertainty component (σl3 s f c), we first calculate an intermediate surface uncertainty component at 25 km2 (σs f c_25km) before upscaling this to 625-km2 resolution. σs f c_25km = v u u t∑σ2(l2) s f c,clr nland (5) σl3 s f c = v u u t∑σ2 s f c_25km n2 land (6) (5) (6) The total uncertainty in a grid cell (σtotal) is given by adding all of the Level 3 uncertainty components in quadrature. The total uncertainty in a grid cell (σtotal) is given by adding all of the Level 3 uncertainty components in quadrature. σtotal = r σ2(l3) ran + σ2(l3) s f c + σ2(l3) atm + σ2(l3) sys (7) (7) where the Level 3 large-scale systematic uncertainty (σsys) is equal to 0.03 K as this is fully correlated across the gridded domain. The retrieval uncertainties (not including cloud detection uncertainties) for the ten scenes in the cloud clearing inter-comparison are shown in Figure 3. They are calculated having applied the manual cloud mask to the data, and the uncertainties are set to zero for entirely cloudy grid cells. We see that typically, the minimum retrieval uncertainty at this resolution is ∼1 K, increasing in some regions to 2–3 K. This is particularly evident over Florida (Scene 3), across parts of the U.K. (Scene 4), over Uruguay (Scene 5) and across Mauritania (Scene 9). Remote Sens. 2018, 10, 616 10 of 20 10 of 20 Figure 3. Level 3 LST retrieval uncertainties for the 10 submission scenes in the cloud clearing inter-comparison (Table 1) at 625-km2 resolution. Individual plot headings give the scene number, date and orbit start time. Figure 3. Level 3 LST retrieval uncertainties for the 10 submission scenes in the cloud clearing inter-comparison (Table 1) at 625-km2 resolution. 3. Uncertainties in LST Retrieval Regions of large cloud masking uncertainty do not always correlate with regions of high retrieval uncertainty, for example over the U.K. (Scene 4) and Mauritania (Scene 9). Remote Sens. 2018, 10, 616 11 of 20 Figure 4. Cloud detection uncertainties for the 10 submission scenes in the cloud clearing inter-comparison (Tables 1 and 2) at 625-km2 resolution. Individual plot headings give the scene number, date and orbit start time. White grid cells denote agreement between all masks on cloudy conditions. Black grid cells denote regions classified as entirely cloudy by the manual mask, but clear-sky in some of the contributing masks. Figure 4. Cloud detection uncertainties for the 10 submission scenes in the cloud clearing inter-comparison (Tables 1 and 2) at 625-km2 resolution. Individual plot headings give the scene number, date and orbit start time. White grid cells denote agreement between all masks on cloudy conditions. Black grid cells denote regions classified as entirely cloudy by the manual mask, but clear-sky in some of the contributing masks. Figure 5. Combined Level 3 LST retrieval and cloud detection uncertainties for the 10 submission scenes in the cloud clearing inter-comparison (Figures 3 and 4) at 625-km2 resolution. Individual plot headings give the scene number, date and orbit start time. White grid cells denote agreement between all masks on cloudy conditions. Black grid cells denote regions classified as entirely cloudy by the manual mask, but clear-sky in some of the contributing masks. Figure 5. Combined Level 3 LST retrieval and cloud detection uncertainties for the 10 submission scenes in the cloud clearing inter-comparison (Figures 3 and 4) at 625-km2 resolution. Individual plot headings give the scene number, date and orbit start time. White grid cells denote agreement between all masks on cloudy conditions. Black grid cells denote regions classified as entirely cloudy by the manual mask, but clear-sky in some of the contributing masks. 4. Understanding Cloud Detection Uncertainties Cloud detection uncertainty shows some spatial correlation with the location of cloudy features (Figure 2), but also has a spatial variability related to the land surface cover. We would expect cloud detection uncertainty to be positively correlated with the cloud fraction. Where fewer clear-sky pixels are available, these are less likely to be representative of the gridded mean LST, as the per-pixel values generally show a degree of heterogeneity. The spatial variability related to land surface cover is driven by several factors affecting the individual cloud masks within the ensemble: (1) Some cloud masks, particularly the threshold based methods, are sensitive to land features such as riverbeds. It is very difficult to select globally-applicable thresholds for cloud detection tests, and inevitably, those chosen work better in some regions than in others. Erroneous flagging of surface features will generate discrepancies in the mean LST between different cloud masks, increasing the cloud detection uncertainty. (2) The variability in the land cover classification within a given grid cell is also 12 of 20 Remote Sens. 2018, 10, 616 important. Where the land surface biome has a large variability, the LST is likely to vary more rapidly spatially, and the difference in sub-sampling of the cell introduced by the different cloud detection schemes (i.e., variations in the definition of the cloud edge) will have a more significant impact. In this situation, the spread of differences in LST with respect to the manual cloud mask will increase. Under these conditions, the distribution of different biomes within a grid cell will also be important, e.g., with a steady gradient in LST across the grid cell potentially giving a different uncertainty to a more heterogeneous LST field. Assuming a perfect cloud mask under these conditions, sampling uncertainties would also be higher here, than in more homogeneous regions [8]. Given these factors, it may be expected that we can parametrise Level 3 uncertainties as: (1) a function of the percentage of clear-sky pixels within a grid cell and (2) as a function of the underlying variability in LST. We examine both, using the manual mask as ‘truth’ for the percentage of clear-sky pixels, and the number of land biomes within a given grid cell as a proxy for LST variability, as we do not have representative LST’s for cloudy pixels. Figure 6 shows the number of clear-sky pixels in each 625-km2 grid cell. 4. Understanding Cloud Detection Uncertainties Figure 8a shows the relationship between the percentage of clear-sky pixels over land and the cloud detection uncertainty at 625-km2 resolution. The percentage of clear-sky pixel data on the x-axis is binned in 10% increments, and panel (b) shows the number of grid cells in each bin. The cloud detection uncertainty is the mean value across all grid cells in a given bin, and the error bars represent the standard error. The solid line shows all of the data from the eight scenes (excluding Scenes 5 and 7), indicating a negative correlation between the percentage of clear-sky pixels and cloud detection uncertainty. For a 10–20% clear-sky fraction, we see average cloud detection uncertainties of 2 K, which fall to 0.65 K for a clear-sky fraction between 90 and 100%. Of the scenes included in the analysis, four are daytime observations and four night-time observations, and we calculate the cloud detection uncertainty under these conditions independently. We see from the number of grid cells that this still gives ≥50 observations within each bin under different solar illuminations. The cloud detection uncertainties during the day are of larger magnitude than at night, but the slope of the relationship between uncertainty and clear-sky fraction is consistent with that shown for all of the data. At clear-sky fractions of 10–20%, cloud detection uncertainties are on average 2.5 K during the day and 1.6 K at night, falling to 1 K and 0.38 K respectively for clear-sky fractions of 90–100%. The magnitude of the day-night difference in cloud detection uncertainties for any given clear-sky fraction is of the order 0.6–0.8 K. We would not expect the cloud detection uncertainties to be 0 K at 100% clear-sky as this definition is applicable to the manual mask only. We also consider the cloud detection uncertainty as a function of the number of biomes present in the Level 3 grid cell, as a measure of the heterogeneity in the background against which the cloud detection must operate, e.g., surface temperature, emissivity and reflectance (Figure 9). We present results for grid cells with between four and twelve different biomes as this is the typical range for the majority of observations. Again, we plot all data and then day and night-time observations independently. 4. Understanding Cloud Detection Uncertainties Comparing this with Figure 4, we see some spatial correlations between lower percentages of clear-sky pixels and high cloud detection uncertainties, notably around the cloud features over China (Scene 1), Russia (Scene 2), Algeria (Scene 6) and in the top half of the Mauritania image (Scene 9). We also see regions where larger cloud detection uncertainties do not correspond to lower clear-sky percentages, for example over Canada (Scene 10, to the left of the image) and over the Antarctic Peninsula (Scene 7). Figure 7 shows the number of biomes in each 625-km2 grid cell for each scene. Here, we see that the number of biomes correlates positively with cloud detection uncertainty over China (Scene 1, to the right of the image) and over Florida (Scene 3). Figure 6. Clear-sky fraction for the 10 submission scenes in the cloud clearing inter-comparison (Table 1) at 625-km2 resolution. Individual plot headings give the scene number, date and orbit start time. Figure 6. Clear-sky fraction for the 10 submission scenes in the cloud clearing inter-comparison (Table 1) at 625-km2 resolution. Individual plot headings give the scene number, date and orbit start time. We examine the relationship between cloud detection uncertainty and the percentage of clear-sky pixels as defined by the manual mask across the ten scenes, excluding Scene 5 (high dust aerosol loading) and Scene 7 (Antarctic Peninsula). These data are excluded for the following reasons: (1) firstly because aerosol affects the retrieved LST, and the most appropriate treatment of aerosol affected pixels is dependent on the data application (see the discussion section for further details); (2) secondly because most cloud detection algorithms show very poor skill over ice, tending to flag all ice as cloud, or on occasion all ice as clear-sky, but failing to discriminate well between snow or ice surfaces and cloud. The cloud detection uncertainties in these particularly challenging regions are considered further at the end of this section. Remote Sens. 2018, 10, 616 13 of 20 13 of 20 Figure 7. Number of land biomes for the 10 submission scenes in the cloud clearing inter-comparison (Table 1) at 625-km2 resolution. Individual plot headings give the scene number, date and orbit start time. Figure 7. Number of land biomes for the 10 submission scenes in the cloud clearing inter-comparison (Table 1) at 625-km2 resolution. Individual plot headings give the scene number, date and orbit start time. 4. Understanding Cloud Detection Uncertainties We find a general increase in the cloud detection uncertainty with the number of biomes, although the relationship is not as consistent as that found with the clear-sky fraction. There will be a dependence on the biome type that folds into this analysis. For example, if all the biomes are different types of forest vegetation, the LST variability will be much lower than where the biome set includes more diverse biome types, e.g., bare soil and forest. At night, there seems to be a stronger relationship between the cloud detection uncertainty and the number of biomes and a much weaker one during the day. There is a time of day-dependent difference in cloud detection uncertainty where the number of biomes is few: 0.5 K at night and 1.5 K during the day for four biomes. For 11–12 biomes in a given grid cell, we see convergence in the uncertainties, which are typically of order 1.7 K under all conditions (for 11 biomes per grid cell). Remote Sens. 2018, 10, 616 14 of 20 14 of 20 0 20 40 60 80 100 Clear-Sky Pixels / % 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Cloud Detection Uncertainty / K (a) All Day Night 0 20 40 60 80 100 Clear-Sky Pixels / % 0 100 200 300 400 500 Number of L3 Grid Cells (b) All Day Night Figure 8. Cloud detection uncertainties as a function of clear-sky fraction (a) at 625-km2 resolution. The following are shown: all data (solid line), night-time only (dot-dash line) and daytime only (dashed line). The linear regression for cloud detection uncertainty as a function of clear-sky percentages is shown for all data. Panel (b) shows the number of observations in each bin. 0 20 40 60 80 100 Clear-Sky Pixels / % 0 100 200 300 400 500 Number of L3 Grid Cells (b) All Day Night 0 20 40 60 80 100 Clear-Sky Pixels / % 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Cloud Detection Uncertainty / K (a) All Day Night Figure 8. Cloud detection uncertainties as a function of clear-sky fraction (a) at 625-km2 resolution. The following are shown: all data (solid line), night-time only (dot-dash line) and daytime only (dashed line). The linear regression for cloud detection uncertainty as a function of clear-sky percentages is shown for all data. Panel (b) shows the number of observations in each bin. 4. Understanding Cloud Detection Uncertainties 4 5 6 7 8 9 10 11 12 Number of Biomes in L3 Grid Cell 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Cloud Detection Uncertainty / K (a) All Day Night 4 5 6 7 8 9 10 11 12 Number of Biomes in L3 Grid Cell 0 100 200 300 400 500 Number of L3 Grid Cells (b) All Day Night Figure 9. Cloud detection uncertainties as a function of number of biomes in each grid cell (a) at 625-km2 resolution. The following are shown: all data (solid line), night-time only (dot-dash line) and daytime only (dashed line). Panel (b) shows the number of observations in each bin. 4 5 6 7 8 9 10 11 12 Number of Biomes in L3 Grid Cell 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Cloud Detection Uncertainty / K (a) All Day Night 4 5 6 7 8 9 10 11 12 Number of Biomes in L3 Grid Cell 0 100 200 300 400 500 Number of L3 Grid Cells (b) All Day Night Figure 9. Cloud detection uncertainties as a function of number of biomes in each grid cell (a) at 625-km2 resolution. The following are shown: all data (solid line), night-time only (dot-dash line) and daytime only (dashed line). Panel (b) shows the number of observations in each bin. Finally, we examine whether this dataset can reveal any differences in the cloud detection uncertainties under different land cover classifications. To do this, we consider all ten scenes and look for grid cells that have a dominant biome, defined as where 80% or more of the land pixels within the grid cell have a single land cover classification. Within our scenes, we found five biomes for which we had 40+ grid cells in which they were the dominant land cover, as shown in Table 3. For each land cover classification considered here, the observations had a range of clear-sky fractions, upon which cloud detection uncertainty has a significant dependence (as shown in Figure 8). We therefore standardise all the results to the average percentage of clear-sky pixels across the five biomes (74.2%). We compare the model slope from a linear regression of all data against the clear-sky fraction (Figure 8) against the linear regressions of data points falling within each of the five identified biomes. 4. Understanding Cloud Detection Uncertainties We find that the slope of the model is strongly biome dependent (Figure 10a) and that a biome-specific correction to a clear-sky fraction of 74.2% is required. The linear regression gives each data point an equal weighting, with the assumption that we have the same confidence in the cloud detection uncertainty at every location, in the absence of any other information. The uncertainty in the regression 15 of 20 Remote Sens. 2018, 10, 616 model is calculated as the standard deviation of the statistical differences between the model and the data points (Table 3). The mosaic vegetation has the best constrained fit with a standard deviation in the model-data differences of 0.11 K. For bare soils, the standard deviation is ∼0.34 K, for forests 0.57 K and for permanent snow and ice 1 K. This means that there is a greater uncertainty in the regression model for forest and permanent snow and ice classes. Table 3. Land cover classification numbers, descriptions, number of observations and regression model uncertainties corresponding to the data shown in Figure 10. Land Cover Classification Description Number of Observations Regression Model Uncertainty 4 Mosaic vegetation/cropland 40 0.11 K 9 Open needle-leaved deciduous or evergreen forest 48 0.57 K 21 Bare soil (entisols-orthents) 352 0.33 K 22 Bare soil (shifting sand) 92 0.35 K 27 Permanent snow and ice 178 0.996 K Table 3. Land cover classification numbers, descriptions, number of observations and regression model uncertainties corresponding to the data shown in Figure 10. Figure 10 shows relative cloud detection uncertainties between the biomes. Panel (b) shows the mean standardised cloud detection uncertainty at 74.2% clear-sky fraction plotted for each biome, including the standard error on the mean (note that the absolute magnitude of these will vary with clear-sky fraction). Panel (c) shows the number of observations available for each land cover classification. We see here that the magnitude of the cloud detection uncertainty shows a significant dependence on the underlying biome. The largest cloud detection uncertainties are seen for permanent snow and ice (biome 27) with a magnitude of 1.95 K. Cloud detection uncertainties over open needle-leaved deciduous or evergreen forest (Biome 9) had a magnitude of 1.2 K. Two categories of bare soil were included: entisol-orthents and shifting sand (Biomes 21 and 22), and these give similar magnitudes of cloud detection uncertainty, 0.9 K and 1.0 K, respectively. 4. Understanding Cloud Detection Uncertainties The lowest cloud detection uncertainty was found over mosaic cropland/vegetation, with an average value of 0.09 K, suggesting that the land cover in this sample was very homogeneous in comparison with other vegetated biomes, e.g., forest (Biome 9). It is also important to note that the sample size available for these analyses is limited, particularly for the mosaic cropland and forest biomes (Table 3), and with a larger sample size, these estimates may vary. 0 20 40 60 80 100 Clear-Sky Pixels / % 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Cloud Detection Uncertainty / K (a) All data Veg (4) Forest (9) Soil (21) Sand (22) Snow (27) 4 9 21 22 27 Land Cover Classification 0.0 0.5 1.0 1.5 2.0 Cloud Detection Uncertainty / K (b) 0 1 2 3 4 5 6 Land Cover Classification 0 50 100 150 200 250 300 350 400 Number of L3 Grid Cells (c) Figure 10. Biome-specific linear regression models for cloud detection uncertainty dependence on clear-sky fraction (a). Standardised cloud detection uncertainties as a function of land cover classification (b). Panel (c) shows the number of observations for each land cover classification, with land cover classifications defined in Table 3. 0 1 2 3 4 5 6 Land Cover Classification 0 50 100 150 200 250 300 350 400 Number of L3 Grid Cells (c) 0 20 40 60 80 100 Clear-Sky Pixels / % 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Cloud Detection Uncertainty / K (a) All data Veg (4) Forest (9) Soil (21) Sand (22) Snow (27) 0 20 40 60 80 100 Clear-Sky Pixels / % 0 5 0 5 0 5 0 5 0 (a) All data Veg (4) Forest (9) Soil (21) Sand (22) Snow (27) 4 9 21 22 27 Land Cover Classification 0.0 0.5 1.0 1.5 2.0 Cloud Detection Uncertainty / K (b) 4 9 21 22 27 Land Cover Classification 0.0 0.5 1.0 1.5 2.0 Cloud Detection Uncertainty / K (b) Figure 10. Biome-specific linear regression models for cloud detection uncertainty dependence on clear-sky fraction (a). Standardised cloud detection uncertainties as a function of land cover classification (b). Panel (c) shows the number of observations for each land cover classification, with land cover classifications defined in Table 3. 5. Discussion and Conclusions The use of a cloud mask ensemble enables us to gain insight into the magnitude of cloud detection uncertainties in Level 3 data. There is a strong dependence on the percentage of clear-sky pixels in a given grid cell, with larger uncertainties in daytime data than at night. This is to be expected, as diurnal warming of the land surface during the day results in a more heterogeneous LST field, as 16 of 20 Remote Sens. 2018, 10, 616 16 of 20 the local rate of warming depends on the underlying land surface. Cloud shadowing will also affect LST, so where clouds are more abundant, giving fewer clear-sky pixels, shadowing may increase the LST variability and increase cloud detection uncertainty due to imperfect masking. At night, the land surface cools rapidly, and temperatures across neighbouring pixels will generally show less variation. One exception to this is boundaries between forest and grass or bare soil. Forests stay warmer at night, whilst grass and bare soil cool more quickly. The number of biomes is also a weak indicator of cloud detection uncertainty, used as a measure of heterogeneity in the background against which the cloud detection must operate. The correlation between the number of biomes and clear-sky fraction is stronger at night where it is the more dominant factor in LST variability, as other factors such as cloud shadowing are less important. As mentioned in Section 4, the appropriate classification of aerosol-affected observations is dependent on the application of the data, e.g., for retrievals of cloud properties, aerosol should be classified as ‘not cloud’, whilst for surface retrievals, it is often preferable for optically-thick aerosol to be classified as ‘not clear’. In the infrared, optically-thick aerosol can obscure the signal from the land surface, and optically-thin aerosol will do so in part. The relative temperature of the aerosol with respect to the land surface will depend on the aerosol composition and altitude, and aerosol is likely to modify the underlying surface temperature (unobserved by infrared satellite measurements) by modifying the atmospheric radiative transfer relative to clear-sky conditions. For these reasons, we excluded the large area of dust aerosol affected observations from the analysis of cloud detection uncertainty as a function of clear-sky fraction and the number of biomes. 5. Discussion and Conclusions These regions were included in the biome-dependent cloud detection uncertainty (Figure 10), with aerosol affecting a significant proportion of the observations over entisols-orthents bare soil (Biome 21). The cloud detection uncertainties were slightly larger here than for shifting sand (Biome 22). We would expect a more varied response in the cloud detection algorithms in aerosol-affected areas depending on their primary application, but find that the spread in the data is smaller than over shifting sand. There are however a number of additional factors that could affect these results, including variations in surface emissivity and cloud shadowing, which it is not possible to separate out using these data. Within this paper, we have considered only data gridded at 625-km2 resolution, but it could be expected that cloud detection uncertainty would vary as a function of gridding resolution. To address this point, we plot the cloud detection uncertainty as a function of both clear-sky fraction and number of biomes for the same data regridded at 25-km2, commensurate with a resolution of 0.05◦at the tropics. This resolution is commonly used in the provision of Level 3 uncollated data from polar orbiting satellite instruments. We assume that the locally systematic surface component of the uncertainty budget is correlated at this resolution, but otherwise follow the methodology described in Section 3. As the gridded resolution is increased, there is a significant increase in the number of Level 3 grid cells included in the analysis. Figure 11 shows that the overall magnitude of the cloud detection uncertainty is lower at 25-km2 than at 625-km2 resolution. Cloud detection uncertainties are still larger for daytime observations than for night-time observations, but at this resolution do not show such a strong correlation with the clear-sky fraction. During the day, cloud detection uncertainties are of a magnitude of ∼1.7 K for clear-sky fractions <80%, falling to ∼1.2 K for >80% clear-sky. Night-time cloud detection uncertainties fall more consistently with increasing clear-sky fraction, from ∼1 K at 0–40% clear-sky to ∼0.4 K at >80% clear-sky. Comparing against the 625-km2 data, the cloud detection uncertainties are similar for large clear-sky fractions, but lower at small clear-sky fractions. This may reflect less variability in the underlying LST for data gridded at a higher resolution. The difference in magnitude between day and night-time cloud detection uncertainties is similar, ranging between ∼0.6 and 1 K. 17 of 20 17 of 20 Remote Sens. 5. Discussion and Conclusions 2018, 10, 616 0 20 40 60 80 100 Clear-Sky Pixels / % 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Cloud Detection Uncertainty / K (a) All Day Night 0 20 40 60 80 100 Clear-Sky Pixels / % 1000 2000 3000 4000 5000 6000 7000 8000 Number of L3 Grid Cells (b) All Day Night 4 5 6 7 8 Number of Biomes in L3 Grid Cell 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Cloud Detection Uncertainty / K (c) All Day Night 4 5 6 7 8 Number of Biomes in L3 Grid Cell 0 2000 4000 6000 8000 10000 12000 Number of L3 Grid Cells (d) All Day Night Figure 11. Cloud detection uncertainties as a function of clear-sky fraction (a) and number of biomes (c) at 25-km2 resolution. Right-hand panels show the number of observations in each clear-sky bin (b) and for each number of biomes represented in a given grid cell (d). The following are shown: all data (solid line), night-time only (dot-dash line) and daytime only (dashed line). 0 20 40 60 80 100 Clear-Sky Pixels / % 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Cloud Detection Uncertainty / K (a) All Day Night 4 5 6 7 8 Number of Biomes in L3 Grid Cell 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Cloud Detection Uncertainty / K (c) All Day Night N mber of L3 Grid Cells 0 20 40 60 80 100 Clear-Sky Pixels / % 1000 2000 3000 4000 5000 6000 7000 8000 Number of L3 Grid Cells (b) All Day Night 4 5 6 7 8 Number of Biomes in L3 Grid Cell 0 2000 4000 6000 8000 10000 12000 Number of L3 Grid Cells (d) All Day Night Number of L3 Grid Cells Figure 11. Cloud detection uncertainties as a function of clear-sky fraction (a) and number of biomes (c) at 25-km2 resolution. Right-hand panels show the number of observations in each clear-sky bin (b) and for each number of biomes represented in a given grid cell (d). The following are shown: all data (solid line), night-time only (dot-dash line) and daytime only (dashed line). Figure 11. Cloud detection uncertainties as a function of clear-sky fraction (a) and number of biomes (c) at 25-km2 resolution. References 1. Merchant, C.J.; Paul, F.; Popp, T.; Ablain, M.; Bontemps, S.; Defourny, P.; Hollmann, R.; Lavergne, T.; Laeng, A.; de Leeuw, G.; et al. Uncertainty information in climate data records from Earth Observation. Earth Syst. Sci. Data 2017, 9, 511–527. [CrossRef] 2. Freitas, S.C.; Trigo, I.F.; Bioucas-Dias, J.M.; Göttsche, F.M. Quantifying the Uncertainty of Land Surface Temperature Retrievals From SEVIRI/Meteosat. EEE Trans. Geosci. Remote Sens. 2010, 48, 523–534. [CrossRef] 2. 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Global Land Surface Temperature from the along-Track Scanning Radiometers. J. Geophys. Res. Atmos. 2017, 122, 12167–12193. [CrossRef] 6. Corlett, G.; Atkinson, C.; Rayner, N.; Good, S.; Fiedler, E.; McLaren, A.; Hoeyer, J.; Bulgin, C.E. CCI Phase 1 (SST): Product Validation and Intercomparison Report (PVIR); ESA SST CCI Project, SST_CCI-PVIR-UoL-001; 2015. Available online: www.esa-sst-cci.org/PUG/pdf/SST_CCI-PVIR-UoL- 201-Issue_1-signed.pdf (accessed on 17 April 2018). 7. Merchant, C.J.; Ghent, D.; Kennedy, J.; Good, E.; Hoyer, J. Common Approach to Providing Uncertainty Estimates Across All Surfaces; EUSTACE (640171) Deliverable 1.1; 2015. Available online: https://www.eustaceproject. eu/eustace/static/media/uploads/Deliverables/eustace_d1-2.pdf (accessed on 17 April 2018). 8. Bulgin, C.E.; Embury, O.; Merchant, C.J. Sampling Uncertainty in Global Area Coverage (GAC) and G Sea Surface Temperature Products. Remote Sens. Environ. 2016, 177, 287–294. [CrossRef] 9. Zavody, A.M; Mutlow, C.T.; Llewellyn-Jones, D.T. Cloud Clearing over the Ocean in the Processing of Data from the Along-Track Scanning Radiometer (ATSR). J. Atmos. Ocean. Technol. 2000, 17, 595–615. [CrossRef] 10. Thomas, G.E.; Poulsen, C.A.; Siddans, R.; Carboni, E.; Grainger, R.G.; Povey, A.C. Algorithm Theoretical Basis Document (ATBD) AATSR Optimal Retrieval of Aerosol and Cloud (ORAC). 2015. Version 2.1, ESA Climate Change Initiative, Aerosol_cci_ORAC_ATBD_V2.1.pdf. Available online: http://www.esa-aerosol- cci.org/?q=webfm_send/1022/1 (accessed on 1 November 2017). 11. 5. Discussion and Conclusions at a gridded resolution of 625 km2), LST data producers are encouraged to quantify and provide cloud-related uncertainties in gridded data products. Acknowledgments: We acknowledge the work of G. H. Griffiths (Institute for Environmental Analytics, University of Reading) in developing the bespoke manual cloud clearing tool for the cloud clearing inter-comparison exercise. We also acknowledge NCEO funding, supporting the preparation of the cloud clearing inter-comparison datasets, and ESA Data User Element (DUE) GlobTemperature funding for conducting this research. For further information and possible access to the datasets used please contact the corresponding author. Author Contributions: The lead author, Claire Bulgin, undertook the majority of this work with input from Christopher Merchant. Darren Ghent, Lars Klüser, Thomas Popp, Caroline Poulsen and Larissa Sogacheva all provided data used in the analysis by taking part in the GlobTemperature cloud mask inter-comparison exercise. Conflicts of Interest: The authors declare no conflict of interest. 5. Discussion and Conclusions Right-hand panels show the number of observations in each clear-sky bin (b) and for each number of biomes represented in a given grid cell (d). The following are shown: all data (solid line), night-time only (dot-dash line) and daytime only (dashed line). When considering the cloud detection uncertainty as a function of the number of biomes, the range is smaller at 25-km2 resolution than at 625-km2 resolution. Here, we find that the number of biomes typically ranges between one and eight, with cloud detection uncertainties remaining fairly constant between one and four biomes and increasing thereafter. This increase happens at lower numbers of biomes in a given grid cell than for 625-km2 resolution data, where cloud detection uncertainty tends to increase for 7+ biomes. As in the comparison against the clear-sky fraction, uncertainties in the daytime data are of a larger magnitude than at night, with a typical difference of ∼0.6–1 K. For pixels containing 4–8 biomes, where a direct comparison can be made with the 625-km2 data, the cloud detection uncertainties are lower in the 25-km2 data, typically by a magnitude of ∼0.5 K. These analyses all provide insight into the magnitude of cloud detection uncertainties for Level 3 data gridded at different resolutions, but do not provide a mathematical model of cloud detection uncertainty for a single given cloud mask, which is usually all that is available for any given LST product. The uncertainty in LST arising from any single mask will be dependent on the tendency of that mask to over-flag or under-flag, and whether this varies as a function of the underlying land cover classification and vegetation fraction. This study does not separate cloud detection uncertainties due to omission (missed cloud) from those due to commission (unnecessarily flagged clear-sky pixels), although it could be hypothesised that the former may typically be larger in magnitude across many land cover classifications, as clouds are often significantly colder than the underlying land surface. 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Potential for Genetic Improvement of Sugarcane as a Source of Biomass for Biofuels
Frontiers in bioengineering and biotechnology
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Abbreviations: AFLP, amplified fragment length polymorphism; BAC, bacterial artificial chromosome; CAD, cinnamyl alcohol dehydrogenase (EC 1.1.1.195); cDNA, complementary DNA; COMT, caffeic acid O-methyltransferase (EC 2.1.1.68); DArT, diversity array technology; EST, expressed sequence tag; Gb/Mb, gigabase/megabase; LD, linkage disequilibrium; Lignin G, lignin guaiacyl; Lignin H, lignin hydroxyphenyl; Lignin S, lignin syringyl; NGS, next-generation sequencing; QTL, quantitative trait locus; RFLP, restricted fragment length polymorphism; RNAi, RNA interference; S/G ratio, syringyl/guaiacyl ratio; SNP, single nucleotide polymorphism; SUCEST, sugarcane EST database; TF, transcription factor; TIGR, the institute for genome research. Edited by: P. C. Abhilash, Banaras Hindu University, India Edited by: P. C. Abhilash, Banaras Hindu University, India Reviewed by: Yu-Shen Cheng, National Yunlin University of Science and Technology, Taiwan Tianju Chen, Chinese Academy of Sciences, China *Correspondence: Nam V. Hoang hoang.nam@uq.net.au Reviewed by: Yu-Shen Cheng, National Yunlin University of Science and Technology, Taiwan Tianju Chen, Chinese Academy of Sciences, China *Correspondence: Nam V. Hoang hoang.nam@uq.net.au Specialty section: This article was submitted to Bioenergy and Biofuels, a section of the journal Frontiers in Bioengineering and Biotechnology Potential for Genetic Improvement of Sugarcane as a Source of Biomass for Biofuels 1 Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St. Lucia, QLD, Australia, 2 College of Agriculture and Forestry, Hue University, Hue, Vietnam, 3 Sugar Research Australia, Indooroopilly, QLD, Australia, 4 Joint BioEnergy Institute, Emeryville, CA, USA Sugarcane (Saccharum spp. hybrids) has great potential as a major feedstock for biofuel production worldwide. It is considered among the best options for producing biofuels today due to an exceptional biomass production capacity, high carbohydrate (sugar + fiber) content, and a favorable energy input/output ratio. To maximize the conver- sion of sugarcane biomass into biofuels, it is imperative to generate improved sugarcane varieties with better biomass degradability. However, unlike many diploid plants, where genetic tools are well developed, biotechnological improvement is hindered in sugarcane by our current limited understanding of the large and complex genome. Therefore, understanding the genetics of the key biofuel traits in sugarcane and optimization of sugarcane biomass composition will advance efficient conversion of sugarcane biomass into fermentable sugars for biofuel production. The large existing phenotypic variation in Saccharum germplasm and the availability of the current genomics technologies will allow biofuel traits to be characterized, the genetic basis of critical differences in biomass composition to be determined, and targets for improvement of sugarcane for biofuels to be established. Emerging options for genetic improvement of sugarcane for the use as a bioenergy crop are reviewed. This will better define the targets for potential genetic manipulation of sugarcane biomass composition for biofuels. REVIEW REVIEW published: 17 November 2015 doi: 10.3389/fbioe.2015.00182 published: 17 November 2015 doi: 10.3389/fbioe.2015.00182 Keywords: sugarcane, biofuels, biomass for biofuels, biofuel traits, association studies INTRODUCTION Plant biomass from grasses such as sugarcane or woody species contains mostly cellulose, hemicel- lulose, and lignin (also referred to as lignocellulosic biomass), which can be converted to biofuels as a source of renewable energy. At the moment, plant biomass-derived biofuels have great potential in countries that have limited oil resources because they reduce the dependence on fossil fuel, mitigate air pollution by cutting down greenhouse gas emissions, and can be produced from a wide range Received: 25 July 2015 Accepted: 26 October 2015 Published: 17 November 2015 Biology and Genetics At the moment, the cost of bagasse pretreatment (to remove or separate its recal- citrant components before converting to biofuels) and microbial enzymes contributes mostly to the total production cost, resulting in reducing the incentive to transition from first generation to second generation of biofuels in sugarcane (Yuan et al., 2008). To maximize the efficiency of conversion of sugarcane biomass into biofuels, it is imperative to generate improved sugarcane cultivars with not only high biomass yield and fiber content but also better biomass degradability for conversion to biofuels in addition to improving the pretreatment and enzyme digestion technologies. Hybrid sugarcane was derived from crosses between a female S. officinarum (2n = 80) and a male S. spontaneum (2n = 40–128). Due to the female restitution phenomenon, at first, the F1 hybrid conserves the whole S. officinarum chromosome set and half of the S. spontaneum which was 2n + n, then a few backcrosses later, this hybrid breaks down to n + n, establishing the hybrid chromosome set of modern sugarcane hybrid (Bremer, 1961). For this reason, current sugarcane cultivars (Saccharum spp. hybrids) have a combination of a highly aneuploid and interspecific set of chromosomes. By using genomic in situ hybridization (GISH) and fluorescent in situ hybridization (FISH), it is revealed that among chromosomes in the nucleus of modern hybrid sugarcane, approximately 80% are contributed by S. officinarum, 10–20% from S. spontaneum, and less than 5–17% from recombination of chromosomes of the two species (D’Hont et al., 1996; Piperidis et al., 2001; Cuadrado et al., 2004). Modern sugarcane hybrids are normally crosses between varieties/clones, which makes the combination of the chromosomes in each offspring unique and unpredictable due to the random sorting of the chromosomes in the genome (Grivet and Arruda, 2002). The first sugarcane breed- ing program, which started more than one century ago, generated a few interspecific hybrids and constitutes the basic germplasm used by sugarcane breeding programs (Ming et al., 2010). Modern sugarcane cultivars are derived from the basic germplasm, but This review focuses on the potential for the genetic improve- ment of sugarcane as a source of biomass for biofuels, exploring the beneficial characteristics of sugarcane, the available genetic resources and germplasm, the potential of cell wall modifi- cation by breeding and biotechnology, and the potential of whole genome/transcriptome sequencing applications in dis- secting important biofuel traits to improve sugarcane biomass composition. Biology and Genetics Taxonomically, sugarcane belongs to the genus Saccharum (estab- lished by Carl Linnaeus in 1753), in the grass family Poaceae (or Gramineae), subfamily Panicoideae, tribe Andropogoneae, sub- tribe Sacharinae, under the group Saccharastrae and has a very close genetic relationship to sorghum and other grass family members such as Erianthus and Miscanthus (Amalraj and Bala- sundaram, 2006). Typically, the genus is divided into six different species namely Saccharum barberi, Saccharum edule, Saccharum officinarum, Saccharum robustum, Saccharum sinense, and Sac- charum spontaneum (Daniels and Roach, 1987; Amalraj and Bala- sundaram, 2006), in which S. spontaneum and S. robustum are wild species; S. officinarum, S. barberi, and S. sinense are early cultivars while S. edule is a marginal specialty cultivar. All genotypes of the Saccharum genus are reported to be polyploid with the ploidy level ranging from 5× to 16× and are considered as among the most complex plant genomes (Manners et al., 2004). The cytotype (2n, the number of chromosomes in the cell) was reported to be different in each species as follows: S. officinarum (2n = 80), S. spontaneum (2n = 40–128), S. barberi (2n = 111–120), S. sinense (2n = 81–124), S. edule (2n = 60–80), and S. robustum (2n = 60, 80); hence, the basic chromosome number (x, the monoploid set of chromosomes in the cell) ranges from 5, 6, 8, 10 to 12 (Sreenivasan et al., 1987). The basic chromosome number of S. spontaneum is 8 (even though a number of very variable cytotype is observed) and of S. officinarum and S. robustum is 10 [Panje and Babu, 1960, D’Hont et al. (1998), and Piperidis et al. (2010)]. For the other three species, S. sinense, S. barberi, and S. edule, due to the fact that these are early interspecific hybrid cultivars, there have not been a consensus reported, but a study by Ming et al. (1998) suggested that the basic chromosome number for these three species could also be 10. Conventionally, sugarcane bagasse is usually burned to produce fertilizer or steam and electricity to fuel the boilers in sugar mills (Pandey et al., 2000). Recently, it has been used for biofuel production; however, the production cost of biofuels from ligno- cellulosic biomass is still considered to be relatively high, which makes it difficult to be price-competitive and commercialized on a large scale (Halling and Simms-Borre, 2008). Citation: Hoang NV, Furtado A, Botha FC, Simmons BA and Henry RJ (2015) Potential for Genetic Improvement of Sugarcane as a Source of Biomass for Biofuels. Front. Bioeng. Biotechnol. 3:182. doi: 10.3389/fbioe.2015.00182 November 2015 | Volume 3 | Article 182 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 1 Sugarcane Genetic Improvement for Biofuels Hoang et al. SUGARCANE AT A QUICK GLANCE of abundant sources (Matsuoka et al., 2009). Biofuels generated from plant lignocellulosic biomass (also known as the second generation of biofuels) have been shown to be advantageous over the first generation (from plant starches, sugar, and oil) in terms of net energy and CO2 balance and, more importantly, they do not compete with food industries for supplies (Yuan et al., 2008). To date, producing bioethanol from the sugar in sugarcane has been one of the world’s most commercially successful biofuel pro- duction systems, with the potential to deliver second-generation fuels with a high positive energy balance and at a relatively low production cost (Yuan et al., 2008; Botha, 2009; Matsuoka et al., 2009). The rapid growth and high yield of sugarcane compared to other grasses and woody plants makes it a good candidate for ethanol processing platform and the second generation of biofuels in general (Pandey et al., 2000). Sugarcane has an exceptional ability to produce biomass as a C4 plant with the potential of a perennial grass crop allowing harvest four to five times by using ratoons without requiring replanting (Verheye, 2010), resulting in a lower cost of energy production from sugarcane than for most of the other potential sources of biomass (Botha, 2009). Brazil is the world’s first country to launch a national fuel alcohol program (ProAlcooL). This program is based on the use of sugarcane and substitutes the usage of gasoline by ethanol (Dias De Oliveira et al., 2005). Approximately, 23.4 billion liters (6.19 billion U.S. liquid gallons) of ethanol was produced in Brazil in the year 2014 (Renewable Fuels Association, 2015). As of 2009, sugarcane bagasse contributes to about 15% of the total electricity consumed in Brazil, and it is predicted that energy generated from sugarcane stalks could supply more than 30% of the country energy needs by 2020 and will be equal to or more than the electricity produced from hydropower (Matsuoka et al., 2009). The Nature of a Complex, Polyploid, and Repetitive Genome Biofuels from sugarcane can be produced extensively not only from its soluble sugar but also from main residues in sugar- cane production, bagasse and trash, on the same production area (Seabra et al., 2010; Alonso Pippo et al., 2011a,b; Macrelli et al., 2012). The total estimated available lignocellulosic biomass from sugarcane worldwide was 584 million dry tons per year, with an average lignocellulosic biomass yield of 22.9 dry tons per hectare per year (Van Der Weijde et al., 2013). Sugarcane bioethanol yield from bagasse is estimated at about 3,000 L per hectare in a total yield of 9,950 L per hectare from sugar and bagasse (Somerville et al., 2010). Biology and Genetics This will define the targets for potential genetic manipulation and better exploitation of sugarcane biomass for biofuels. Frontiers in Bioengineering and Biotechnology | www.frontiersin.org November 2015 | Volume 3 | Article 182 2 Sugarcane Genetic Improvement for Biofuels Hoang et al. TABLE 1 | Average lignocellulosic biomass yield (dry matter) from sugar- cane compared to other sources. Plant name Yield (tons/ha/year) Reference Sugarcane 22.9a Van Der Weijde et al. (2013) Switch grass 7–35 Reviewed in Hattori and Morita (2010) Miscanthus 12–40 Reviewed in Hattori and Morita (2010) Erianthus 40–60 Reviewed in Hattori and Morita (2010) Eucalyptus 15–40 Reviewed in Johansson and Burnham (1993) aAverage total cane biomass dry matter is 39 tons/ha/year (Moore, 2009). TABLE 1 | Average lignocellulosic biomass yield (dry matter) from sugar- cane compared to other sources. there has been only a few generations for chromosome recombi- nation opportunities (the number of meiosis that chromosomes have undergone is mainly about 2–7) as the sugarcane breeding processes normally take between 10 and 15 years (Raboin et al., 2008; Ming et al., 2010). As a result, the modern sugarcane popu- lation has a narrow genetic basis and high linkage disequilibrium (Roach, 1989; Lima et al., 2002; Raboin et al., 2008). The Nature of a Complex, Polyploid, and Repetitive Genome Globally, sugarcane is the most important crop in about 100 countries with a produc- tion area of 26.9 million hectares, total production of ~1.9 billion tons, and yield of 70.9 tons of fresh cane per hectare (FAOSTAT, 2015). At present, Brazil is the world’s largest sugarcane producer followed by India, China, Thailand, Pakistan, Mexico, Colombia, Indonesia, Philippines, U.S., and Australia (FAOSTAT, 2015). In sugarcane internodal tissue, sucrose concentration ranges from 14 to 42% of the dry weight (Whittaker and Botha, 1997), while the rest of dry biomass comes from the cell wall lignocellulose, mostly containing cellulose, hemicellulose, lignin, and ash (Pereira et al., 2015). Biofuels from sugarcane can be produced extensively not only from its soluble sugar but also from main residues in sugar- cane production, bagasse and trash, on the same production area (Seabra et al., 2010; Alonso Pippo et al., 2011a,b; Macrelli et al., 2012). The total estimated available lignocellulosic biomass from sugarcane worldwide was 584 million dry tons per year, with an average lignocellulosic biomass yield of 22.9 dry tons per hectare per year (Van Der Weijde et al., 2013). Sugarcane bioethanol yield from bagasse is estimated at about 3,000 L per hectare in a total yield of 9,950 L per hectare from sugar and bagasse (Somerville et al., 2010). et al., 2009; Moore, 2009; Henry, 2010a). At present, the most suitable energy crop is probably sugarcane because of its high biomass yield and the potential for production on other than prime agricultural land avoiding competing with the land used for food industries (Waclawovsky et al., 2010). Globally, sugarcane is the most important crop in about 100 countries with a produc- tion area of 26.9 million hectares, total production of ~1.9 billion tons, and yield of 70.9 tons of fresh cane per hectare (FAOSTAT, 2015). At present, Brazil is the world’s largest sugarcane producer followed by India, China, Thailand, Pakistan, Mexico, Colombia, Indonesia, Philippines, U.S., and Australia (FAOSTAT, 2015). In sugarcane internodal tissue, sucrose concentration ranges from 14 to 42% of the dry weight (Whittaker and Botha, 1997), while the rest of dry biomass comes from the cell wall lignocellulose, mostly containing cellulose, hemicellulose, lignin, and ash (Pereira et al., 2015). Existing Variations within Saccharum Germplasm Genetically diverse sugarcane germplasm may play a key role in improving sugarcane for biofuels through breeding and biotech- nological approaches. Genetic variation may be found in biomass yield, fiber content, and sugar composition in the Saccharum germplasm. This includes the diversity among the cultivars within one species and also diversity among species within the genus. A relatively high genetic variability within sugarcane hybrid cultivars was reported thanks to their heterozygosity and high polyploidy despite their originating from a few clones of a narrow genetic base (Aitken and McNeil, 2010). There is also great genetic and morphological diversity within Saccharum species, Miscanthus species, and Erianthus species to be poten- tially exploited and incorporated to broaden the genetic base in The Nature of a Complex, Polyploid, and Repetitive Genome aAverage total cane biomass dry matter is 39 tons/ha/year (Moore, 2009). p The complex and polyploid genome of sugarcane makes the pro- cess of analyzing and understanding difficult by normal methods applied to diploid plants. The size of the sugarcane genome is about 10 Gb while its genome complexity is due to the mixture of euploid and aneuploid chromosome sets with homologous genes present in from 8 to 12 copies (Souza et al., 2011). The estimated monoploid genome size is about 750–930 Mb (the monoploid genome size of the two parental species, S. officinarum and S. spon- taneum, are 930 Mb and 750 Mb, respectively), not much larger than the sorghum genome (~730 Mb) and about twice the size of the rice genome (~380 Mb) (D’Hont and Glaszmann, 2001). On the other hand, studies revealed that despite this complex and polyploid genome, sugarcane showed synteny with other grasses, especially sorghum (collinear, due to the limited divergence time) and maize (orthologous but altered loci collinearity) [reviewed in Grivet and Arruda (2002)]. It was thought that the sugarcane genome contains roughly the same amount of repetitive DNA as in the sorghum genome (Jannoo et al., 2007); however, studies on BAC-end sequences by Wang et al. (2010), Figueira et al. (2012), and Kim et al. (2013) suggested that there is less repet- itive content in the sugarcane genome (e.g., 45.2% and 42.8% repetitive sequences observed in large BAC collections in com- parison to 61% in the sorghum genome). More recently, using the k-mer approach, Berkman et al. (2014) found that the repet- itive proportion in three sugarcane hybrid cultivars ranges from 63.74 to 78.37% and higher than that in the sorghum genome (55.5%) using the same approach. The authors postulated that the increased proportion could be attributed to ploidy level rather than repetitive content in the sugarcane genome. A high gene- copy number, the integration of two chromosome sets from two different species, and a significant repeat content hinder the understanding of how the genome functions and obtaining a gen- uine assembled monoploid genome (Souza et al., 2011; Figueira et al., 2012). et al., 2009; Moore, 2009; Henry, 2010a). At present, the most suitable energy crop is probably sugarcane because of its high biomass yield and the potential for production on other than prime agricultural land avoiding competing with the land used for food industries (Waclawovsky et al., 2010). Candidate Crop for Future Biofuels These collections could be potentially selected and utilized for breeding purpose to improve sugarcane germplasm for new biofuel traits (Todd et al., 2014). The wild sugarcane species show wider variability in comparison to the domesticated species. In the Saccharum genus, S. spontaneum has the widest range of morphological variability, ratoon yielding, as well as biotic and abiotic stress tolerance (Tai and Miller, 2001; Aitken and McNeil, 2010; Govindaraj et al., 2014). The coefficient of variation (CV%) for some of the traits such as internode length, midrib width, leaf width, plant height, and stalk height studied by Govindaraj et al. (2014) were reported to be between 15 and 30%, which indicates a very high variability within the collection. It has been shown that the diversity within modern sugarcane hybrids was mostly contributed by the introgression from S. spontaneum (D’Hont et al., 1996). On the other hand, S. robustum also possesses a large amount of phenotypic variations in many traits studied (Aitken and McNeil, 2010). Sugarcane parental species (S. officinarum, S. spontaneum, and S. robustum), Miscanthus species, Erianthus species, and sorghum species with their diversity in genome con- tent, structure, and tremendous allelic variation are a valuable and significant genetic reservoir which could be exploited for improving sugarcane biomass. Therefore, it is difficult to allocate these markers into linkage groups or cosegregation groups or sugarcane expected chromo- some number (Souza et al., 2011). More detailed linkage maps of S. officinarum cultivar IJ76-545 (534 markers in 123 linkage groups) and cultivar Green German (615 markers in 72 linkage groups); S. spontaneum cultivar IND (536 markers in 69 linkage groups); and the hybrid cultivars R570 and Q165 (with 2,000 markers placed in more than 100 linkage groups) have been constructed (Souza et al., 2011; Aitken et al., 2014). Most recently, using Diversity Array Technology (DArT), Aitken et al. (2014) integrated DArT markers, RFLPs, AFLPs, SSRs, and SNPs into the largest marker collection for sugarcane, which contains 2,467 single-dose mark- ers for the cross between Q165 and IJ76-514 (a S. officinarum accession) and 2,267 markers from the cultivar Q165. These were placed into 160 linkage groups and eight homology groups, with some uncategorized linkage groups indicating that more markers are required. There is still a need to develop high-throughput marker arrays for sugarcane association studies, to generate more markers, and also to make use of the available markers. Candidate Crop for Future Biofuels These markers will be a valuable resource in facilitating and unraveling the complex genome structure of sugarcane. It is worth consider- ing that information on DNA-based molecular markers of progen- itor plants can potentially reveal available genetic polymorphism for the analysis of their progenies (Henry et al., 2012). This could be a useful strategy in the case of sugarcane, where the genomes of the parental species are less complex than that of the hybrids. g p p ( g , ) There are two world largest collections of germplasm of Sac- charum species, one is located in Florida (USA) while the other is in Kerala (India), containing approximately 1,200 accessions collected from 45 countries (Tai and Miller, 2001; Todd et al., 2014). These collections could be potentially selected and utilized for breeding purpose to improve sugarcane germplasm for new biofuel traits (Todd et al., 2014). The wild sugarcane species show wider variability in comparison to the domesticated species. In the Saccharum genus, S. spontaneum has the widest range of morphological variability, ratoon yielding, as well as biotic and abiotic stress tolerance (Tai and Miller, 2001; Aitken and McNeil, 2010; Govindaraj et al., 2014). The coefficient of variation (CV%) for some of the traits such as internode length, midrib width, leaf width, plant height, and stalk height studied by Govindaraj et al. (2014) were reported to be between 15 and 30%, which indicates a very high variability within the collection. It has been shown that the diversity within modern sugarcane hybrids was mostly contributed by the introgression from S. spontaneum (D’Hont et al., 1996). On the other hand, S. robustum also possesses a large amount of phenotypic variations in many traits studied (Aitken and McNeil, 2010). Sugarcane parental species (S. officinarum, S. spontaneum, and S. robustum), Miscanthus species, Erianthus species, and sorghum species with their diversity in genome con- tent, structure, and tremendous allelic variation are a valuable and significant genetic reservoir which could be exploited for improving sugarcane biomass. Transcriptome Sequences and Transcription Factors p Expressed sequence tags (ESTs) and complementary DNA (cDNA) sequences provide direct evidence of the genes present in the samples, and this sequence information is very useful for genome exploration, gene prediction/discovery, genome structure identification, SNP characterization, and transcriptome and pro- teome analysis (Nagaraj et al., 2007). As of May 2015, the GenBank EST database (dbEST) was composed of 75,906,308 ESTs from different organisms of which 284,818 hits were detected under the search term sugarcane (“S. officinarum” or “Saccharum hybrid cultivar” or sugarcane). In the last 20 years, sugarcane ESTs have been used for gene discovery, BAC clone selection, and dissecting the coding regions of the genome, involving many projects in South Africa, France, U.S., Australia, and Brazil (Carson and Botha, 2000, 2002; Vettore et al., 2001; Casu et al., 2003, 2004; Grivet et al., 2003; Pinto et al., 2004; Bower et al., 2005). The largest collection of sugarcane ESTs was generated by SUCEST, which is composed of approximate 238,208 ESTs from 26 diverse cDNA libraries of different tissues of sugarcane cultivars, e.g., SP80-3280, SP70-1143, RB845205, RB845298, and RB805028 (Vettore et al., 2001, 2003; Souza et al., 2011). These sequences were assembled into 42,982 sugarcane assembled sequences representing more than 30,000 unique genes (~90% of the estimated genes, about 43,141, of S. officinarum) (Vettore et al., 2003; Hotta et al., 2010; Grassius: Grass Regulatory Information Server, 2015). There are other sugarcane EST collections containing less EST entries gen- erated by Casu et al. (2003, 2004) (8,342 ESTs), Ma et al. (2004) (7,993 ESTs), Gupta et al. (2010) (~35,000 ESTs) and small number of ESTs by Carson and Botha (2000, 2002). Frontiers in Bioengineering and Biotechnology | www.frontiersin.org Candidate Crop for Future Biofuels To date, sugarcane is among the most efficient crops in the world together with other C4 grasses such as switch grass (Panicum virgatum), Miscanthus species (Miscanthus x giganteus), and Eri- anthus species (Erianthus arundinaceus Retz.) in terms of con- verting solar energy into stored chemical energy and biomass accumulation (Tew and Cobill, 2008; Furtado et al., 2014). In gen- eral, C4 plants outperform C3 plants in biomass yield, including grain, stem, and leaf yield (Jakob et al., 2009; Wang and Pater- son, 2013). Sugarcane and other C4 grasses are the highest yield potential feedstocks (Table 1), and for sugarcane, the potential yield can exceed 100 tons dry matter per hectare per year (Jakob Frontiers in Bioengineering and Biotechnology | www.frontiersin.org November 2015 | Volume 3 | Article 182 3 Sugarcane Genetic Improvement for Biofuels Hoang et al. breeding programs (Harvey et al., 1994; Aitken and McNeil, 2010). To date, the genetic diversity of S. officinarum has been exploited in breeding programs; however, the diversity of S. spontaneum and other species have not been used much (Aitken and McNeil, 2010). Saccharum species have also been shown to have varied genome size, S. officinarum genome is about 7.50–8.55 Gb, S. robustum ranging from 7.56 to 11.78 Gb, and S. spontaneum ranging from 3.36 to 12.64 Gb, whereas the other three species – S. sinense, S. barberi, S. edule – and modern sugarcane are interspecific hybrids whose genome size depends upon each cross (Zhang et al., 2012). breeding programs (Harvey et al., 1994; Aitken and McNeil, 2010). To date, the genetic diversity of S. officinarum has been exploited in breeding programs; however, the diversity of S. spontaneum and other species have not been used much (Aitken and McNeil, 2010). Saccharum species have also been shown to have varied genome size, S. officinarum genome is about 7.50–8.55 Gb, S. robustum ranging from 7.56 to 11.78 Gb, and S. spontaneum ranging from 3.36 to 12.64 Gb, whereas the other three species – S. sinense, S. barberi, S. edule – and modern sugarcane are interspecific hybrids whose genome size depends upon each cross (Zhang et al., 2012). There are two world largest collections of germplasm of Sac- charum species, one is located in Florida (USA) while the other is in Kerala (India), containing approximately 1,200 accessions collected from 45 countries (Tai and Miller, 2001; Todd et al., 2014). Sorghum bicolor Genome as the Closest Related Reference Genome Due to the homology between genomes, genome-wide map- ping of ESTs of one species provides an important framework for the genome structure of other related species (Sato et al., 2011). However, it is noteworthy that the discovery of the ESTs may be restricted to specific cultivars, as within sugarcane germplasm each cultivar has been shown to have different gene expres- sion level [reviewed in Hotta et al. (2010)]. Moreover, for bio- fuel trait analysis, the TFs regulating monolignol biosynthesis in lignin pathway have received attention as understanding this allows reducing and modifying lignin content and composition which are essential in addressing the recalcitrant problem in biomass conversion (Santos Brito et al., 2015). It is shown that the lignin regulation can be species specific and information on TFs obtained from model plants such as Arabidopsis may require to be validated in other species (Santos Brito et al., 2015). A limited number of TFs in grass and sugarcane have been preliminarily characterized recently including those involved in monolignol biosynthesis, for example, in grass (Handakumbura and Hazen, 2012), rice (Yoshida et al., 2013), sorghum (Yan et al., 2013), and sugarcane (Santos Brito et al., 2015). Gene discovery of sugarcane has progressed to some extent despite the complex- ity of the genome. The valuable information of ESTs, TFs, full- length cDNAs, and BACs provides an understanding of allelic variations in the genome while a full-genome sequence is not available. Sorghum is the most closely related species to sugarcane (Grivet et al., 1994; Dillon et al., 2007). The sorghum genome sequencing project was initiated and completed in 2007 with the total genome size of ~730 Mb, and 34,496 protein-coding loci, at the coverage of 8.5× using whole-genome shotgun sequencing by standard Sanger methodologies (Paterson et al., 2009). The sequenced genome is composed of 10 pairs of chromosomes and 3,294 supercontigs (most of these have been placed into chunks on 10 chromosomes), covering 90% of the genome and 99% of protein- coding regions (including the majority of available non-repetitive markers, known sorghum protein-coding genes, and the major- ity of ESTs) (Paterson et al., 2009). The sorghum genome has approximately 61% repetitive DNA, a low level of gene duplication compared to other C4 grasses, and a high degree of gene paral- lelism with sugarcane, even though the sugarcane genome is much more polyploid (Paterson et al., 2009, 2010). Sorghum bicolor Genome as the Closest Related Reference Genome Microcollinearity between sugarcane and sorghum genomes indicated that sorghum is suitable as the template for sugarcane genome assembly (Ming et al., 1998; Wang et al., 2010; Figueira et al., 2012). It has been suggested that the sugarcane genome could be 20–30% smaller than that of sorghum despite the estimated monoploid genome size of sugarcane being about 760–930 Mb, at approximately the size of the sorghum genome (Figueira et al., 2012). BIOMASS-DERIVED BIOFUELS AND THE CHALLENGING ISSUES IN BIOMASS CONVERSION TO BIOFUELS g Sugarcane cultivar R570 and other cultivars including ones from the parental species S. officinarum and S. spontaneum have been used for constructing of bacterial artificial chromosome (BAC) libraries (Hotta et al., 2010). BAC libraries from the sugarcane include hybrid cultivar R570 (103,296 clones, average insert size of 130 kb and two other libraries of 100,000 clones) (Tomkins et al., 1999; Grivet and Arruda, 2002), S. spontaneum cultivar SES208 (38,400 clones, average insert size of 120 kb), and S. officinarum cultivar LA Purple (74,880 clones, average insert size of 150 kb) generated from different restriction enzymes, e.g., HindIII and BamH1 [reviewed in Souza et al. (2011)]. BAC sequencing in sugarcane is currently based on the sequencing of BAC clones anchored to an available physical map. Even though it requires a higher cost compared to the whole-genome shotgun sequencing (using high-throughput platforms, Illumina, for example), it is a reliable approach for reference construc- tion, especially, for highly repetitive genomes which cannot be sequenced and resolved by a short-read method (Eversole et al., 2009; Steuernagel et al., 2009). This BAC sequencing approach has been used successfully in sequencing of Arabidop- sis, rice, and maize genomes and producing the barley reference genome [reviewed in Steuernagel et al. (2009)]. The ongoing Sugarcane Genome Sequencing Initiative (SUGESI) has selected 5,000 BAC clones for sequencing from a library by Tomkins et al. (1999) of cultivar R570, the most intensively character- ized cultivar to date, to help assembly of the monoploid cov- erage (monoploid tiling path) of the sugarcane genome using the sorghum sequence as the guide (Souza et al., 2011; Sugesi, 2015). Genetic Markers and Maps To support the effort of understanding the sugarcane genome, many physical maps, molecular markers, and resources such as RFLP, RAPD, AFLP, SSR, and ESTs have been developed over time. These common markers have been applied for genetic stud- ies such as diversity, mapping, quantitative trait loci (QTL), and synteny definition; however, these systems have been developed mostly for well-established diploid species and are less effective for polyploidy plants (Garcia et al., 2013). Markers like AFLP, SSR, and RFLP are unable to estimate the number of allelic copies and level of polyploidy in complicated genomes such as potato, strawberry, and sugarcane (Garcia et al., 2013). More recently, the use of SNPs markers, which are distributed at high density across the genome, for complex genomes can allow estimation of the number of allelic copies and the ploidy level of genomes (Zhu et al., 2008; Hall et al., 2010). The currently available genetic maps and markers have been generated for sugarcane by using low- throughput methods, providing limited information on genome organization due to the low density of markers and coverage (most of them have less than 1,000 markers) (Aitken et al., 2014). Frontiers in Bioengineering and Biotechnology | www.frontiersin.org November 2015 | Volume 3 | Article 182 4 Sugarcane Genetic Improvement for Biofuels Hoang et al. Dealing with the Conversion Issues Dealing with the Conversion Issues Even though sugarcane biomass is less resistant to enzymatic digestion compared to that from woody plants, it is reported that biomass recalcitrant components impede the efficiency of the conversion to ethanol (Jung, 1989; Anterola and Lewis, 2002; Chen and Dixon, 2007; Himmel et al., 2007; Balat et al., 2008; Li et al., 2013). Biomass recalcitrance is caused by many factors such as the presence of epidermal and sclerenchyma tissues, vas- cular bundle density and arrangement, degree of lignification, heterogeneity and complexity of cell wall constituents, insolu- ble matter, natural inhibitors, and cellulose crystallinity (Him- mel et al., 2007). Most approaches for producing biofuels from biomass at the moment rely on the disruption of the biomass, to separate lignocellulose and remove lignin in the biomass, and then conversion using microbial enzymes (Sticklen, 2006). In general, overcoming the recalcitrant issue can be addressed by physical, chemical, and genetic approaches. Physical and chemical strategies deal mainly with the pretreatment and involve loosening the cell wall structure, lowering the biomass heterogeneity, pro- viding the enzymes access to the cellulose, cleaving the crossing linkages, and removing enzymatic inhibitors (Balat et al., 2008; Saathoff et al., 2011). To make the physical and chemical changes in plant biomass, pretreatment processing conditions must be tailored to the specific chemical and structural composition of the various and variable sources of lignocellulosic biomass (Mosier et al., 2005). Currently available physical and chemical pretreat- ment methods are varied and can be listed as uncatalyzed steam explosion, flow-through acid, liquid hot water, pH-controlled hot water, dilute acid, ammonia, lime and, more recently, the method using ionic liquids (Mosier et al., 2005; Shi et al., 2013; Sun et al., 2013). Genetic approaches involve genetic enhancement, molecular biology, and plant breeding efforts to improve biomass sources by having crops with less lignin, modified lignin, crops that self-produced enzymes, and crops with increased cellulose and biomass overall [reviewed in Sticklen (2006)]. The Second Generation of Biofuels – Cell Walls for Fuels Due to the depletion of fossil fuel sources, the potential for oil to become more expensive, and the raising awareness of the negative impact of fossil fuels on the environment, biomass- derived biofuels have been investigated and developed recently as an alternative source of renewable, sufficient, and clean energy (Botha, 2009). The demand for renewable biofuels is predicted to be increasing (Fedenko et al., 2013). The first generation of biofuels from plant biomass involved the process of conversion of stored polysaccharides, non-structural carbohydrates, and oils from plants (starchy, sugary, and oily parts of plants such as corn starch, sugarcane molasses, soybeans, canola seeds, and palm oil) into fuels like ethanol and diesel (Schubert, 2006; Yuan et al., 2008). However, these sources are also used as food supplies and are limited due to the increasing demand from the growing world’s population (Schubert, 2006). The second generation of biofuels can be generated by using the non-food parts of plants such as cell walls, composed of structural polysaccharides, such as cellulose and hemicellulose (Schubert, 2006; Yuan et al., 2008; Henry, 2010a). This is considered to be advantageous over the first generation of biofuels as it has a higher energy production potential, lower cost, sustainable CO2 balance, no competition with the food production, and a wide range of plant biomass sources are available at costs affordable to a biorefinery (Yuan et al., 2008; Henry, 2010a). As of 2009, sugarcane biomass as November 2015 | Volume 3 | Article 182 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 5 Sugarcane Genetic Improvement for Biofuels Hoang et al. and the inner part of stem has higher syringyl/guaiacyl (S/G) ratio than the outer part. Polysaccharides found in sugarcane leaf and culm walls were similar but different in the proportions of xyloglu- can and arabinoxylan (Souza et al., 2013). The major monosaccha- rides released from sugarcane cell walls were glucose, xylose, and arabinose (Loureiro et al., 2011; Rabemanolontsoa and Saka, 2013; Souza et al., 2013). Understanding the fine structure and detailed composition of sugarcane cell wall will assist in optimizing the tissue pretreatment and cell wall hydrolysis protocol. The Second Generation of Biofuels – Cell Walls for Fuels At present, converting sugarcane lignocellulosic biomass to ethanol includes (1) pretreatment to remove the lignin and other recalcitrant cel- lular constituents (or hemicellulose) to free cellulose, (2) enzyme- mediated action to depolymerize carbohydrates to simple sugars, and (3) fermentation of sugars and distillation of ethanol as the end product (Canilha et al., 2012). sucrose accounted for about 40% of biofuels feedstock worldwide for first-generation biofuel production (Lam et al., 2009). Using sugarcane bagasse as a feedstock for second-generation biofuels would lead to doubling the current output of biofuel production from sugarcane (Halling and Simms-Borre, 2008). Frontiers in Bioengineering and Biotechnology | www.frontiersin.org Sugarcane Cell Wall and Biomass Composition Physically, sugarcane biomass can be divided into four major frac- tions, whose content depends on the industrial process: fiber (het- erogeneous organic solid fraction), non-soluble solids (inorganic substances), soluble solids (sucrose, waxes, and other chemicals), and water (Canilha et al., 2012; Shi et al., 2013). Second generation of biofuels focuses on using the fiber fraction especially the cell wall constituents of the plant to produce biofuels (Schubert, 2006; Henry, 2010a). This approach may be made more efficient by opti- mizing the composition of the biomass source for biofuel produc- tion. This could be achieved by advances in pretreatment methods or biotechnological modification of cell wall synthesis pathways to create a biomass that can be more efficiently processed (Sims et al., 2006; Yuan et al., 2008; Simpson, 2009; Viikari et al., 2012). Three major components make up the fiber fraction of sugarcane, namely, cellulose, hemicellulose (or non-cellulosic polysaccharide components), and lignin. Cellulose constitutes around 50% of the dry weight sugarcane bagasse while hemicellulose and lignin each account for about 25% (Loureiro et al., 2011). These three com- ponents are biosynthesized through different complex pathways (Higuchi, 1981; Whetten and Ron, 1995; Saxena and Brown, 2000; Mutwil et al., 2008; Harris and DeBolt, 2010; Pauly et al., 2013). Cellulose and hemicellulose molecules form the cell walls which act as the skeleton of plants and are strengthened by lignin and phenolic cross-linkages (Carpita, 1996; Henry, 2010b). The com- plex interlinking between cell wall components plays an important role in grass defense and yet challenges the biofuel production by requiring the pretreatment to separate them (De O. Buanafina, 2009). Dealing with the Conversion Issues The costs of the enzymatic pretreatment of cellulosic biomass (which accounts for about 25% of total processing expenses), biomass conversion, and microbial tanks limit the price-competitiveness of biofuel from lignocellulosic biomass in comparison to fossil fuel (Gnansounou and Dauriat, 2010; Macrelli et al., 2012, 2014; Van Der Weijde The sugarcane and grass cell wall are categorized as type II cell wall, which differs from the type I and type III cell walls of other plants [reviewed in Souza et al. (2013)]. In general, there is little pectin, less lignin, and less structural proteins in grass cell walls than that in the non-grasses (Carpita, 1996; Henry, 2010b; Saathoff et al., 2011). There is similar cellulose content between grass and non-grass primary and secondary cell walls; however, hemicellulose composition is different between two groups. Grass cell walls have four to eight times more xylans, higher mixed linkage glucans, and lower levels of xyloglucans, mannans, gluco- mannans, and pectin in primary cell wall, but higher phenolics and lignin in the secondary cell wall (Loureiro et al., 2011). Grassy lignin is composed of three monolignols (lignin syringyl – S, lignin guaiacyl – G and lignin hydroxyphenyl – H subunits) forming various ratios of them and normally has more H subunit (more coumaryl derivatives) than in non-grasses (Vogel, 2008). A recent study by Bottcher et al. (2013) showed that sugarcane lignin content and composition are varied depending on tissue types and stem positions on the plant. Within one plant, the bottom internode has higher lignin accumulation than the top internode, November 2015 | Volume 3 | Article 182 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 6 Sugarcane Genetic Improvement for Biofuels Hoang et al. the genetic diversity of the germplasm (Tabasum et al., 2010; Berkman et al., 2012). The use of markers associated with the desired traits in combination with the advances in next-generation sequencing (NGS) technology, bioinformatics tools, and high- throughput phenotyping methods will significantly improve the sugarcane breeding programs (Lam et al., 2009). NGS will allow a great number of markers such as SNPs to be generated, which could be used to obtain a high density of marker at high coverage across the genome, to dissect the important traits they associate with. Dealing with the Conversion Issues These sources of markers will be essential in breeding programs for screening of the parental plants from germplasm collection and of progenies derived from the crosses, selecting traits where the phenotypic methods are not practical (Berkman et al., 2012). High-throughput phenotyping methods will collect data from a large number of samples to overcome the small effects of genes, especially the QTL, controlling the traits (Lam et al., 2009). et al., 2013). This emphasizes the value of genetic improvement of biomass composition to reduce processing costs. POTENTIAL IMPROVEMENT BY MOLECULAR GENETICS FOR BIOFUELS The competitiveness of biofuels over other options relies on biotechnology advancement. Efficient conversion of plant biomass to biofuels requires the supply of appropriate feedstocks that can be sustainably produced in large quantities at high yields. The efficient conversion of the biomass in these feedstocks will be facilitated by having a composition that is optimized for efficient processing to deliver high yields of the desired end products. Manipulating of the carbohydrates of the cell walls is the key of improving the biomass composition for biofuels (Harris and DeBolt, 2010). Powerful tools of biotechnology could aim to produce genetically modified sugarcane plants with a favorable ratio of cellulose to non-cellulose content; with in planta enzymes that can digest the biomass or degrade the lignin prior to its conversion to ethanol; with pest and disease resistance, flower inhibition, abiotic resistance; or incorporate them into elite sugarcane cultivars for better agronomic performance (Sticklen, 2006; Yuan et al., 2008; Matsuoka et al., 2009; Arruda, 2012). To facilitate the second generation of biofuels, sugarcane breed- ing programs need to be focusing not only on important traits such as total biomass yield, sugar yield adaptability to local environ- ment, and resistance to major pathogens but also on biofuel traits (e.g., less lignin, improve biomass composition for conversion) as a whole (Matsuoka et al., 2009; Waclawovsky et al., 2010). In sugarcane breeding, to maximize heterosis, the parents are usually selected from divergent genotypes of genetic background (Taba- sum et al., 2010). Increasing sugarcane biomass yield and produc- tivity is getting more and more difficult to achieve by conventional methods; hence, broadening the sugarcane genetic basis by intro- gression of its ancestors or closely related species such as Miscant- hus and Erianthus is being explored in sugarcane improvement [reviewed in Dal-Bianco et al. (2012) and De Siqueira Ferreira et al. (2013)]. This is normally done by crossing S. officinarum and Erianthus, Miscanthus, or backcrossing the hybrids to S. spon- taneum (Matsuoka et al., 2009). Dual-purpose cane and energy cane, sugarcane lines for lignocellulosic biomass production, have been derived from two sugarcane species, S. spontaneum and S. robustum, by crossing to develop lines with a high ability to accu- mulate fiber and high biomass content in addition to accumulat- ing soluble sugars (De Siqueira Ferreira et al., 2013). Another case is Miscane, which was the result of crossing between Saccharum x Miscanthus. POTENTIAL IMPROVEMENT OF SUGARCANE BY BREEDING FOR BIOFUELS The complex and highly polyploid genome of sugarcane poses a great challenge in unraveling and studying its functions. Each cross of modern sugarcane cultivar has a unique set of chromo- somes due to the random sorting of chromosomes and recombi- nation of alleles from two progenitor species (Grivet and Arruda, 2002). There are several distinct alleles at each locus in sugar- cane chromosomes, making the characteristics of the offspring unpredictable and requiring evaluation of thousands of lines from many parents to gather sufficient information in breeding pro- grams (Matsuoka et al., 2009). In conventional breeding, after crossing and obtaining the F1 generation, hundreds of thousands of F1 seedlings are used for screening for the desired traits such as disease resistance, sugar content, agronomic characteristics, and adaptability (Matsuoka et al., 2009). The process is normally repeated for some vegetatively propagated generations to obtain the required stability of the traits. For industrial purpose, after a long process of selection, from hundreds of thousands seedlings at the beginning, breeders normally end up at a limited number of clones for release as commercial lines or cultivars. POTENTIAL IMPROVEMENT BY MOLECULAR GENETICS FOR BIOFUELS This produces cane varieties with more biomass (lignocellulose and total fermentable sugars), disease resistance, and cold tolerance. This effectively adapts Miscanthus to a tropical climate and expands sugarcane production to temperate, dry, and cold conditions (Alexander, 1985; Burner et al., 2009; Lam et al., 2009). Recently, using molecular markers in sugarcane breeding program (marker-assisted selection) allows the direct comparison of DNA genetic diversity and provides a precise tool in assessing Among the grasses potentially used for biofuel production such as sugarcane, switch grass, Miscanthus, and Erianthus, sugarcane has been used more for gene transformation studies (Falco et al., 2000; Manickavasagam et al., 2004; Basnayake et al., 2011) and the first transgenic sugarcane was established by Bower and Birch (1992). The current status of improving sugarcane biomass by using the genetic tools is hindered by its genome complexity, low transformation efficiency, transgene inactivation (gene silencing and regulation), somaclonal variation, and difficulty in backcross- ing (Ingelbrecht et al., 1999; Hotta et al., 2010; Arruda, 2012; Dal-Bianco et al., 2012). Targets tackled so far on sugarcane include sucrose and biomass yield increase [i.e., in Ma et al. (2000) and Botha et al. (2001)], downregulation of lignin con- tent or monolignol changes in lignin to lower biomass recalci- trance (described later), expression and accumulation of microbial cellulosic enzymes in leaf [i.e., in Harrison et al. (2011)], her- bicide tolerance [i.e., in Gallo-Meagher and Irvine (1996) and Enríquez-Obregón et al. (1998)], disease or pest resistance [i.e., in Joyce et al. (1998), Arencibia et al. (1999), and Zhang et al. Frontiers in Bioengineering and Biotechnology | www.frontiersin.org November 2015 | Volume 3 | Article 182 7 Sugarcane Genetic Improvement for Biofuels Hoang et al. wall constituent biosynthesis (Jung et al., 2012; Bottcher et al., 2013). (1999)], flowering inhibition [reviewed in Matsuoka et al. (2009) and Hotta et al. (2010)], and drought tolerance [i.e., in Zhang et al. (2006)]. Genetically modified sugarcane has great potential to contribute to biofuel production, with new varieties incorporating these characteristics (Arruda, 2012). Unexploited genes not only from the Saccharum germplasm but also in other related species, such as cold-tolerant genes in S. spontaneum and Miscanthus or drought-tolerant genes in sorghum, once identified would allow their integration into the sugarcane genome, facilitating the pro- duction of more sugarcane biomass in temperate areas or under dry conditions (Lam et al., 2009). POTENTIAL IMPROVEMENT BY MOLECULAR GENETICS FOR BIOFUELS Producing enzymes in planta is another way to cut the cost of biofuel production as it reduces the expense of enzymes and enzyme treatment. Cellulase has been produced within the plant (in the apoplast) of Arabidopsis, rice, and maize without effects on the growth and development of the host plants [reviewed in Sticklen (2006)]. In planta enzyme expression in sugarcane is still in its infancy; however, a high-yield biofuel plant such as sugarcane must be a target for the production of enzymes within the biomass. Recombinant protein enzymes have been targeted to organelles such as chloroplasts, vacuoles, and the endoplasmic reticulum to separate the enzymes produced and their substrates (Harrison et al., 2011). In sugarcane, thanks to its well-established transformation methods via Agrobacterium, the expression of enzymes in leaves and other tissues is feasible (Manickavasagam et al., 2004; Taylor et al., 2008). Endoglucanases and exoglu- canases have been overexpressed in sugarcane leaves by using the maize PepC promoter achieving an accumulation level of 0.05% of total soluble proteins (endoglucanase, in chloroplast) and less of exoglucanases without altering the phenotype (Harrison et al., 2011). In the future, enzymes might be synthesized in specific energy cane plants that could be coprocessed with other biomass sources from sugarcane for sugar and biomass production (e.g., bagasse from sugar mills) (Arruda, 2012). Increasing plant cellulose and total biomass content may be achieved by using approaches such as manipulation of growth regulators or key nutrients, increasing the ability of the plant to fix carbon by increasing atmospheric CO2 and also manipulat- ing some key metabolic enzymes in biomass synthesis pathways [reviewed in Sticklen (2006)]. Reduction of the cross-links of the maize cell walls (including ferulate and diferulate cross-links; benzyl ether and ester cross-links) has been shown to increase the initial hydrolysis of its cell wall polysaccharides by up to 46% (Grabber, 2005). In general, selection of grasses with less ferulate cross-linking or potent microbial xylanases by breeding or engineering tools are more attractive than pretreatment of the cell wall with a feruloyl esterase (Grabber, 2005). y Lignin content accounts for about 25% of sugarcane total lig- nocellulosic biomass and is probably the main obstacle affecting the efficiency of saccharification during conversion to ethanol (Canilha et al., 2012, 2013). Lignin and other recalcitrant com- ponents in cell walls prevent cellulase accessing the cellulose molecules and need to be removed before further processing (Sticklen, 2006). POTENTIAL IMPROVEMENT BY MOLECULAR GENETICS FOR BIOFUELS Lignin biosynthesis pathways are complicated and at least 10 different enzymes have been found involved in the lignin pathway in sugarcane (Higuchi, 1981; Whetten and Ron, 1995) and a total of 28 unigenes associated with monolignol biosynthesis were identified in sugarcane using SUCEST database and annotated genes from closely related species such as sorghum, maize, and rice (Bottcher et al., 2013). Tailoring sugarcane biomass composition for biofuels can be achieved by manipulating some of the key genes in lignin pathway (downregulation of some key enzymes), mostly targeting genes which encode the terminal enzymes such as caffeic acid O-methyltransferase (COMT) and cinnamyl alcohol dehydrogenase (CAD), to minimize the impact of the modifications on growth and development of the plant [as reviewed in Sticklen (2006), Jung et al. (2012), and Furtado et al. (2014)]. Not only lignin content but also the lignin S/G ratio is a very important aspect to consider in terms of modifying the lignin content because these two are both associated with biomass recalcitrance (Chen and Dixon, 2007; Li et al., 2010). Sugarcane lignin content was reduced by 3.9–13.7% using RNA interference (RNAi) suppression to downregulate the COMT gene [which has at least 31 different ESTs involved (Ramos et al., 2001)] by 67–97% and at the same time, the lignin S/G ratio was reduced from 1.47 to 1.27–0.79 (Jung et al., 2012). This resulted in an increase of up to 29% in total sugar yield without pretreatment (34% with dilute acid pretreatment). This study suggests that RNAi- mediated gene suppression is a promising method for suppres- sion of target genes not only in lignin pathway but also for cell POTENTIAL OF SUGARCANE WHOLE GENOME AND TRANSCIPTOME SEQUENCING FOR BIOFUELS The advent of NGS technology and a sharp reduction in per-base cost in the past decade [as reviewed in Van Dijk et al. (2014)] allows us to sequence the whole genome of a species, even a complex genome such as sugarcane, at a relatively low price within a relatively short time. At present, the cost of sequencing of a human genome at 30× coverage using the latest Illumina’s Hiseq X is around US $1,000. Since the first plant genome was com- pletely sequenced (Arabidopsis thaliana in 2000) using the tradi- tional Sanger sequencing platform, the sequencing strategies have moved to high-throughput and cost-effective approaches (Henry et al., 2012). High-throughput genome sequencing platforms have recently advanced and facilitated improved genotyping, allowing huge data output to be generated for polymorphism detection (especially SNPs) and marker discovery. Potential Strategies in Dissection of Biofuel Traits in Sugarcane Due to the limited number of generations, low recombination rate between chromosomes, and strong founder effect, it is expected that sugarcane has an extensive linkage disequilibrium despite the large number of chromosomes and being an outcrossing species (Huang et al., 2010). In fact, attaining a F2 population (such as inbred backcrosses or recombinant inbred lines and double haploid lines) in sugarcane is not practical due to its clonal prop- agation, high heterozygosity, and inbreeding depression (Aitken and McNeil, 2010; Sreedhar and Collins, 2010). Therefore, more commonly, a segregating F1 population from biparental crosses or self-pollinated progenies from heterozygous parents (as the pseudo F2 population) are used, and hence, most of sugarcane linkage maps (as AFLP, RAPD, isozyme, and SSR) were developed on this type of F1 population (Sreedhar and Collins, 2010). To date, most of these maps have low coverage and a limited number of markers because of the genome complexity and high cost of marker generation (Aitken et al., 2014). The high redundancy of the chromosomes in the sugarcane genome implies that with conventional approaches only the single-dose markers (present on only one of the homologous/homoelogous haplotype) can be used to obtain a high-resolution mapping (Hoarau et al., 2002; Le Cunff et al., 2008). The potential applications of the current genotyping technolo- gies to sugarcane association studies employ both whole-genome is restricted to genes which are likely thought to be associated with traits of interest based on prior knowledge (Hirschhorn and Daly, 2005; Ingvarsson and Street, 2011). At present, whole-genome sequencing based on the random sequencing of fragments of whole genomic DNA has been successfully applied to medium- size genomes with limited amount of repetitive elements, genome resequencing with the guide of a reference sequence, or de novo assembly of small genomes (Steuernagel et al., 2009; Henry et al., 2012; Xu et al., 2012; Edwards et al., 2013). The large genome size of sugarcane is partially attributable to sugarcane being a poly- ploid and the genome having a significant amount of repetitive sequences (Berkman et al., 2014). As a result, the current short reads generated from NGS technologies cannot resolve completely the challenges in the sugarcane genomes. For highly repetitive genomes, the genomic complexity will be lost or reduced by using the de novo assembly approaches of NGS-derived short reads as the identical repeat sequences in the genome will be col- lapsed (Green, 2002). Potential Strategies in Dissection of Biofuel Traits in Sugarcane Therefore, it is required to develop efficient genotyping strategies using whole-genome sequencing data for sugarcane system to overcome the challenges. Moreover, whole transcriptome sequencing gives details of the entire transcript expressed in the samples across the whole genome and could be applicable to the sugarcane genome in identifying biological significant variations (SNPs) between different developmental stages, between varieties, or for transcripts de novo assembly and gene discovery (Henry et al., 2012). g y y For large and polyploid genomes, there are still requirements to enrich the genomic DNA to capture the coding regions to ensure the depth of coverage, resolve the variable short reads, and lessen the effect of repetitive sequences in the genome on discovery of polymorphisms (Bundock et al., 2012; Henry et al., 2012). Selective sequencing of genomic loci of interest (genes or exomes) can reduce the cost compared to whole-genome sequencing and therefore simplify the data interpretation since non-coding regions are not abundant in the data. The enrich- ment techniques can be hybrid capture (e.g., Agilent SureSelect, NimbleGen, FlexGen) or selective circularization (e.g., Selector probes) or PCR amplification (e.g., Raindance). Hybrid capture supported by a microarray platform has been applied to sugarcane and other complex genomes due to its high capacity to enrich large regions of interest (1–50 Mb), the possibility of multiplexing, the availability of kits, and a the small amount of input DNA required (<1–3 μg) (Mertes et al., 2011). This approach uses a selection library of fragmented DNA or RNA representing the targets (normally oligonucleotides from 80 to 180 bases produced from known information such as gene indices, ESTs) to capture the cDNA fragments from a shotgun DNA library based on the hybridization and then sequence the captured fragments (Mertes et al., 2011; Bundock et al., 2012). Bundock et al. (2012) conducted the solution-based hybridization (Agilent SureSelect) to capture the exome regions of sugarcane using sorghum and sugarcane coding probes, enriched the genome 10–11 folds, and detected 270,000–280,000 SNPs in each genotype of the material tested. At the moment, a great number of SNPs from a genome or haplo- type can be generated by using high-capacity genome sequencing instruments or high-density oligonucleotide arrays (Zhu et al., 2008). The continuous advancement in genotyping technology The potential applications of the current genotyping technolo- gies to sugarcane association studies employ both whole-genome sequencing and whole transcriptome sequencing technologies. Potential Strategies in Dissection of Biofuel Traits in Sugarcane At present, a whole-genome sequence of sugarcane is not avail- able to support its biofuel trait analysis. However, a strategy to overcome this using the currently available resources, for dis- secting biofuel traits, for example, in sugarcane biomass, is to carry on the association studies, in which a population of genetic variability is selected, phenotyped, and genotyped. Association studies use the molecular markers from the genetic variability to detect the association between markers and traits of inter- est in order to validate the location of the genes, especially for November 2015 | Volume 3 | Article 182 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 8 Sugarcane Genetic Improvement for Biofuels Hoang et al. quantitative traits (Huang et al., 2010). This strategy has been used for human and animal genetic studies since it was first established and more recently also for plants. To date, associ- ation studies have been applied successfully to many different plants including Arabidopsis, wheat, barley, rice, cotton, maize, potato, soybean, sugar beet, Pinus, Eucalyptus, ryegrass [also Zhu et al. (2008); for a review, see Hall et al. (2010)], and sugar- cane (Aitken et al., 2005; Wei et al., 2006) for important traits like pathogen resistance, flowering time, grain composition, and quality. Association studies differ from traditional QTL stud- ies, where in QTL analysis the linkage disequilibrium between markers and QLTs from a segregating population is established in a cross of different genotypes, whereas in association stud- ies a non-structured population is used (Neale and Savolainen, 2004; Ingvarsson and Street, 2011). Therefore, association studies investigate variations of the whole population not just variations between parents. Association studies analyze the direct linkage disequilibrium between genetic markers and traits to overcome the limitations of the traditional QTL in sample size, low varia- tion, and recombination in the population (Ingvarsson and Street, 2011). In sugarcane, association studies are a powerful method for understanding the complex traits which are controlled by many loci and dosage effects (i.e., Ming et al., 2001; Wei et al., 2006; Banerjee et al., 2015). In general, association studies involve pop- ulation selection, phenotyping, genotyping, population structure, and statistical testing for the association. For these, there is a requirement to have a population with genetic variability and high linkage disequilibrium; and for sugarcane, the most important aspect of doing association studies is having marker data and a breeding population of elite varieties (Huang et al., 2010). Frontiers in Bioengineering and Biotechnology | www.frontiersin.org CONCLUSION Some of the association studies have been carried out on sugar- cane recently such as those for QTLs which control the Pachymetra root rot and brown rust resistance on 154 genotypes (McIntyre et al., 2005); genetics of root rot, leaf scald, Fiji leaf gall, cane sugar, and yield using 1,068 AFLP, 141 SRR (on 154 genotypes), and 1,531 DArT markers (on 480 genotypes) (Wei et al., 2006, 2010); smut and eldana stalk borer using 275 RFLP and 1,056 AFLP markers on 77 genotypes (Butterfield, 2007); resistance to sugarcane yellow leaf virus using 3,949 polymorphic markers (DArT and AFLP) on 189 genotypes (Debibakas et al., 2014); markers agro-morphological traits, sugar yield disease resistance, and bagasse content using 3,327 DArT, AFLP, and SSR markers on 183 genotypes (Gouy et al., 2015); and sucrose and yield contributing traits using 989 SSR markers on 108 genotypes (Banerjee et al., 2015). Using the Affymetrix GeneChip Sugar- cane Genome Array, Casu et al. (2007) identified 119 transcripts associated with enzymes of cell wall metabolism and development on sugarcane variety Q177. These promising preliminary studies were carried out on small sample sizes and limited numbers of markers (even though a small number of significant associations have been identified) while the polyploid sugarcane genome and small effect of quantitative traits requires larger sample sizes and more markers (e.g., genome-wide markers) so that significant association can be detected (Huang et al., 2010; Gouy et al., 2015). Sugarcane has been shown to be a good candidate for use as a lignocellulosic biomass feedstock for second-generation biofuel production. However, its genome complexity still remains a great bottleneck restricting the dissection of biofuel traits. The most significant achievements in improving sugarcane biomass for bio- fuels so far have been the establishment of the high fiber cane vari- eties to generate more lignocellulosic biomass, and preliminary results in modifying biomass with more cellulose, less lignin con- tent, a preferable lignin S/G ratio, and enzyme expressed in planta (in leaves) for easy conversion to biofuels. The improvement of sugarcane biomass has been by traditional breeding, molecu- lar genetics approaches and, more recently, accelerated with the use of NGS technology. The future of second-generation biofuel production using sugarcane lignocellulosic biomass will depend greatly on advances in understanding of the key biofuel traits required to deliver more efficient and price-competitive biofuels. This objective will be facilitated once the whole genome of sug- arcane is fully sequenced. Potential Strategies in Dissection of Biofuel Traits in Sugarcane Genotyping is normally either by analysis candidate genes or genome-wide approaches, in which the candidate gene approach November 2015 | Volume 3 | Article 182 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 9 Sugarcane Genetic Improvement for Biofuels Hoang et al. resources such as the sorghum gene indices (sorghum gene mod- els), sugarcane gene indices (DFCI Sugarcane Gene Index version 3.0, an integrated collection of sugarcane ESTs, complete cDNA sequences, non-redundant data of all sugarcane genes and their related information), transcription factors (TFs), and sugarcane tentative consensus/assembled sequences. For example, in the study mentioned earlier, Bundock et al. (2012), based on the gene sequences in the sorghum genome and sugarcane gene indices, captured the exomic regions of two sugarcane genotypes Q165 and IJ76-514, detected SNPs present in 13,000–16,000 targeted genes from Illumina short read data of these samples, and 87–91% of SNPs were validated and confirmed by 454 sequencing. For transcript profiling, the reference transcriptome sequence can be constructed for specific tissues using de novo assembly such as in Vargas et al. (2014) and Cardoso-Silva et al. (2014) and validated to find suitable reference gene sets to be used for gene expression normalization as in Guo et al. (2014). The currently available resources, on the other hand, are also utilized. Park et al. (2015) used the Sugarcane Assembled Sequences from SUCEST-FUN database as reference sequences in a study on cold-responsive gene expression profiling of sugarcane hybrids and S. spontaneum and found that more than 600 genes are differentially expressed in each genotype after applying stress. allows generation of up to 1 million SNPs spanning across the entire genome in one reaction (e.g., using SNP chip), and the newest SNP chip can measure the copy number as well as the allelic variation. Examples of available platforms are Affymetrix (e.g., Affymetrix Genome-Wide Human SNP Array 6.0) and Illu- mina (e.g., Illumina’s WGGT Infinium BeadChips). Due to the multiple chromosomes in the homologous groups of sugarcane genome and the number of alleles at each locus (and the SNPs numbers consequently), an allele would likely to be defined by a combination of SNPs, not just a single SNP (McIntyre et al., 2006, 2015). SNP genotyping including SNP calling and statistical methods to estimate the ploidy level and allele dosage within homologous groups have been developed for sugarcane by Garcia et al. (2013) to allow in-depth association analysis of the genome. Potential Strategies in Dissection of Biofuel Traits in Sugarcane In this study, SNPs were developed by SEQUENOM iPLEX Mas- sARRAY and capture primers and then discovered by QualitySNP software, mass-based procedures, and the SuperMASSA software. For whole transcriptome sequencing, Cardoso-Silva et al. (2014) identified 5,106 SSRs and 708,125 SNPs from the unigenes assem- bled from RNA-seq data of contrasting sugarcane varieties. These advances in sugarcane genotyping technology, together with well- developed high-throughput phenotyping methods for biofuel traits [reviewed in Lupoi et al. (2013) and Lupoi et al. (2015)] and bioinformatics tools, could accelerate sugarcane analysis while a reference genome is not available. CONCLUSION Optimizing sugarcane lignocellulosic bagasse composition may result in biomass with better digestibil- ity, modified carbohydrates, and reduction of cross-linking or self-produced enzymes (in planta). Currently available sugarcane genetic resources include diverse germplasm in the genus Sac- charum, genetic markers and maps, ESTs, and the sequence of a closely related species genome. However, novel strategies need to be developed to overcome the challenges posed by the com- plex genetics. Traditional approaches using breeding and molec- ular genetics have potential for wider use improving sugarcane while the advent of NGS technology and high-throughput phe- notyping technologies will accelerate the process of dissection of biofuel traits, genome-wide. By using these approaches, the loci of interest will be defined for use to improve sugarcane The Reference Sequence Matters REFERENCES Botha, F. 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Once a better understanding of the genes controlling cell wall biosynthesis is achieved, breeding programs will be able to accelerate the selection and development of varieties with opti- mized biomass composition to generate better sugarcane biomass sources to meet the demand of biofuel production. We are grateful to the Australian Agency for International Devel- opment (AusAID) for financial support through an Australian Development Scholarship (ADS) to NVH and to the Queensland Government for funding this research. This work was part of the DOE Joint BioEnergy Institute (http://www.jbei.org) supported by the U. S. Department of Energy, Office of Science, Office of Bio- logical and Environmental Research, through contract DE-AC02- 05CH11231 between Lawrence Berkeley National Laboratory and the U. S. Department of Energy. The Reference Sequence Matters As mentioned earlier, construction of a sugarcane nuclear genome reference sequence is an important objective, even though it might take some time to finish. However, in the meantime, sugarcane genome analysis still can exploit the currently available genetic November 2015 | Volume 3 | Article 182 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 10 Sugarcane Genetic Improvement for Biofuels Hoang et al. AUTHOR CONTRIBUTIONS NVH wrote the paper. AF, FCB, BAS, and RJH discussed and edited the manuscript. All authors read and approved the final manuscript. NVH wrote the paper. AF, FCB, BAS, and RJH discussed and edited the manuscript. All authors read and approved the final manuscript. Botha, F. (2009). “Energy yield and cost in a sugarcane biomass system,” in: Proceedings Australian Society Sugarcane Technologists. 1–9. REFERENCES biortech.2010.02.009 Cuadrado, A., Acevedo, R., Moreno Diaz De La Espina, S., Jouve, N., and De La Torre, C. 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Micro- collinearity between autopolyploid sugarcane and diploid Sorghum genomes. BMC Genomics 11:261. doi:10.1186/1471-2164-11-261 Conflict of Interest Statement: The authors declare that the research was con- ducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Wang, X., and Paterson, A. (2013). “Comparative genomic analysis of C4 photosyn- thesis pathway evolution in grasses,” in Genomics of the Saccharinae, ed. A. H. Paterson (New York, NY: Springer), 447–477. Copyright © 2015 Hoang, Furtado, Botha, Simmons and Henry. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Copyright © 2015 Hoang, Furtado, Botha, Simmons and Henry. 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. Wei, X., Jackson, P. A., Hermann, S., Kilian, A., Heller-Uszynska, K., and Deomano, E. (2010). Simultaneously accounting for population structure, genotype by environment interaction, and spatial variation in marker-trait associations in sugarcane. Genome 53, 973–981. doi:10.1139/g10-050 Wei, X., Jackson, P. A., McIntyre, C. L., Aitken, K. S., and Croft, B. (2006). Associations between DNA markers and resistance to diseases in sugarcane and November 2015 | Volume 3 | Article 182 Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 15
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Cervical Cancer Prevention in El Salvador: Gains to Date and Challenges for the Future
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Article Cervical Cancer Prevention in El Salvador: Gains to Date and Challenges for the Future Article Karla Alfaro 1, Montserrat Soler 1,2,*, Mauricio Maza 3, Mauricio Flores 4, Leticia López 1, Juan C. Rauda 1, Andrea Chacón 5, Patricia Erazo 5, Nora Villatoro 5, Eveline Mumenthaler 1, Rachel Masch 1, Gabriel Conzuelo 1, Juan C. Felix 6 and Miriam Cremer 1,2 1 Basic Health International, San Salvador, C.P. 01101El Salvador and Pittsburgh, 15205, USA; kalfaro@basi- chealth.org (K.A.); llopez@basichealth.org (L.L.); jcrauda@basichealth.org (J.C.R.); emumenthaler@basi- chealth.org (E.M.); rmasch@basichealth.org (R.M.); gconzuelo@basichealth.org (G.C.); mcremer@basi- chealth.org (M.C.) 1 Basic Health International, San Salvador, C.P. 01101El Salvador and Pittsburgh, 15205, USA; kalfaro@basi- chealth.org (K.A.); llopez@basichealth.org (L.L.); jcrauda@basichealth.org (J.C.R.); emumenthaler@basi- chealth.org (E.M.); rmasch@basichealth.org (R.M.); gconzuelo@basichealth.org (G.C.); mcremer@basi- chealth.org (M.C.) g 2 Ob/Gyn and Women’s Health Institute, Cleveland Clinic, Cleveland, OH 44195, USA 3 Cancer Program, Pan American Health Organization, Washington, DC, 20037 USA; mazamau@paho.org g g g p g 4 Comité Asesor de Prácticas en Inmunizaciones de El Salvador, San Salvador, C.P. 01101, El Salvador; mernesto.flores@salud.gob.sv g 5 Unidad de Cáncer, Ministerio de Salud de El Salvador, San Salvador, C.P. 01101, El Salvador; marina.chacon@salud.gob.sv (A.C.); mayra.erazo@salud.gob.sv (P.E.); nora.villatoro@salud.gob.sv (N.V. g ( ) y g ( ) g ( 6 Department of Pathology, Medical College of Wisconsin, Milwaukee, WI 53226, USA; jcfelix@mcw.edu * Correspondence: msoler@basichealth.org. * Correspondence: msoler@basichealth.org. Simple Summary: Cervical cancer is a leading cause of death for women in low-resource areas around the world. However, the disease can be prevented through vaccination, screening, and early treatment. Recently, the World Health Organization released a strategy with the goal of eliminating cervical cancer through a combination of these three preventive strategies. In El Salvador, a screen- ing program has been in place for some years, and vaccination is in its early stages. Here we describe the Salvadoran experience and discuss successes to date and challenges for the future. We also pro- vide recommendations that may be helpful for other countries working to meet the WHO elimina- tion goals. Abstract: Cervical cancer is preventable through vaccination, early detection, and treatment of pre- cancerous lesions. However, global inequalities mean that the disease remains a leading cause of cancer death around the world, with over 80% of new cases and 90% of deaths occurring in low and middle-income countries (LMICs). In El Salvador, joint efforts between the Ministry of Health (MoH) and the non-profit organization Basic Health International (BHI) have been in place since 2008 with the goal of reducing the country’s disease burden. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 May 2022 doi:10.20944/preprints202205 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 May 2022 doi:10.20944/preprints202205 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 May 2022 doi:10.20944/preprints202205.0285.v1 Article Cervical Cancer Prevention in El Salvador: Gains to Date and Challenges for the Future While the World Health Organization’s (WHO) call to action to eliminate cervical cancer provided worldwide momentum to implement new public health initiatives, the COVID-19 pandemic disrupted ongoing programs and jeopard- ized plans for the future. The purpose of this manuscript is to describe the progress that El Salvador has achieved in improving cervical cancer prevention, the impact of the pandemic on current strat- egies, and potential solutions that can help the country meet the WHO’s strategic targets by 2030 to accelerate the elimination of cervical cancer. Keywords: El Salvador; cervical cancer; cervical precancer; human papillomavirus (HPV); screen- and-treat Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 May 2022 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 May 2022 doi:10.20944/preprints202205.0285.v1 2 of 8 LMICs, and these numbers are expected to grow in the next decade [2]. In light of these considerations, the World Health Organization (WHO) released a call to action in 2018 to eliminate cervical cancer as a public health problem [3]. The WHO strategy sets three global targets to be reached by 2030 in order to accelerate elimination: (1) vaccinating 90% of girls by age 15 with the human papillomavirus (HPV) vaccine, the causal agent of the disease, (2) screen 70% of women at age 35 and again at age 45 with a high-performance test, and (3) treat 90% of women with underlying cervical disease [4]. Meeting these goals would save 300,000 lives by 2030, 13.4 million lives by 2070, and 62 million lives by 2120 [5]. While the WHO’s strategy presents challenges to resource-poor countries with com- peting health priorities, the call to action has provided momentum to increase access to cervical cancer preventive services. El Salvador is well-positioned to demonstrate an ef- fective implementation of cervical cancer control programs. The country has historically high rates of incidence and mortality (13.1 and 7.43 in 100,000, age-adjusted, respectively) [1]. The sociocultural context complicates access to cancer prevention services. Almost 40% of the population lives in rural areas, and gang-related violence, poverty and out- migration remain pervasive problems. The Ministry of Health operates a network of mu- nicipal clinics that offer free-of-cost primary care services, although medications must of- ten be purchased out of pocket. More specialized care can only be found at regional hos- pitals in urban areas or in expensive private facilities. For many women, transportation to clinics and hospitals is a significant hurdle. Since 2008, a collaboration between the Ministry of Health (MoH) and Basic Health International (BHI), a non-profit organization, has resulted in a range of initiatives to strengthen cervical cancer prevention in the country [6]. Previously, the national screening program relied on colposcopy and/or cytology (Pap test), which involved multiple visits for each patient and resulted in very low national coverage (estimates range from 19% [7] to 44% [8]). In 2012, a partnership between the MoH and BHI resulted in a donation of low-cost HPV tests (careHPV, Qiagen, Gaithersburg, MD, USA) to the country. The tests were utilized to conduct a demonstration project that eventually reached 28,000 women. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 May 2022 In the first stage, 2000 women were assigned to either screen-and-treat or colposcopy man- agement [9]. Screen-and-treat approaches, where all screen-positive women are offered treatment without biopsy-confirmed diagnosis, are recommended by the WHO in low- resource settings [10]. In El Salvador, the initial pilot was gradually expanded to an addi- tional 8000 women. Outcomes revealed significant differences in loss to follow-up, with 88.3% of HPV positive women in the screen-and-treat group completing treatment in 6 months, compared to 44.2% of HPV positive women who were referred to colposcopy management within the same time period [11]. Following the success of this project, the screen-and-treat program was scaled-up in the Paracentral region [12]. Cost-effectiveness analyses demonstrated the long-term ad- vantages of this approach in the Salvadoran context [13,14]. Findings were presented to public health officials and resulted in the incorporation of HPV testing into the national cervical cancer control guidelines [15], making El Salvador one of the first countries to do so in the region. In 2020, the COVID 19 pandemic disrupted health systems worldwide, but secondary prevention services continued as local regulations allowed. The purpose of this manuscript is to describe the progress of cervical cancer prevention programs in El Salvador, review the challenges presented by the COVID-19 pandemic, and outline po- tential future directions for the country. 1. Background Cervical cancer is a disease of inequality. Wealthy countries have access to preventive strategies that have significantly reduce incidence and mortality rates in the last 50 years, but low-and-middle-income countries (LMICs) continue to bear a large burden of disease [1]. It is estimated that 80% of new cases and 90% of deaths from cervical cancer occur in © 2. The HPV Screening Program in El Salvador In 2017, after the initial success of a primary HPV-based screen-and-treat demonstra- tion project, El Salvador was able to obtain grant funding for further HPV tests in addition to HPV vaccines. The MoH included the procurement of HPV tests in its 2022 annual budget, cementing the screen-and-treat program as part of the national cervical cancer control effort. Today, HPV screening is available in a network of 750 primary care units Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 May 2022 doi:10.20944/preprints202205.0285.v1 3 of 8 distributed in all five regions of the country (Figure 1). Provider trainings for implemen- tation in the fifth and final region, which includes the metropolitan area of San Salvador, began in March 2022. Since its inception, the program was designed to rely on the existing public health infrastructure. As technical advisors, BHI has followed a “train the trainer” approach with MoH personnel to ensure long-term sustainability. distributed in all five regions of the country (Figure 1). Provider trainings for implemen- tation in the fifth and final region, which includes the metropolitan area of San Salvador, began in March 2022. Since its inception, the program was designed to rely on the existing public health infrastructure. As technical advisors, BHI has followed a “train the trainer” approach with MoH personnel to ensure long-term sustainability. Figure 1. Gradual scale-up of the primary HPV screen-and-treat program in El Salvador. Figure 1. Gradual scale-up of the primary HPV screen-and-treat program in El Salvador. Community health teams, or ECOs in Spanish (Equipos Comunitarios de Salud), form the backbone of the HPV screening program. ECOs consist of healthcare providers at three levels: general practitioners that head municipal health clinics, nurses at the clin- ics, and health promoters who are usually community members with no formal clinical training. Health promoters are the first line of advocacy and education. They are respon- sible for contacting women due for screening to set up appointments at local clinics. Cur- rently, El Salvador guidelines recommend HPV testing for women aged 30–59 and cytol- ogy for those between 20 to 29 and over 59 years of age. Participants of the HPV program receive an educational session taught by nurses on arrival at the local clinic. Women then undergo a provider sample collection using careHPV, which detects 14 types of high-risk HPV (pooled, not genotyped). Screened women are given an appointment to return after 30 days for their test results. 2. The HPV Screening Program in El Salvador Since not all clinics offer cryotherapy, some women must schedule the second appointment at the nearest treatment center. There are 74 such cen- ters strategically distributed throughout the country. On the second visit, those who test positive are offered an immediate pelvic exam to visually assess eligibility for ablation treatment and, if eligible, gas-based cryotherapy. HPV testing, visual assessment, and treatment are performed by trained general practitioners or gynecologists. Women who are deemed ineligible for treatment during the visual assessment are referred to col- poscopy and biopsy by colposcopists at regional hospitals. Criteria for ablation treatment ineligibility are drawn from WHO guidelines and include a lesion that is larger than 75% of the cervix, enters the endocervical canal, or is suspicious for cancer, or cases where the squamocolumnar junction is not fully visible. To date, over 4500 healthcare providers have been trained as part of the screen-and- treat program (Table 1) and 145,000 women have been tested. The HPV positivity rate is approximately 14%. Women who test negative return for re-testing after five years. Those who test positive return at one year after ablation treatment, and if they test positive again, they are referred to colposcopy and biopsy. Although we know that approximately 88% Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 May 2022 doi:10.20944/preprints202205.0285.v1 4 of 8 of HPV-screened women have received cryotherapy treatment to date, data on treatment refusals, adherence to colposcopy and biopsy, or treatment follow-up are not currently available. While El Salvador began to add outcomes of its cervical cancer prevention pro- grams to its epidemiological surveillance system since 2012, data entry across health units and hospitals is not consistent. Efforts are underway to improve surveillance, including training of data entry specialists. Figure 2.. ECO team members train in El Salvador as part of the national HPV screening program. Figure 2.. ECO team members train in El Salvador as part of the national HPV screening program. After the WHO call for elimination was released, the MoH engaged in further efforts to maximize the impact of this opportunity in El Salvador with two important outcomes. First, the MoH released a new screening target to increase the number of eligible women screened to 15% per year for the next 5 years, which would achieve the WHO target of 70% of eligible women screened by 2030. 2. The HPV Screening Program in El Salvador Second, after a series of stakeholder meetings that included MoH and BHI personnel, changes are planned to the national cervical cancer guidelines. BHI had successfully completed several projects on the feasibility of HPV self- collection in underserved areas [16–18]. Self-collection allows women to collect their own sample using a small brush, circumventing the need for a speculum exam. BHI had also participated in the review of WHO guidelines for thermal ablation [19], an alternative treatment to cryotherapy that does not require a consumable (i.e., compressed gas) and can be applied with a portable, battery-operated device. The BHI team introduced thermal ablation to El Salvador through two ongoing research protocols (NCT NCT03084081 and NCT03429582). Both HPV self-sampling and thermal ablation are expected to be included in guidelines to be released in 2022. These innovations have the potential to increase access to screening and treatment, particularly among rural and hard-to-reach populations. After the WHO call for elimination was released, the MoH engaged in further efforts to maximize the impact of this opportunity in El Salvador with two important outcomes. First, the MoH released a new screening target to increase the number of eligible women screened to 15% per year for the next 5 years, which would achieve the WHO target of 70% of eligible women screened by 2030. Second, after a series of stakeholder meetings that included MoH and BHI personnel, changes are planned to the national cervical cancer guidelines. BHI had successfully completed several projects on the feasibility of HPV self- collection in underserved areas [16–18]. Self-collection allows women to collect their own sample using a small brush, circumventing the need for a speculum exam. BHI had also participated in the review of WHO guidelines for thermal ablation [19], an alternative treatment to cryotherapy that does not require a consumable (i.e., compressed gas) and can be applied with a portable, battery-operated device. The BHI team introduced thermal ablation to El Salvador through two ongoing research protocols (NCT NCT03084081 and NCT03429582). Both HPV self-sampling and thermal ablation are expected to be included in guidelines to be released in 2022. These innovations have the potential to increase access to screening and treatment, particularly among rural and hard-to-reach populations. 4. The Impact of COVID-19 In March 2020, as COVID-19 cases began to rise nationally, non-emergency health services were suspended throughout the El Salvador public health system. Most MoH personnel, including the ECOs that implemented the HPV screening program, were re- directed to pandemic containment efforts. Thousands of women who had been screened as part of the HPV screen-and-treat program had follow-up care delayed for several months. In addition to this growing backlog of patients, a large number of HPV tests were left in storage and expired during the lockdown period, rendering them unusable. The planned April start date for the new HPV vaccination campaign was delayed to Novem- ber 2020 when it was hoped in-person schooling would return. However, as non-essential medical services slowly resumed in late 2020 and early 2021, an urgent COVID-19 vac- cination campaign was prioritized. Thus, screening and vaccination initiatives for cervical cancer were further interrupted. The true impact of the pandemic on routine medical services has only recently begun to surface. In the Occidental and Oriental regions of El Salvador, the MoH estimated that approximately 5000 HPV-positive women were awaiting follow-up care since the begin- ning of lockdowns in 2020. Thus, in collaboration with BHI, a funding opportunity was identified and a project initiated with the goal of locating these women and providing follow-up care. Since MoH providers and laboratories remained at capacity with COVID- related care, private colposcopists and pathology laboratories were engaged to conduct this intervention. From March 2021 to March 2022, 3017/5000 (60%) screen-positive women have been identified and efforts are ongoing to locate the remaining 1983/5000 (40%). Of those accounted for, 702/3017 (23%) had already received follow-up care at pri- vate clinics, 210/3017 (7%) refused further care, and 96/3017 (3%) were lost to follow-up (had moved, migrated out of the country, died, etc.). The remaining 2009/3017 (66.5%) have undergone colposcopy and biopsy as part of this project. Laboratory analysis have been completed for 1990/2009 (99%). Results show alarmingly high rates of high-grade precancer at 23% (452/1990) and invasive cancer at 1.3% (26/1990). Although there are no comparable pre-pandemic figures available, findings from the original HPV screen-and- treat demonstration project provide a frame of reference. Among HPV positive women in the colposcopy management group who completed biopsy within 6 months, 56/385 (15%) were diagnosed with high-grade precancer and 1 (<1%) with invasive cancer [11].These women were similarly recruited from a semi-rural screening population. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 May 2022 doi:10.20 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 May 2022 doi:10.20944/preprints202205.0285.v1 5 of 8 and the MoH settled on a school-based strategy targeting 9-year old girls for a first dose, with a second dose after six months. Quadrivalent Gardasil (Merck &Co., Kenilworth, NJ, USA) was the vaccine of choice. Usually, childhood vaccines are given at local health units, but given the need for two doses and the target ages, it was determined that a school-based approach would result in better follow-up. HPV vaccination was projected to start in April 2020 during the Americas Vaccination Week with the goal of vaccinating approximately 55,000 girls the first year. The HPV vaccine was also included in El Salva- dor’s vaccination schedule, ensuring HPV vaccine doses would be included in future na- tional budgets. Finally, the vaccine data collection system was prepared to receive general outcomes of the HPV vaccination effort. As the program was about to begin, the COVID- 19 was declared a pandemic by the WHO. HPV screening and vaccination programs were immediately halted. 3. HPV Vaccination in El Salvador The MoH planned to introduce HPV vaccination into the country since 2015. The WHO call to action coincided with a donation from the World Bank, providing the neces- sary momentum to begin implementation efforts. An advisory committee was convened 5. Challenges for the Future El Salvador has achieved significant gains over the last decade to reduce its cervical cancer burden. There is now a scalable screening program in all regions of the country and, for the first time in 2022, HPV tests were purchased solely with government funds. The WHO call for elimination has provided additional motivation to set defined screening targets and begin the implementation of a new HPV vaccination program despite the chal- lenges of the pandemic. However, significant barriers remain, many of which represent broader issues that can impede elimination goals globally. Although HPV screening is highly acceptable to patients and providers, there are limited low-cost options. In El Sal- vador, budgetary constraints currently allow for the procurement of only 100,000 test kits per year, which is projected to cover 15% of the eligible population. In addition, many existing HPV tests require complex equipment or long processing times. Inexpensive, rapid HPV tests are in development, but none are yet available that allow for the mainte- nance of a nation-wide, single-day screen-and-treat program. There is also an urgent need for affordable and accessible triage options that can minimize overtreatment and more efficiently channel resources. Better follow-up options for women ineligible for ablation, who may be at greater risk for invasive cancer, are also lacking. Innovative and affordable point-of-care screening and treatment alternatives will be necessary to meet elimination goals. g In terms of primary prevention, the HPV vaccination program in El Salvador is still in early adoption. It is a positive sign that the vaccine has been included in the national vaccination schedule, which will greatly assist in ensuring its sustainability. However, a recent survey in two regions of the country revealed that only 60% of adult respondents would vaccinate a daughter or close relative between the ages of 9 and 12, even if such a program was free of cost [20]. Thus, even if vaccines become widely available effective interventions to overcome vaccine hesitancy will be essential. On the other hand, if a sin- gle-dose schedule is eventually introduced, this would facilitate implementation by re- ducing costs and avoiding loss to follow-up. The COVID-19 pandemic has also highlighted the need to strengthen health systems globally, and cervical cancer programs in particular. In El Salvador, the return of screening has been gradual and irregular due to recurring infection waves, and there is a significant backlog of women waiting for results or treatment. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 May 2022 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 May 2022 doi:10.20944/preprints202205.0285.v1 6 of 8 HPV vaccine in the national budget means that procurement of HPV doses in the next few years is assured. This, in turn, will facilitate meeting elimination goals. HPV vaccine in the national budget means that procurement of HPV doses in the next few years is assured. This, in turn, will facilitate meeting elimination goals. 5. Challenges for the Future While other health emergencies will always exist (for example, at the time of writing El Salvador was undergoing a dengue breakout that suspended training for cervical cancer screening providers), there are strat- egies that can help mitigate these challenges. For example, HPV self-collection can allow for at-home testing and including HPV vaccination in national schedules can help priori- tize cervical cancer prevention. 4. The Impact of COVID-19 Thus, unless rapid action is taken, the suspension of routine screening services is expected to negatively impact the achievement of elimination goals. The HPV vaccination effort has also faced challenges. Since public schools were shut down for most of 2020 and part of 2021, the MoH decided to shift its strategy to an at- home approach. The age range was also expanded to encompass girls aged 9 to 11 years. Despite these adjustments, the total number of first doses provided was 15,286, 28% of the original target. Although these figures fall short of initial projections, the inclusion of the Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Conflicts of Interest: M.C. is a speaker and on the advisory board of Merck & Co. and Organon International and the President and Founder of Basic Health International. M.S. is a paid consultant for Basic Health International. 5. Brisson, M.; Kim, J.J.; Canfell, K.; Drolet, M.; Gingras, G.; A Burger, E.; Martin, D.; Simms, K.T.; Bénard, E.; Boily, M.-C.; et al. Impact of HPV vaccination and cervical screening on cervical cancer elimination: A comparative modelling analysis in 78 low- income and lower-middle-income countries. Lancet 2020, 395, 575–590. https://doi.org/10.1016/S0140-6736(20)30068-4. 6. Maza, M.; Alfaro, K.; Garai, J.; Velado, M.M.; Gage, J.C.; Castle, P.E.; Felix, J.; Luciani, S.; Campos, N.; Kim, J.; et al. Cervical cancer prevention in El Salvador (CAPE)—An HPV testing-based demonstration project: Changing the secondary prevention paradigm in a lower middle-income country. Gynecol. Oncol. Rep. 2017, 20, 58–61. https://doi.org/10.1016/j.gore.2017.02.011. p g y y p p g j g 7. Murillo, R.; Almonte, M.; Pereira, A.; Ferrer, E.; Gamboa, O.A.; Jerónimo, J.; Lazcano-Ponce, E. Cervical cancer screening pro- grams in Latin America and the Caribbean. Vaccine 2008, 26 (Suppl. 11), L37–L48. https://doi.org/10.1016/j.vaccine.2008.06.013. 4. World Health Organization. Global Strategy to Accelerate the Elimination of Cervical Cancer as a Public Health Problem; World Health Organization: Geneva, Switzerland, 2020. 6. Conclusions In order for LMICs to meet the WHO’s elimination goals by 2030, more affordable and accessible vaccination and screening modalities are necessary but not sufficient. It is also essential to follow evidence-based strategies and recommendations to implement and sustain population-level programs. Our experiences in El Salvador have taught us valua- ble lessons in this regard. First and foremost, it is crucial to engage stakeholders at the early planning stages of any project. At the local level, a joint BHI and MoH team orga- nized meetings and workshops with other MoH staff, professional organizations, and other actors working in the cervical cancer space in order to build interest in a pilot demonstration. We also activated an international network of academic connections, which proved valuable in bringing expertise to the design and implementation of the pro- ject. The MoH has been extremely receptive of these contributions and this has helped to Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 May 2022 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 May 2022 doi:10.20944/preprints202205.0285.v1 7 of 8 build in-country capacity across multiple sectors. We would encourage any government team involved in screening or vaccination initiatives to seek out partnerships with re- search or non-profit groups that can bring flexible skills and resources to the development of a new initiative. Once a program is ready for implementation, it is crucial to design an education and awareness plan, not only at the community level, but also targeting the staff that will run it on a day-to-day basis. In El Salvador, there are separate training and refresher for clini- cians and health promoters, which ensures that providers at all levels are knowledgeable and motivated to recruit participants. On the more practical side, negotiating with local distributors to supply tests and necessary materials can be helpful in avoiding customs delays and cutting down costs. Finally, surveillance and data collection, at any scale that is feasible, are absolutely necessary to gauge program success and detect areas where more work is needed. In our case, data have also been essential to effect guideline change and ensure meaningful and sustainable impact. The WHO call for elimination has placed cervical cancer prevention at the front and center of global health. It is crucial that gov- ernments, funding agencies, researchers, and other stakeholders take advantage of the current moment to meet elimination targets and ensure that future generations are free of this preventable disease. Author Contributions: Conceptualization, K.A., M.S., M.M., M.F., A.C., P.E., N.V., R.M., J.C.F. and M.C.; Data curation, M.S., L.L., J.C.R., A.C. and G.C.; Formal analysis, M.S. and G.C.; Funding ac- quisition, K.A., M.M., E.M., R.M. and M.C.; Investigation, K.A., M.S., M.M., L.L., E.M., R.M., G.C., J.C.F. and M.C.; Project administration, K.A., L.L., J.C.R., A.C., P.E. and N.V.; Supervision, K.A., M.M., M.F., L.L., J.C.R., A.C., P.E., N.V., R.M., J.C.F. and M.C.; Writing—original draft, M.S.; Writ- ing—review & editing, K.A., M.M., M.F., J.C.R., A.C., P.E., N.V., E.M., R.M., G.C., J.C.F. and M.C. All authors have read and agreed to the published version of the manuscript. Funding: The Freedom Philanthropic Fund, Einhorn Collaborative. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. References 1. Sung, H.; Ferlay, J.; Siegel, R.L.; Laversanne, M.; Soerjomataram, I.; Jemal, A.; Bray, F. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA A Cancer J. Clin. 2021, 71, 209–249. https://doi.org/10.3322/caac.21660. p g 2. Hull, R.; Mbele, M.; Makhafola, T.; Hicks, C.; Wang, S.; Reis, R.M.; Mehrotra, R.; Mkhize‑Kwitshana, Z.; Kibiki, G.; Bates, D.O.; et al. Cervical cancer in low and middle.income countries (Review). Oncol. Lett. 2020, 20, 2058–2074. https://doi.org/10.3892/ol.2020.11754. p g 3. World Health Organization. WHO Director-General Calls for All Countries to Take Action to Help End the Suffering Caused by Cervical Cancer. Published 2018. Available online: https://www.who.int/reproductivehealth/call-to-action-elimination-cer- vical-cancer/en/ (accessed on 29 August 2021). p g 3. World Health Organization. WHO Director-General Calls for All Countries to Take Action to Help End the Suffering Caused by Cervical Cancer. Published 2018. Available online: https://www.who.int/reproductivehealth/call-to-action-elimination-cer- vical-cancer/en/ (accessed on 29 August 2021). g 4. World Health Organization. Global Strategy to Accelerate the Elimination of Cervical Cancer as a Public Health Problem; World Health Organization: Geneva, Switzerland, 2020. 5. Brisson, M.; Kim, J.J.; Canfell, K.; Drolet, M.; Gingras, G.; A Burger, E.; Martin, D.; Simms, K.T.; Bénard, E.; Boily, M.-C.; et al. Impact of HPV vaccination and cervical screening on cervical cancer elimination: A comparative modelling analysis in 78 low- income and lower-middle-income countries. Lancet 2020, 395, 575–590. https://doi.org/10.1016/S0140-6736(20)30068-4. 6. Maza, M.; Alfaro, K.; Garai, J.; Velado, M.M.; Gage, J.C.; Castle, P.E.; Felix, J.; Luciani, S.; Campos, N.; Kim, J.; et al. Cervical cancer prevention in El Salvador (CAPE)—An HPV testing-based demonstration project: Changing the secondary prevention paradigm in a lower middle-income country. Gynecol. Oncol. Rep. 2017, 20, 58–61. https://doi.org/10.1016/j.gore.2017.02.011. p g y y p p g j g 7. Murillo, R.; Almonte, M.; Pereira, A.; Ferrer, E.; Gamboa, O.A.; Jerónimo, J.; Lazcano-Ponce, E. Cervical cancer screening pro- grams in Latin America and the Caribbean. Vaccine 2008, 26 (Suppl. 11), L37–L48. https://doi.org/10.1016/j.vaccine.2008.06.013. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 May 2022 doi:10.20944/preprints202205.0285.v1 8 of 8 8. Maza, M.; Matesanz, S.; Alfaro, K.; Alonzo, T.; Masch, R.; Calderon, S.; Velis, J.; Jamshidi, R.; Cremer, M. Adherence to recom- mended follow-up care after high-grade cytology in El Salvador. Int. J. Healthc. 2016, 2, 31–36. https://doi.org/10.5430/ijh.v2n2p31. p // g/ / j p 9. Cremer, M.L.; Maza, M.; Alfaro, K.M.; Kim, J.J.; Ditzian, L.R.; Villalta, S.; Alonzo, T.A.; Felix, J.C.; Castle, P.E.; Gage, J.C. References Intro- ducing a high-risk HPV DNA test into a public sector screening program in El Salvador. J. Low. Genit. Tract Dis. 2016, 20, 145– 150. https://doi.org/10.1097/LGT.0000000000000188. p g 10. World Health Organization. WHO Guidelines for Screening and Treatment of Precancerous Lesions for Cervical Cancer Pre- vention. Published Online 2013. Available online: https://apps.who.int/iris/bitstream/han- dle/10665/94830/9789241548694_eng.pdf?sequence=1 (accessed on 29 August 2021). 11. Cremer, M.; Maza, M.; Alfaro, K.; Velado, M.M.; Felix, J.; Castle, P.E.; Kim, J.; Gage, J.C. Scale-up of an human papillomavirus testing implementation program in El Salvador. J. Low. Genit. Tract Dis. 2017, 21, 26–32. https://doi.org/10.1097/LGT.0000000000000280. p g 12. Alfaro, K.; Maza, M.; Felix, J.C.; Castle, P.E.; Alonzo, T.A.; Chacón, A.; González, E.; Soler, M.; Conzuelo-Rodriguez, G.; et al. Outcomes for step-wise implementation of a human papillomavirus testing-based cervical screen-and-treat program in El Sal- vador. JCO Glob. Oncol. 2020, 6, 1519–1530. https://doi.org/10.1200/GO.20.00206. p g 13. Campos, N.G.; Maza, M.; Alfaro, K.; Gage, J.C.; Castle, P.E.; Felix, J.C.; Masch, R.; Cremer, M.; Kim, J.J. The cost-effectiveness of implementing HPV testing for cervical cancer screening in El Salvador. Int. J. Gynecol. Obstet. 2019, 145, 40–46. https://doi.org/10.1002/ijgo.12773. p g jg 14. Campos, N.G.; Maza, M.; Alfaro, K.; Gage, J.C.; Castle, P.E.; Felix, J.C.; Cremer, M.L.; Kim, J.J. The comparative and cost-effec- tiveness of HPV-based cervical cancer screening algorithms in El Salvador. Int. J. Cancer 2015, 137, 893–902. https://doi.org/10.1002/ijc.29438. p g j 15. Ministerio de Salud Gobierno de El Salvador. Lineamientos Técnicos Para la Prevención y Control Del Cáncer Cérvico Uterino y de Mama. Published Online 2015. Available online: http://asp.salud.gob.sv/regulacion/pdf/lineamientos/lineamientos_pre- vencion_cancer_cervico_uterino_y_de_mama_v3.pdf (accessed on 29 August 2021). 16. Rosenbaum, A.J.; Gage, J.C.; Alfaro, K.M.; Ditzian, L.R.; Maza, M.; Scarinci, I.C.; Felix, J.C.; Castle, P.E.; Villalta, S.; Miranda, E.; et al. Acceptability of self-collected versus provider-collected sampling for HPV DNA testing among women in rural El Salva- dor. Int. J. Gynecol. Obstet. 2014, 126, 156–160. https://doi.org/10.1016/j.ijgo.2014.02.026. y p g j jg 17. Maza, M.; Melendez, M.; Masch, R.; Alfaro, K.; Chacon, A.; Gonzalez, E.; Soler, M.; Conzuelo-Rodriguez, G.; Gage, J.; Alonzo, T.; et al. Acceptability of self-sampling and human papillomavirus testing among non-attenders of cervical cancer screening programs in El Salvador. Prev. Med. 2018, 114, 149–155. https://doi.org/10.1016/j.ypmed.2018.06.017. p g p g j yp 18. Laskow, B.; Figueroa, R.; Alfaro, K.M.; Scarinci, I.C.; Conlisk, E.; Maza, M.; Chang, J.C.; Cremer, M. References A pilot study of community- based self-sampling for HPV testing among non-attenders of cervical cancer screening programs in El Salvador. Int. J. Gynecol. Obstet. 2017, 138, 194–200. https://doi.org/10.1002/ijgo.12204. p g jg 19. Guidelines Review Committee. WHO Guidelines for the Use of Thermal Ablation for Cervical Pre-Cancer Lesions. Who.int. Published Online September 2019. Available online: https://www.who.int/publications/i/item/9789241550598 (accessed on 29 August 2021). p g jg 19. Guidelines Review Committee. WHO Guidelines for the Use of Thermal Ablation for Cervical Pre-Cancer Lesions. Who.int. Published Online September 2019. Available online: https://www.who.int/publications/i/item/9789241550598 (accessed on 29 August 2021). 20. Conocimientos, Actitudes y Practicas Sobre El Cáncer Cervicouterino, El Virus Del Papiloma Humano y Sus Formas de Preven- ción, En Los Departamentos de Santa Ana y Sonsonate, El Salvador, 2014. Published 2014. Available online: https://www.iccp- portal.org/system/files/resources/Estudio%20CAPs%20sobre%20el%20CCU%20%20-%20El%20Salvador%202014%202.pdf (ac- cessed on 4 April 2022). 20. Conocimientos, Actitudes y Practicas Sobre El Cáncer Cervicouterino, El Virus Del Papiloma Humano y Sus Formas de Preven- ción, En Los Departamentos de Santa Ana y Sonsonate, El Salvador, 2014. Published 2014. Available online: https://www.iccp- portal.org/system/files/resources/Estudio%20CAPs%20sobre%20el%20CCU%20%20-%20El%20Salvador%202014%202.pdf (ac- cessed on 4 April 2022).
https://openalex.org/W2018711649
https://www.scielo.br/j/paz/a/q5KtqfwhN3wRGLTjbYq4SnB/?lang=en&format=pdf
English
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Historical Biogeography of a Clade of Liolaemus (Iguania: Liolaemidae) based on ancestral areas and dispersal-vicariance analysis (DIVA)
Papéis Avulsos de Zoologia
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cc-by
7,652
Cátedra de Anatomía Comparada. Facultad de Ciencias Naturales, Universidad Nacional de Salta. Avda. Bolivia 5150, 4400, Salta, Argentina. E-mail: jmdiaz@unsa.edu.ar CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas). IBIGEO (Instituto de Bio y Geociencias), IBIGEO. HISTORICAL BIOGEOGRAPHY OF A CLADE OF LIOLAEMUS (IGUANIA: LIOLAEMIDAE) BASED ON ANCESTRAL AREAS AND DISPERSAL-VICARIANCE ANALYSIS (DIVA) JUAN MANUEL DÍAZ GÓMEZ FERNANDO LOBO ABSTRACT Although many phylogenetic studies have been made on the genus Liolaemus, they are inappropriate for a biogeographic study because of incomplete taxon sampling. Here we develop a biogeographic analysis using a new phylogenetic study of the chiliensis group, which differs from the other studies in having his members fully represented. The biogeographic analysis presented here consists in reconstructing the ancestral area of this clade, under three different methods of analysis: Fitch Optimisation, Dispersal Vicariance Analysis and Weighted Ancestral Area Analysis. The analyses were made on phylogenies from parsimony under equally weighted characters, as well as under implied weighting. The equally weighted tree recovers most of the groupings proposed in previous studies. The strict consensus of the implied weighting trees recovers fifteen groups, but there is no agreement of the relationships between those groups. The ancestral area analyses agree in an Andean-Patagonian origin for the chiliensis clade, and are congruent with previous hypotheses and paleontological data. KEYWORDS: Liolaemus, chiliensis-group, biogeography, ancestral area, DIVA, Fitch, Patagonia, Andes. Volume 46(24):261-274, 2006 Volume 46(24):261-274, 2006 INTRODUCTION orous climates, in very high latitudes or at high altitudes (over 4000 m above sea level). This diversity and wide distribution has allowed the development of very di- verse biological characteristics, like oviparous and vivipa- rous species (perhaps related to cold conditions, Schulte et al. 2000) and a diverse diet, existing omnivorous, her- bivorous and insectivorous species. The genus Liolaemus includes about one hundred and sixty species (Espinoza et al., 2004), and is the sec- ond most specious genus of Lizards. Its distribution extends from Tierra del Fuego (Liolaemus magellanicus) in the southernmost extreme of South America, to the Peruvian Andes (L. walkeri). Liolaemus species occupy diverse habitats, including terrestrial and arboreal, some are saxicolous, some arenicolous, others live in very rig- Some authors included several statements regard- ing the distributional patterns of this genus in their taxonomic studies. One of them is Hellmich (1951) DÍAZ GÓMES, J.M. & LOBO, F.: ANCESTRAL AREA ANALYSIS ON LIOLAEMUS 262 of dispersal events could be just a problem of taxon sampling. who described the distribution of six species groups from Chile, which occupy very different areas. Hellmich explained these current distributions as a consequence of expansions and contractions of the original distri- butions. According to him, this happened during the interglacial and glacial periods of the Tertiary, and this would have given rise to the ‘races’ recognized. For Hellmich, the final explanation was dispersal, a state- ment which was also used by Lamborot et al. (1981) to explain the chromosomic variation in the monticola spe- cies complex. Although in the last years many works have been focused on the phylogeny of Liolaemus (Ortiz, 1981; Etheridge, 1995, Halloy et al. 1998; Young-Downey, 1998; Etheridge, 2000; Schulte et al. 2000), they are not useful for a biogegraphic analysis because they are fo- cused on subgroups or the genus is not fully repre- sented. The cladistic analysis on the chiliensis group re- cently performed by Lobo (2001, 2005) differs from the other studies in having the chiliensis group, which is half the genus Liolaemus, fully represented. Laurent (1983, 1985, 1992) also included biogeo- graphic comments when dividing the genus into two major subgroups (subgenus Eulaemus or Argentine group and subgenus Liolaemus or Chilean group), divi- sion attributed to the uplift of the Andes. INTRODUCTION In this paper we present a new cladistic analysis of the chiliensis group, based on already published data as well as evidence not previously considered. We in- clude species recently described and ethological charac- ters, and use parsimony to obtain a new hypothesis for the clade, and study its historical biogeography based on the topologies obtained. We apply three different methods of ancestral area analysis: Fitch optimisation (Ronquist, 1994), Weighted Ancestral Area Analysis (WAAA, Hausdorf, 1998) and Dispersal Vicariance Analysis (DIVA, Ronquist, 1997). The results are com- pared with previous hypothesis, and the methods are discussed. Cei (1979) described the composition of the Patagonian and Andean herpetofauna, characterising these regions by their high endemicity, and described centres of speciation and dispersal by using paleonto- logical and paleoclimatic data. Lobo (2001) described the areas of distribution on the phylogeny of the chiliensis group, following the biogeographic characterization of Roig-Juñent (1994) and adding areas not considered previously (i.e: Puna). There are only two works where the historical bio- geography of Liolaemus is discussed using some cladis- tic methodology. Young-Downey (1998) performed a Brooks Parsimony Analysis by using areas defined by Cabrera & Willink (1980) and Brown & Gibson (1983), and compared her results with the hypothesis of Solbrig (1976) on the origin and evolution of those areas. Schulte et al. (2000) proposed a hypothesis in which the species inhabiting three general areas, Andes, West of the Andes and East of the Andes were monophyletic. Using a molecular-based phylogeny, they optimized the distri- butions of the species, and ultimately rejected that hy- pothesis as the shortest phylogenetic estimate required multiple invasions of each of the areas. The pattern of distribution of Liolaemus is explained by recurring vicariance with subsequent dispersal between the areas that allowed further vicariance. Both studies had the problem that the areas of distribution were represented by biogeographic units (provinces) or arbitrarily defined, and in many cases, those units were not fully occupied by the species. Using areas much larger than the ranges of the species used in the analysis, as is the case with biogeographic provinces, will cause a loss of resolution in the analysis, because sometimes species separated by hundreds of kilometres will be included in the same province. Even worse, the sampling of species in the cited studies was not complete, then, the identification MATERIALS AND METHODS Taxa and Characters Biogeographic Analysis Ancestral Area Analysis was proposed by Bremer (1992) as a way for identifying the area of distribution of the ancestor of a monophyletic taxon, which he named the ancestral area. The analytical meth- ods for ancestral areas are based on the assumption that there is information about the ancestral area of a taxon in the topology of its area cladogram, that is, the probability for an area of being part of the ances- tral area increases with its presence in plesiomorphic branches and its general presence in the cladogram (Hausdorf, 1998). We applied three methods pro- posed in the last years: Fitch optimisation (Ronquist, 1994), Weighted Ancestral Area Analysis (WAAA) (Hausdorf, 1998) and Dispersal Vicariance Analysis (DIVA) (Ronquist, 1997). All these methods use optimisations with reversible parsimony for estimat- ing ancestral areas. Fitch Optimisation was proposed by Ronquist (1994) to avoid the problems of Camin- Sokal (irreversible) parsimony originally proposed by Bremer (1992). WAAA uses Fitch parsimony with a weighting scheme that weights favourably areas lo- cated in plesiomorphic branches, and areas more com- mon as terminals. In this method, a Probability In- dex (PI) is calculated to give a measure of the likeli- hood of a particular area of being part of the ances- tral area. The index is calculated for each area from the ratio of gains (assuming that the area was not part of the ancestral area) over losses (assuming that the area Cladistic Analysis was part of the ancestral area). DIVA searches ances- tral areas using a three dimensional cost matrix that gives different costs to events, minimizing the dis- persal events needed for explaining the distributions. In this approach, the vicariance events have no cost, while the dispersals and extinctions cost one per area unit added to the distribution. Parsimony analysis was made under implied weighting (Goloboff, 1993). This weighting method weights each character according to the number of extra or homoplasious steps in a tree, using a concave func- tion that gives lower weights to the characters with high homoplasy (Kitching et al. 1998). For the implied weighting analysis 6 concavity values were used (K, 1 to 6). Also we made a parsimony analysis under equal weights. For both analyses TNT version 1.0b4 (Goloboff et al. 2003) was used. We searched for opti- mal trees using TBR branch swapping, making 500 replications saving up to 10 trees for both the equally weighted and implied weights analysis. A strict consen- sus tree was generated using the topologies obtained from the implied weights. Continuous characters were coded using the gap weighting method (Thiele, 1993). Characters 0-20, 22-34, 37-82, 115 and 116 were coded as additive. Ancestral area analysis has been considered by some authors as a reimplementation of the centre of origin concept, and recently criticized on the basis that it utilizes paralogy (widespread taxa and redundant dis- tributions) instead of reducing it, as is the usual proce- dure for methods of cladistic biogeography (Ebach, 1999). However, ancestral area methods rely on the fact that all taxa have an area where their ancestor was dis- tributed, and the search for that area is a valid proce- dure. The ancestral area of a taxon needs not to be smaller than the actual distribution of its descendants, it can be larger or equal (Bremer, 1992), and dispersal needs not to be postulated as the only possible expla- nation. Ancestral area analysis tries to identify areas that may have been part of the ancestral area, and to distin- guish vicariance events from possible dispersals (Hausdorf, 1998). As Hovenkamp (1997) pointed out, methods that try to elucidate Earth history search for common patterns in cladograms, discard the incongru- ences, opposed to methods that try to search for taxon histories (as ancestral area methods), which have differ- ent methodologies and does not need to discard paralogies. Taxa and Characters The biogeographic analyses are based on a new hypothesis for the chiliensis group, and include all the species currently known for this group (83 species). We constructed a matrix using the morphological data from Lobo (2005), which was updated by adding three new species and 35 characters. The new species are Liolaemus yanalcu, (Martinez Oliver & Lobo, 2002), L. puna and L. chaltin (Lobo & Espinoza, 2004). Three new charac- ters are original to this study: gravid coloration, plant- microhabitat preference and lateral black spots; character states are included in appendix 1. We also included in the morphological matrix the allozyme data set of Young-Downey (1998) (32 characters for 26 taxa of the chiliensis group); the species L. alticolor of Young- Downey (op.cit.) analysis is actually L. puna (Lobo & Espinoza, 2004). Sp 1 is a new Liolaemus under descrip- tion (Vega et al. pers. comm), Sp 2 is a new species (Lobo et al. submitted). Being Phymaturus the closest living rela- tive to Liolaemus (Espinoza et al. 2004), we used Phymaturus indisctintus as outgroup. Data matrix is avail- able at www.unsa.edu.ar/acunsa/index02.html. PAP. AVULS ZOOL. 46(24), 2006 263 Selection of Areas The areas used were taken from Roig-Juñent (1994). To better represent the distribution of several species, we decided to include some new areas not con- sidered in that study like Puna, Atacama, Cordillera Ori- ental and Andes. This last area was divided into Andes, Andes Centrales and Andes Meridionales. We also di- vided the Monte area from Roig-Juñent (1994) into the new areas Sierras Pampeanas and Sierras Subandinas. The areas used are (Fig. 1): Andes, Andes Centrales, Andes Meridionales, Atacama, Coquimbo, Cordillera Oriental, Maulina, Monte, Patagonia, Puna, Selva Valdiviana, Sierras Pampeanas, Sierras Subandinas, and Valle Central. To make comparisons with a previous biogeo- graphic hypothesis, we also replicated the topology from Schulte et al. (2000) for the chiliensis group, and applied the three methods replacing the areas used by Schulte et al. with the areas used in this study. DÍAZ GÓMES, J.M. & LOBO, F.: ANCESTRAL AREA ANALYSIS ON LIOLAEMUS 264 FIGURE 1: Areas used in the Ancestral Area Analysis. AND: Andes; ANDC: Andes Centrales; ANDM: Andes Meridionales; ATA: Atacama; COQ: Coquimbo; COR: Cordillera Oriental; MAU: Maulina; MON: Monte; PAT: Patagonia; PUN: Puna; SEV: Selva Valdiviana; SPA: Sierras Pampeanas; SSA: Sierras Subandinas; VAC: Valle Central TABLE 1: List of groups and species belonging to each group according to the parsimony analysis under equal weights and implied weighting. The species not included were found not belonging to any group. g g y g p Group Equally Implied weighted weighting analysis analysis alticolor L. chaltin L. alticolor L. alticolor L. paulinae L. puna L. puna L. walkeri L. variegatus L. tacnae L. walkeri bibronii L. exploratorum L. hernani L. bibronii L. yanalcu L. ramirezae L. bitaeniatus L. chaltin capillitas L. capillitas L. buergueri L. heliodermis L. heliodermis L. dicktracyi L. capillitas L. umbrifer L. dicktracyi L. umbrifer ceii-kriegi L. ceii L. ceii L. kriegii L. kriegii L. buergueri elongatus L. petrophilus L. austromendocinus L. elongatusRN gravenhorsti L. gravenhorsti L. brattstroemi L. cyanogaster L. cyanogaster L. schroederi L. gravenhorsti L. schroederi leopardinus L. leopardinus L. leopardinus L. ramonensis L. ramonensis L. valdesianus nigroviridis L. maldonadae L. campanae L. nigroviridis nigromaculatus L. donosoi L. platei L. monticola L. atacamensis L. tacnae L. silvai L. platei L. sieversi L. velosoi L. nigromaculatus L. atacamensis L. kuhlmanni L. ater L. copiapensis L. bisignatus L. bisignatus L. copiapensis L. ater L. nigromaculatus L. sieversi L. zapallarensis L. kuhlmanni L. hellmichi L. silvai pictus L. pictus L. Liolaemus systematics Four different topologies were obtained using the six concavity values (K). The strict consensus indicates that these four topologies have in common thirteen groups (Fig. 2). The analysis under equal weights yielded one tree (Fig. 3). Eight groups of species are recovered. The list of groups and the species included are in Table 1. Selection of Areas talcanensis L. argentinus L. chiloensis robertmertensi L. robertmertensi L. curicensis L. chiliensis L. chiliensis L. nitidus L. nitidus L. robertmertensi L. saxatilis L. gracilis L. sp 2 tenuis L. tenuis L. coerulus L. punctatissimus L. neuquensis L. argentinus L. punctatissimus L. pictus L. tenuis L. talcanensis L. brattstroemi L. chiloensis L. coerulus L. neuquensis FIGURE 1: Areas used in the Ancestral Area Analysis. AND: Andes; ANDC: Andes Centrales; ANDM: Andes Meridionales; ATA: Atacama; COQ: Coquimbo; COR: Cordillera Oriental; MAU: Maulina; MON: Monte; PAT: Patagonia; PUN: Puna; SEV: Selva Valdiviana; SPA: Sierras Pampeanas; SSA: Sierras Subandinas; VAC: Valle Central Liolaemus biogeography The results for the three methods utilized are re- sumed on Table 2. These assignations refer to the most basal node of the tree. Fitch Optimisation. The Fitch optimization on the con- sensus tree (Fig. 2) assigns the most basal node Andes as the ancestral area. The Fitch optimisation on the equally weighted tree (Fig. 3) assigns for the most basal node the areas Patagonia, Andes or Monte as the ances- tral area. PAP. AVULS ZOOL. 46(24), 2006 265 2: Strict consensus from the four topologies obtained with the implied weights analysis. The area assignations are as rmal, Fitch optimization; Italics, Weighted Ancestral Area Analysis. FIGURE 2: Strict consensus from the four topologies obtained with the implied weights analysis. The area assignations are as follow: Normal, Fitch optimization; Italics, Weighted Ancestral Area Analysis. DÍAZ GÓMES, J.M. & LOBO, F.: ANCESTRAL AREA ANALYSIS ON LIOLAEMUS 266 FIGURE 3: Tree from the equally weighted analysis. The area assignations are as follow: Normal, Fitch optimization; Italics, Weighted Ancestral Area Analysis; Bold, DIVA (constrained to 2 maximum areas) FIGURE 3: Tree from the equally weighted analysis. The area assignations are as follow: Normal, Fitch optimization; Italics Weighted Ancestral Area Analysis; Bold, DIVA (constrained to 2 maximum areas) Dispersal Vicariance analysis (DIVA). The DIVA on the equally weighted tree achieved an exact solution, with 6 optimal reconstructions, requiring 45 dispersals each. (Table 2). For the most basal node, the areas Puna, Atacama, Monte, Andes, Valle Central, Coquimbo, Sier- ras Pampeanas, Andes Meridionales y Andes Centrales are repeated in all reconstructions. Maulina appears in four of six reconstructions, and Selva Valdiviana in three. The only areas that don’t appear in any reconstruction are Sierras Subandinas and Patagonia. The DIVA could not be done on the consensus tree, as the software cannot handle trees with polytomies. We selected for DIVA the trees corresponding to K = 1 and 2, and K = 5 and 6, because those hypothesis repre- sented two different weighting schemes: a stronger for K = 1,2 and weaker for K = 5, 6. Because of time con- PAP. AVULS ZOOL. 46(24), 2006 267 TABLE 2: Results of the ancestral area analysis. The abbreviations for the areas are from Fig. 1. Liolaemus biogeography Equally Weighted Analysis Consensus K = 1,2 K = 5,6 Fitch PAT, AND, MON AND AND AND WAAA AND, MON AND AND, PAT AND, SPA Constrained Max areas = 2 Max areas = 3 — Max areas = 2,3,4 Max areas = 2,3,4,5,6 DIVA PAT, MON PAT, AND, MON PAT, AND PAT, AND PAT, AND, MON Unconstrained DIVA PUN ATA MON AND ANDM ANDC PUN ATA MON AND ANDM ANDC ATA MON AND ANDM VAC VAC COQ SPA VAC COQ MAU SEV SPA COR COQ MAU SEV SPA COR PUN ATA MON AND ANDM ANDC PAT PUN ATA MON AND ANDM ANDC PAT PUN ATA MON AND ANDM VAC VAC COQ MAU SPA VAC COQ MAU SEV SPA COR COQ MAU SEV SPA COR PUN ATA MON AND ANDM ANDC PUN ATA MON AND ANDM ANDC PUN ATA MON AND ANDM VAC VAC COQ SEV SPA VAC COQ MAU SEV SPA SSA COR COQ MAU SEV SPA SSA COR PUN ATA MON AND ANDM ANDC PAT PUN ATA MON AND ANDM ANDC PAT PUN ATA MON AND ANDM VAC VAC COQ MAU SEV SPA VAC COQ MAU SEV SPA SSA COR COQ MAU SEV SPA SSA COR PUN ATA MON AND ANDM ANDC PUN ATA MON AND ANDM ANDC VAC VAC COQ MAU SPA COR COQ MAU SEV SPA COR PUN ATA MON AND ANDM ANDC PAT PUN ATA MON AND ANDM ANDC VAC VAC COQ MAU SEV SPA COR COQ MAU SEV SPA COR PUN ATA MON AND ANDM ANDC VAC COQ MAU SEV SPA SSA COR PAT PUN ATA MON AND ANDM ANDC VAC COQ MAU SEV SPA SSA COR DÍAZ GÓMES, J.M. & LOBO, F.: ANCESTRAL AREA ANALYSIS ON LIOLAEMUS 268 Implied weighting analysis. The WAAA analysis was made on the strict consensus tree as well as the topologies corresponding to K = 1, 2 and K = 5, 6 (Table 2). The areas with higher PI in the consensus were Andes (0.48) and Coquimbo (0.23). Monte has a low PI of 0.10. The areas with higher PI for the tree with K 1 and 2 are Andes (0.69) and Patagonia (0.30). Again Monte has a very low PI (0.07). Sierras Pampeanas (0.19) and Coquimbo (0.17) are areas that present higher values of PI than the rest. Liolaemus biogeography The tree resulting from K 5 and 6 has only Andes with a high PI of 0.59, the values for other areas are: 0.16 (Atacama), 0.16 (Sierras Pampeanas), 0.16 (Selva Valdiviana) and 0.12 (Coquimbo). straints the solution was heuristic, yielding 4 optimal reconstructions that required 41 dispersals each. These reconstructions are both essentially the same, except that Andes and Sierras Subandinas are not present in three of them: in one reconstruction both areas are missing, and one of them is absent from each of the other two. The last reconstruction includes all the areas. The analy- sis on the tree resulting form K = 5 and 6, also heuris- tic, produced 8 optimal reconstructions, requiring 42 dispersals. One of the optimal reconstructions involves all the areas, the other seven differ in the presence or absence of Patagonia, Sierras Subandinas, and Andes Centrales. These results are summarized in Table 2. Given the number of possible reconstructions, it’s hard to identify unambiguous vicariance events, making diffi- cult to hypothesize a sequence of events for the distri- bution of Liolaemus. Ancestral Area analysis on the topology of Schulte et al. (2000) When constraints were used on the maximum number of areas allowed for the ancestral area, on the equally weighted analysis, for a maximum of 2 areas the reconstruction is Patagonia-Monte or Patagonia-Andes; for a maximum of 3 to 6 areas the reconstruction is Patagonia, Andes, Monte (Table 2). For the trees with implied weights, when making DIVA on the topolo- gies corresponding to K = 1 and 2, the reconstruction for a maximum of 2 to 4 areas is Patagonia and Andes; for K = 5 and 6 with a maximum of 2 to 6 areas, the reconstruction is Patagonia and Andes (Table 2). In all cases, the optimizations for a maximum number of areas superior to 6 showed several ambiguous recon- structions (data not shown). The topology of Schulte et al. (2000) (Fig. 4) has a polytomy with the taxons Liolaemus bellii, L. chiliensis and L. cyanogaster; the polytomy was resolved, and the two resolutions used for the DIVA analysis. For the Fitch optimization (11 steps) on the topology of Schulte et al. (2000) assigns Andes as the ancestral area. Uncon- strained DIVA (8 equally optimal reconstructions) as- signs to the basal node all the areas, requiring 15 dis- persal events. When using constraints to a maximum of 2 areas, it requires 18 dispersal events, with two equally optimal reconstructions between Andes and Sierras Pampeanas. Weighted Ancestral Area Analysis found Andes with the higher PI (0.44) followed by Sierras Pampeanas and Maulina (0.39), and Valle Central in Chile (0.38). Weighted Ancestral Area Analysis (WAAA). The PI val- ues for the WAAA analysis were calculated for the most basal node of the tree, and for the nodes that give rise to groups of species. Liolaemus systematics Equally weighted analysis. The areas with higher PI are Andes and Monte (0.67 and 0.44 respectively), and have more probability of being part of the ancestral area (Table 2). This result is congruent with Fitch optimisation, which has Patagonia, Andes or Monte as equally probable reconstructions for the basal node, and Andes as the ancestral reconstruction for the nodes as we go up in the tree. Nevertheless, the area Patagonia has a very low PI (0.04), discarding it as part of the ancestral area. There are other areas that have low values of PI, but slightly higher than the remaining, these ar- eas are Sierras Pampeanas and Selva Valdiviana (0.13 and 0.12 respectively). This may be reflecting the fact that these areas appear in more plesiomorfic branches, and are given more weight accordingly. Lobo (2005) found six different resolutions among subclades of the chiliensis group, some of those resolutions are recovered here, i.e. the basal position of leopardinus and kriegi groups is recovered in every analysis,and the clade formed by the robertmertensi, gravenhorsti and alticolor groups appears only on the equally weighted tree. The strict consensus of the im- plied weighting analysis recovered 15 groups, most of them found in previous studies, but the relationships among these groups remain unresolved. Cei (1986, 1993) and Avila et al. (1992) defined the bibronii group based on a character combination includ- ing as members of this group the following species: 269 PAP. AVULS ZOOL. 46(24), 2006 FIGURE 4: Tree from Schulte et al. (2000), showing the possible resolutions of the polytomy. The area assignations are as follow: Normal, Fitch optimization; Italics, Weighted Ancestral Area Analysis; Bold, DIVA (constrained to 2 maximum areas). Liolaemus alticolor is actually Liolaemus puna (Lobo & Espinoza, 2004). FIGURE 4: Tree from Schulte et al. (2000), showing the possible resolutions of the polytomy. The area assignations are as follow: Normal, Fitch optimization; Italics, Weighted Ancestral Area Analysis; Bold, DIVA (constrained to 2 maximum areas). Liolaemus alticolor is actually Liolaemus puna (Lobo & Espinoza, 2004). Liolaemus bibronii, L. exploratorum, L. sanjuanensis, L. saxatilis, L. gracilis, L. robertmertensi and L. fuscus. In this study this group is paraphyletic. In the equally weighted analysis L. bibronii is sister taxon of L. sanjuanensis included in a subclade related to the most terminal grouping formed by robertmertensi, gravenhorsti and alticolor groups; L. saxatilis, L. bitaeniatus, L. fuscus and L. Liolaemus systematics gracilis are nested within this clade but not form- ing a monophyletic group. In the Implied weighting analysis species belonging to the traditional bibronii group are separated in two groups which here we redefine as bibronii (including only L. exploratorum, L. bibronii, L. hernani and northern Argentina species: L. bitaeniatus, L. chaltin, L. yanalcu and L. ramirezae) and robertmertensi group including now L. curicensis, L. chiliensis, L. nitidus, L. robertmertensi, L. saxatilis, L. gracilis and L. tandilensis. Liolaemus robertmertensi, L. saxatilis and L. tandilensis are distributed following the mountain arc formed by Sier- ras Pampeanas, and Sierras de Tandil in eastern Argen- tina, L. robertmertensi in the slopes of Sierras of Catamarca and La Rioja, L. saxatilis in Sierras de San Luis and Córdoba, L. tandilensis in Sierra de Tandil. Liolaemus sanjuanensis lives in Sierra Pie de Palo (San Juan province, Argentina) a chain of mountains related to the same system of Sierras Pampeanas but in this analysis is not found related to this group of species, perhaps this is due to problems of taxon sampling, because only two paratypes of this species were available for our study. The alticolor-bibronii group of Espinoza et al. (2004) includes 10 species in two clades one formed by Liolaemus bibronii, L. gracilis, L. robertmertensi, L. yanalcu, L. ramirezae, and other clade with L. bitaeniatus, L. pagaburoi, L. chaltin, L. puna, and L. cf. walkeri, which is actually L. puna according to Lobo & Espinoza (2004). Lobo (2005) also found the alticolor-bibronii group, with between 14 and 17 species in different analysis. In this study the alticolor-bibronii is not recovered. In the equally weighted analysis is only formed by 4 species (Fig. 3), and in the consensus the alticolor and bibronii groups appear separated, with species inhabiting Northern Argentina (L. puna), northeastern Chile (L. paulinae), Puna and high mountains in Bolivia DÍAZ GÓMES, J.M. & LOBO, F.: ANCESTRAL AREA ANALYSIS ON LIOLAEMUS 270 following climate effects (desertification of the Patagonian steppe). Unfortunately, the areas indicated as occupied by the ancestor of Liolaemus are not clearly described by Young-Downey and is difficult to estab- lish direct correspondences with the areas used here. Young-Downey (1998) found Atacama region as more related to Coquimbo and Chilean (Valle Central) in con- gruence with Solbrig (1976). Liolaemus systematics The current distributions of the species and the phylogenetic analysis used here as well as the cladogram of Lobo (2005) suggest different area relationships. The nigromaculatus group from the Northern deserts in Chile (Coquimbo and Atamaca ar- eas) in that analysis is sister taxon of the alticolor group (Puna); interestingly Morrone (1996) found Puna and the Northern deserts forming a monophyletic unit. (L. variegatus and L. alticolor) and Perú (L. tacnae and L. walkeri) forming the alticolor group, and the remain- ing species of alticolor-bibronii which are oviparous (L. ramirezae, L. yanalcu, L. bitaeniatus and L. chaltin) are included in the bibronii group. Liolaemus fuscus and L. lemniscatus form a separated monophyletic group, lemniscatus group (congruent with the fuscus-lemniscatus group of Ortiz (1981), and lemniscatus group of Cei, 1986). The petrophilus group recovered in the analysis with implied weights includes the capillitas group; this is con- gruent with the results of Morando et al. (2003). The morphological information does not support the in- clusion of the ceii-kriegi within the petrophilus group as suggested by the mitochondrial DNA analysis. Comparisons with the distribution of the groups more related with Liolaemus may prove useful. The sis- ter clade of Liolaemus, Phymaturus (Schulte et al. 2003; Espinoza et al. 2004), is distributed in Patagonia and in both sides of the Andes, reaching north to the Puna of Catamarca Province in Argentina, distribution that is shared with many species of Liolaemus and does not contradict the hypothesis of ancestral areas for the chiliensis group. Nevertheless, Ctenoblepharis, the basal taxa for Liolaemidae (Schulte et al. 2003; Espinoza et al. 2004), is found only in coastal deserts of Peru, thus making nec- essary further studies including these two genera. Liolaemus Biogeography The results are summarized in Table 2. There is congruence between the three methods because all sus- tain the hypothesis of an Andean-Patagonian origin for the chiliensis group. The common area to all the analyses is Andes, followed by Patagonia, and Monte. This results show some degree of congruence with previous studies. Cei (1979) sustained that Patagonia was a center of origin for at least four groups of Liolaemus, describing two major faunal regions in Patagonia (see Cei 1979, Fig. 13:3): the northern or an- cient Patagonian region and the southern or Santa Cruz region, the former includes part of the Patagonia and Andes areas used in this study. The first fossil attrib- uted to Liolaemus (Albino, 1998) comes from the Mi- ocene of Patagonia, found in sediments of the Miembro Trelew, Formación Sarmiento, near Gaiman in Chubut Province, this locality is included in the area Patagonia. These studies, although not focused on the chiliensis group but on the entire genus, are congruent with the ancestral areas found here. Recently, Albino (2005) found new fossils attributed to Liolaemus from the late Quater- nary in the Southwest of Buenos Aires Province, in- cluded in the Monte Area of this study. Fitch optimisation. The Fitch optimisation on the equally weighted analysis yielded Andes, Patagonia and Monte. This shows the perhaps excessive effect that Fitch opti- mization places in the more plesiomorfic areas, because, Patagonia only appears again in more terminal branches of the tree. Fitch optimisation. The Fitch optimisation on the equally weighted analysis yielded Andes, Patagonia and Monte. This shows the perhaps excessive effect that Fitch opti- mization places in the more plesiomorfic areas, because, Patagonia only appears again in more terminal branches of the tree. Effect of constraining the number of areas Ronquist (1997) noted that when using DIVA for reconstruct the ancestral area of a taxon, the root node will tend to include all the areas occupied by the terminals, making it very uninformative. The solutions to this suggested by Ronquist are two: the inclusion of more outgroups, thus making the root node no longer the root node, and constraining the maximum number of areas that may form part of the ancestral area. The first of this possible solutions is, at least in this case, not useful: for example, the inclusion of other outgroups related to Liolaemus, i.e. Phymaturus and Ctenoblepharis, will not solve the problem, given that Phymaturus is distributed in the same areas used for Liolaemus, and Ctenoblepharis, distributed in coastal Peru, Because the BPA results of Young-Downey (1998) had incongruences with the hypothesis of Solbrig (1976), she evaluated the area cladogram of Liolaemus regarding ancestral distributions and evaluating possible dispers- als, extinctions, and lack of response to vicariance. Thus, Young-Downey (1998) postulates that the ancestor of Liolaemus inhabited areas comprising the Southern Nothofagus forest (Subantartic Province), Central Argen- tina and Central-Northern Chile, and from there, it vicariated by the appearance of the Andes, and by the PAP. AVULS ZOOL. 46(24), 2006 271 areas, making the results uninformative, given the size of the areas. probably will only add this new area to the ancestral area reconstruction in the root, leaving the ancestral area for Liolaemus without major changes. The second alterna- tive presents the problem that there is no criteria for choosing the number of areas for constraining. The sympatry of sister groups is evidence for dispersal (Hausdorf, 1998), so it can be argued that the ancestral distribution may have been larger o smaller than the present area occupied by the taxon. Examples of such sympatry in sister species are L. chiliensis and L. nitidus, both living in Valle Central (Chile); L. cyanogaster and L. schroederi, both distributed in Maulina (Chile). On this basis, one may assume that the ancestral area may have been smaller in the past, and this provides a ratio- nale for constraining the maximum number of areas allowed in the ancestral distribution, taking into account that this practice remains ad hoc. A last remark is needed about the areas used for this study. Commentaries about Schulte et al. (2000) Schulte et al. (2000) postulated that the ancestral area for the subgenus Liolaemus (equivalent to the chiliensis group) in their reconstruction is equivocal between Andes, western lowlands and eastern lowlands, the three areas used for the analysis. With the exception of Andes, the other areas found in the analysis (Sierras Pampeanas, Maulina and Chile Central) are not hypothesized as an- cestral in the biogeographic analysis of the more com- plete phylogeny herein. This shows the effects of a lim- ited taxon sampling for making biogeographic infer- ences about ancestral areas. There are areas not repre- sented in the analysis, and the species located plesiomorphically on the cladogram have a strong influ- ence in the result of the optimization. There are only three species inhabiting Sierras Pampeanas (L. bitaeniatus, L. robertmertensi and L. capillitas) but ten in Andes, but both areas appear as equally probable ancestral areas. Also, there is an oversimplification of the distribution of the species used by Schulte et al. (2000); any ambigu- ity at any node will involve at least two of the three Effect of constraining the number of areas These areas from Roig-Juñent (1994) are based on distributions of arthropods, and despite they are accurate respect to the distribution of Liolaemus, some regions are missing (i.e. Puna), and others do not repre- sent very well the distributions of Liolaemus, like Monte, and would need a reformulation. The ancestral area of a taxon, as defined by Bremer (1992), needs not to be limited to a single place, it may be equal or larger than the current area occupied by the taxa. However, as implemented by the methods, the results of the ancestral area analysis will be an ancestral area for a taxon at least equal or smaller than the com- bined distributions of his descendants. The only pos- sible way to find an ancestral area larger than the sum of the distributions used would be with the inclusion of fossil groups distributed in areas not currently occupied by the taxa in study. For many groups, this is very diffi- cult. For example, the fossil record may be absent, or the fossils may be found only in the areas already present in the analysis, as is the case with the fossils attributed to Liolaemus (Albino, 1998, 2005). Even in the case that a fossil distribution could be used, the area occupied by the fossil taxa would have to be present in deep branches of the cladogram or in numerous terminals or it would have no influence in the analysis and could not be hy- pothesized as being part of the ancestral area. Thus, although the methods contemplate the possibility of identifying an ancestral area larger than the current distri- bution using data from fossils, is very difficult to this situation to effectively appear in an analysis. When using constrained DIVA (Table 2), con- straining the maximum number of areas that may form the ancestral distribution from 3 to 6 areas, yield as op- timal reconstruction Andes, Patagonia and Monte, the same areas that yield the Fitch optimization on the equally weighted tree (when constraining to 2 areas, the recon- struction is Andes and Monte). Andes also have the highest PI in the WAAA analysis, being Monte the sec- ond. Sadly, there are no criteria for selecting a number of area units by constraining the ancestral distributions, making any further statements speculative. CONCLUSIONS In recent years several studies have dealed with the phylogenetic relationships of the chiliensis group (Schulte et al. 2000; Lobo, 2001; Espinoza et al. 2004; Lobo, 2005; this study). Some groups are recovered in this analysis, but others do not, as the alticolor-bibronii, which appears splitted. The consensus of the implied weighting analy- sis shows that the relationships between the groups are not yet resolved, and would require further studies to clarify the intern relationships inside the chiliensis group. As with any cladistic biogeographic analysis, the results of the ancestral area methods may vary according to the different hypothesis. However, if the degree of varia- tion is only on the terminal groups, the ancestral area assignations will likely not be affected. Nevertheless, some area relationships found in other studies (Morrone, 1996; DÍAZ GÓMES, J.M. & LOBO, F.: ANCESTRAL AREA ANALYSIS ON LIOLAEMUS 272 Lobo, 2005) are recovered here, like the sister relation- ship between Puna and the Northern deserts of Chile (Coquimbo and Atacama). ancestral, resultado este, congruente com hipóteses prévias e com informação paleontológica. ( q ) Of the studies that include biogeographic consid- erations about Liolaemus, only Young-Downey (1998) applied an explicit methodology. However, these stud- ies have the problem of limited taxon sampling and area definitions, making difficult to make comparisons between the results. However, the results of the ances- tral area analysis of this work are congruent with previ- ous studies and paleontological data, hypothesizing an Andean-Patagonian ancestral area for the chiliensis group. A large part of the genus is not included in the chiliensis group (the Eulaemus subgenus or Argentine group), and its inclusion is necessary for the completion of the ancestral area analysis of the genus. This work is under- way and will be published elsewhere. PALABRAS-CHAVE: Liolaemus, chilinesis, Dispersão- Vicariância, Fitch, Áreas Ancestrais Pesadas, Andes, Patagonia. PALABRAS-CHAVE: Liolaemus, chilinesis, Dispersão- Vicariância, Fitch, Áreas Ancestrais Pesadas, Andes, Patagonia. ACKNOWLEDGMENTS We thank Claudia Szumik and Sebastián Quinteros for making suggestions to an earlier draft of the manuscript. We also thank Sonia Kretzschmar for help with the translation of the abstract to Portuguese. Cristian Abdala, Sebastián Quinteros, Federico Arias and Soledad Vadecantos helped with disscussions and with friendship. The senior author would like to thank Elisa Núñez for her invaluable help and patience. This study was supported by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET – PIP Nº 5982). A last consideration is needed regarding the ances- tral area methodologies. Although they erroneously are criticized as an absolute dispersalist approach, they are a valid procedure for studying the historical biogeogra- phy of a taxon. These methods, however, present some problems, as the sometimes uninformative multiple reconstructions of DIVA, or the excessive importance of the basal areas for the Fitch optimization. REFERENCES Avila, L.J.; Cei, J.M.; Martori, R.A. & Acosta, J.C. 1992. A new species of Liolaemus of the bibronii group from granitic ravines of Achiras, Sierra de Comechingones, Córdoba, Argentina (Reptilia: Tropiduridae). Bolletino del Museo Regionale di Scienze Naturali, Torino, 10(1):101-111. This work represents the first study of the his- torical biogeography of the chiliensis group, a major part of the genus Liolaemus using explicit methodology and with a complete taxon sampling. Further studies need to be carried out, especially concerning the areas used, particularly its unambiguous definition, and using geo- morphologic data to contrast the hypothesis produced. z ( ) Albino, A.M. 1998. Primer registro fósil de Liolaemus (Squamata: Iguania: Tropiduridae). In: VII Congreso Argentino de Paleontología y Bioestratigrafía, Bahía Blanca. Actas, p. 57. Albino, A.M. 2005. A late Quartenary lizard assemblage from the Southern Pampean region of Argentina. Journal of Vertebrate Paleontology, 25(1):185-191. Bremer, K. 1992. Ancestral Areas: a cladistic reinterpretation of the center of origin concept. Systematic Biology, 41:436-445. RESUMO puna salteña, Argentina. Cuadernos de Herpetología, 16(1):47-64. Goloboff, P. 1993. Estimating character weights during tree search. Cladistics, 9:83-91. Morando M.; Avila, L.J. & Sites Jr., J.W. 2003. Sampling strategies for delimiting species: genes, individulas, and populations in the Liolaemus elongatus-kriegi complex (Squamata: Liolaemidae) in andean-patagonian South America. Systematic Biology, 52(2):159-185. Goloboff, P.; Farris, S. & Nixon, K. 2003. T.N.T.: Tree analysi Goloboff, P.; Farris, S. & Nixon, K. 2003. T.N.T.: Tree analysis using New Technology. Available at: <www.zmuc.dk./ public/phylogeny>. y using New Technology. Available at: <www.zmuc.dk./ public/phylogeny>. Morrone, J.J. 1996. The biogeographical Andean subregion: a proposal exemplified by Arthropod taxa (Arachnida, Crustacea and Hexapoda). Neotropica, 42:103-114. Halloy, M.; Etheridge, R. & Burghardt, G.M. 1998. To bury in sand: Phylogenetic relationships among lizard species of the boulengeri group, Liolaemus (Reptilia: Squamata: Tropiduridae), based on behavioral characters. Herpetological Monographs, 12:1-37. Ortiz, J.C. 1981. Révision taxonomique et biologique des Liolaemus du groupe nigromaculatus (Squamata, Iguanidae). Université de Paris VII. (Thèse Doctorat d’Etat ès Sciences Naturelles). Hausdorf, B. 1998. Weighted ancestral area analysis and the solution of the redundant distribution problem. Systematic Biology, 47(3):445-456. Roig-Juñent, S. 1994. Historia Biogeográfica de América del Sur Austral. Multequina. 3:167-203. Hellmich, W. 1951. On ecotypic and autotypic characters, a con-tribution to the knowledge of the evolution of the genus Liolaemus (Iguanidae). Evolution, 5:359-369. Ronquist, F. 1994. Ancestral areas and parsimony. Systematic Biology, 43:267-274. g genus Liolaemus (Iguanidae). Evolution, 5:359-369 Ronquist, F. 1997. Dispersal-vicariance analysis: a new approach to the quantification of historical biogeography. Systematic Biology, 46:195-203. Hovenkamp, P. 1997. Vicariance events, not Areas, should be used in Biogeographical analysis. Cladistics, 13:67-79. Kitching, I.J.; Forey, P.L.; Humphries, C.J. & Williams, D.M. 1998. Cladistics: The Theory and Practice of Parsimony Analysis. Oxford University Press, Oxford. Schulte, J.A.; Macey, J.R.; Espinoza, R.E. & Larson, A. 2000. Phylogenetic relationships in the iguanid lizard genus Liolaemus: multiple origins of viviparous reproduction and evidence for recurring andean vicariance and dispersal. Biological Journal of the Linnean Society, 69:75-102. Lamborot, M.; Alvarez, E.; Campos, I. & Espinoza, A. 1981. Karyotypic characterization of three chilean subspecies of Liolaemus monticola. The Journal of Heredity, 72:328-334. Schulte II, J.A.; Valladares, J.P. & Larson, A. 2003. Phylogenetic relationships within Iguanidae using molecular and morphological data and a phylogenetic taxonomy of iguanian lizards. Herpetologica, 59:399-419. Laurent, R.F. 1983. Contribución al conocimiento de la estructura taxonómica del género Liolaemus Wiegmann (Iguanidae). Boletin de la Asociación Herpetologica Argentina, 1:16-18. Solbrig, O.T. 1976. RESUMO The origin and floristic affinities of the South American temperate desert and semidesert regions. In: Goodall, D.W. (Ed.), Evolution of desert biota. University Texas Press, Austin, p. 7-49. Laurent, R.F. 1985. Segunda contribución al conocimiento de la estructura taxonómica del género Liolaemus Wiegmann (Iguanidae). Cuadernos de Herpetología, 1:1-37. Laurent, R.F. 1992. On some overlooked species of the genus Liolaemus Wiegmann (Reptilia Tropidurinae) from Peru. Breviora, 494:1-33. Thiele, K. 1993. The holy grail of the perfect character: the cladistic treatment of morphometric data. Cladistics, 9:275-304. Lobo, F. 2001. A phylogenetic analysis of lizards of the Liolaemus chiliensis group (Iguania: Tropiduridae). Herpetological Journal, 11(4):137-150 Young-Downey, A.R. 1998. Phylogenetic studies on Liolaemus (Sauria: Tropiduridae): an interpretation based on molecular data and a biochemical test of a biogeographic hypothesis. Univestity of Miami, Coral Gables, Florida. (Ph.D. Dissertation). Lobo, F. 2005. Las relaciones filogenéticas dentro del grupo chiliensis (Iguania: Liolaemidae) sumando nuevos caracteres y taxones. Acta Zoológica Lilloana, 49(1-2):67-89. of Miami, Coral Gables, Florida. (Ph.D. Dissertation). Lobo, F. & Espinoza, R.E. 2004. Further Resolution of Purported Reproductive Bimodality in Liolaemus alticolor (Iguania: Tropiduridae) with descriptions of Two New Species from the Puna Region of Argentina and Chile. Copeia, (4):850-867. Recebido em: 21.08.2006 Aceito em: 19.12.2006 Recebido em: 21.08.2006 Aceito em: 19.12.2006 Recebido em: 21.08.2006 Aceito em: 19.12.2006 RESUMO the center of origin concept. Systematic Biology, 41:436-445. Brown, J.H. & Gibson, A.C. 1983. Biogeography. C.V. Mosby, Toronto. Ainda que tenham sido feito muitos estudos filogenéticos no gênero Liolaemus, os mesmos não são apropriados para uma análise biogeográfica, porque não possuem uma amostragem taxonômica adequada. Desenvolve-se neste trabalho, uma análi- se biogeográfica do grupo de L. chiliensis, que se diferencia das contribuições prévias por representar a todos os membros do gru- po. A análise biogeográfica consiste em reconstruir a área ances- tral deste clado, utilizando três métodos diferentes: Otimização de Fitch, Análise de Dispersão-Vicariância e Áreas Ancestrais Pesadas. As análises foram realizadas em filogenias feitas com o critério de parsimônia sob pesos iguais e pesos implicados. A árvore obtida sob pesos iguais, recupera a maioria dos grupos encontrados em análises prévias. O consenso estrito das árvores de pesos implicados, recupera quinze grupos, mas não pode-se estabelecer as relações entre eles. A análise de áreas ancestrais permite postular a região Andino-Patagônica como provável área Cabrera, A.L. & Willink, A. 1980. Biogeografía de América Latina. Secretaría General de la OEA. Washington. Cei, J.M. 1979. The Patagonian Herpetofauna. In: Duellman, W.E. (Ed.), The South American Herpetofauna: Its Origin, Evolution, and Dispersal. Museum of Natural History University of Kansas, Monograph 7, p. 309-339. Cei, J.M. 1986. Reptiles del centro, centro-oeste y sur de la Argentina. Museo Regionale di Scienze Naturali, Torino, Monografie 4. Cei, J.M. 1993. Reptiles del noroeste, nordeste y este de la Argentina. Museo Regionale di Scienze Naturali, Torino, Monografie 14. Ebach, M.C. 1999. Paralogy and the Centre of Origin Concept. Cladistics, 15(4):387-391. Espinoza, R.E.; Wiens, J.J. & Tracy, C.R. 2004. Recurrent evolution of herbivory in small, cold-climate lizards: breaking the ecophysiological rules of reptilian herbivory. Proceedings of the National Academy of Sciences, DOI: 101:16819-16824. Etheridge, R. 1995. Redescription of Ctenoblepharys adspersa Tschudi, 1845, and the taxonomy of Liolaeminae (Reptilia: PAP. AVULS ZOOL. 46(24), 2006 273 Squamata: Tropiduridae). American Museum Novitates, 3142:1-34. Lobo, F; Quinteros, S. & Díaz Gómez, J.M. In press. Description of a new species of the liolaemus alticolor group (Iguania: Liolaemidae) from Cuzco, Peru. Etheridge, R. 2000. A review of lizards of the Liolaemus wiegmanniii group (Squamata, Iguania, Tropiduridae), and a history of morphological change in the sand-dwelling species. Herpertological Monographs, 14:293-352. Martinez Oliver, I. & Lobo, F. 2002. Una nueva especie de Liolaemus del grupo alticolor (Iguania: Liolaemidae) de la puna salteña, Argentina. Cuadernos de Herpetología, 16(1):47-64. 274 DÍAZ GÓMES, J.M. & LOBO, F.: ANCESTRAL AREA ANALYSIS ON LIOLAEMUS APPENDIX 1 New characters used: 1. Gravid Coloration: (0) Absent; (1) Present 2. Plant-Microhabitat: (0) Bunch grass (Festuca sp); (1) Spiny shrub (Parastrephia); (2) Both 3. Lateral Black Spots: (0) Absent; (1) Present New characters used: 1. Gravid Coloration: (0) Absent; (1) Present 1. Gravid Coloration: (0) Absent; (1) Present 2. Plant-Microhabitat: (0) Bunch grass (Festuca sp); (1) Spiny shrub (Parastrephia); (2) Both 3 Lateral Black Spots (0) Absent (1) Present 2. Plant-Microhabitat: (0) Bunch grass (Festuca sp); (1) Spiny shrub (Parastrephia); (2) Both 3. Lateral Black Spots: (0) Absent; (1) Present
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Recent experimental activity on heavy-ion induced reactions within the NUMEN project
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for the NUMEN Collaboration 1Istituto Nazionale di Fisica Nucleare, Laboratori Nazionali del Sud, Catania, Italy 2Dipartimento di Fisica e Astronomia “Ettore Majorana”, Università di Catania, Italy 3Instituto de Fısica, Universidad Nacional Autonoma de Mexico - Mexico City, Mexico 4Instituto Nacional de Investigaciones Nucleares - Ocoyoacac, Mexico 5Department of Physics, Akdeniz University - Antalya, Turkey 6Istituto Nazionale di Fisica Nucleare, Sezione di Torino, Italy 7DISAT, Politecnico di Torino, Italy 8LPC Caen, Normandie Universite, ENSICAEN, UNICAEN, CNRS/IN2P3 Caen, France 9Ankara University, Institute of Accelerator Technologies, Turkey 10Centro Universitario FEI - Sao Bernardo do Campo, Brazil 11Institute of Natural Sciences, Karadeniz Teknik University - Trabzon, Turkey 12Università degli Studi di Enna “Kore” - Enna, Italy 13Instituto de Fisica, Universidade Federal Fluminense - Niteroi, Brazil 14Instituto de Fisica, Universidade de Sao Paulo - Sao Paulo, Brazil 15Instituto de Pesquisas Energeticas e Nucleares IPEN/CNEN - Sao Paulo, Brazil 16Department of Physics and HINP, University of Ioannina - Ioannina, Greece 17IFIN-HH - Magurele, Romania 18Department of Chemistry, National and Kapodistrian University of Athens, Athens, Greece 1Istituto Nazionale di Fisica Nucleare, Laboratori Nazionali del Sud, Catania, Italy 2Dipartimento di Fisica e Astronomia “Ettore Majorana”, Università di Catania, Italy 3Instituto de Fısica, Universidad Nacional Autonoma de Mexico - Mexico City, Mexico 4Instituto Nacional de Investigaciones Nucleares - Ocoyoacac, Mexico 5Department of Physics, Akdeniz University - Antalya, Turkey 6Istituto Nazionale di Fisica Nucleare, Sezione di Torino, Italy 7DISAT, Politecnico di Torino, Italy 8LPC Caen, Normandie Universite, ENSICAEN, UNICAEN, CNRS/IN2P3 Caen, France 9Ankara University, Institute of Accelerator Technologies, Turkey 10Centro Universitario FEI - Sao Bernardo do Campo, Brazil 11Institute of Natural Sciences, Karadeniz Teknik University - Trabzon, Turkey 12Università degli Studi di Enna “Kore” - Enna, Italy 13Instituto de Fisica, Universidade Federal Fluminense - Niteroi, Brazil 14Instituto de Fisica, Universidade de Sao Paulo - Sao Paulo, Brazil 15Instituto de Pesquisas Energeticas e Nucleares IPEN/CNEN - Sao Paulo, Brazil 16Department of Physics and HINP, University of Ioannina - Ioannina, Greece 17IFIN-HH - Magurele, Romania 18Department of Chemistry, National and Kapodistrian University of Athens, Athens, Greece Abstract. The possibility to use a special class of heavy-ion induced direct reactions, such as double charge exchange reactions, is discussed in view of their application to extract information that may be helpful to determinate the nuclear matrix elements entering in the expression of neutrinoless double beta decay half-life. for the NUMEN Collaboration The strategies adopted in the experimental campaigns performed at INFN - Laboratori Nazionali del Sud are briefly described, emphasizing the advantages of the multi-channel approach to nuclear reaction data analysis. 1 Introduction aspect is the possibility to use CE to investigate the spin- isospin-dependent response of nuclei. Charge exchange (CE) reactions are nuclear processes in which the individual balance of neutrons and protons inside the projectile and the target is modified, while keeping the overall number of nucleons unchanged. In the isospin formalism the CE promote isovector excitations in both the projectile and target. CE are thus selective tools to emphasize the role of isospin in both nuclear structure and reaction studies. Relevant aspects of single charge exchange (SCE) induced by light projectile have been discussed in several articles, among which excellent reviews are in ref. [1] [2] [3]. For heavy-ion induced CE we refer to the recent review [4]. An especially appealing Recently, particular interest has been given to double charge exchange (DCE) processes [5] [6] [7] as they can give access to the nuclear response to second order isospin operators, which also promote double beta decays. SCE and DCE studies have a genuine nuclear physics interest, since they reflect a key aspect of nuclear dynamics. However, beyond that, there is an even broader impact of such investigations because of their relevance for nuclear beta decay, thus connecting strong and weak interactions. In this context, the NUMEN and NURE projects at INFN- LNS laboratory in Catania [8] [9] aim at exploring heavy- ion induced single and double charge exchange (DCE) Recent experimental activity on heavy-ion induced reactions within the NUMEN project Francesco Cappuzzello1,2,*, Luis Acosta3, Clementina Agodi1, Carmen Altana1, Paulina Amador-Valenzuela4, Ismail Boztosun5, Giuseppe A. Brischetto1,2, Salvatore Calabrese1,2, Daniela Calvo6, Vittoria Capirossi6,7, Diana Carbone1, Manuela Cavallaro1, Efrain R. Chávez Lomeli3, Irene Ciraldo1,2, Mauro Cutuli1,2, Franck Delaunay1,2,8, Haris Djapo9, Canel Eke5, Paolo Finocchiaro1, Suna Firat5, Maria Fisichella1, Marcilei A. Guazzelli da Silveira10, Aylin Hacisalihoglu11, Felice Iazzi6,7, Gaetano Lanzalone1,12, Laura La Fauci1,2, Roberto Linares13, Nilberto H. Medina14, Mauricio Moralles15, José R. B. Oliveira14, Athena Pakou16, Luciano Pandola1, Horia Petrascu17, Federico Pinna6,7, Giuseppe Russo1, Roberto B. B. Santos10, Onoufrios Sgouros1, Selçuk O. Solakci5, George Souliotis18, Vasileios Soukeras1,2, Alessandro Spatafora1,2, Domenico Torresi1, Salvatore Tudisco1, Aydin Yildirin5, Vinicius A. B. Zagatto13 https://doi.org/10.1051/epjconf/202125204001 https://doi.org/10.1051/epjconf/202125204001 EPJ Web of Conferences 252, 04001 (2021) HINPw6 © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). * Corresponding author: cappuzzello@lns.infn.it DP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 ses/by/4 0/) 3 The experiments A crucial feature is the implementation of a technique of trajectory reconstruction, based on differential algebraic techniques and the accurate mapping of the spectrometer magnetic fields [29] [30] [31] [32], which solves the equation of motion of each detected particle to 10th order [33]. This is a unique characteristic of MAGNEX, which guarantees the above mentioned performances and its relevance in the worldwide scenario of heavy-ion physics. The possible connections of DCE measured cross- sections with double beta decay NMEs and, consequently, with the still unsolved neutrino puzzles are among the goals of the present studies. 3 The experiments The NUMEN experiments are performed at INFN-LNS using the K800 Superconducting Cyclotron to accelerate beams and the MAGNEX large acceptance magnetic spectrometer for the detection of the ejectiles. The NUMEN experiments are performed at INFN-LNS using the K800 Superconducting Cyclotron to accelerate beams and the MAGNEX large acceptance magnetic spectrometer for the detection of the ejectiles. MAGNEX is a large acceptance magnetic device consisting of a large aperture vertically focusing quadrupole and a horizontally bending dipole magnet. A hybrid Focal Plane detector (FPD) featuring a gaseous tracker and a wall of silicon pad sensors is mounted at the focal plane to detect the reaction ejectiles [25] [26]. MAGNEX was designed to investigate processes characterized by very low yields, guaranteeing the identification of heavy ions with quite high mass (ΔA/A∼1/160), angle (Δθ∼0.2°) and energy resolutions (ΔE/E∼1/1000), within a large solid angle (Ω∼50 msr) and momentum range (−14% < Δp/p < +10%). High- resolution measurements for quasi-elastic processes, characterized by differential cross-sections as low as tens of nb/sr, were already performed with this setup [27] [28]. A crucial feature is the implementation of a technique of trajectory reconstruction, based on differential algebraic techniques and the accurate mapping of the spectrometer magnetic fields [29] [30] [31] [32], which solves the equation of motion of each detected particle to 10th order [33]. This is a unique characteristic of MAGNEX, which guarantees the above mentioned performances and its relevance in the worldwide scenario of heavy-ion physics. MAGNEX is a large acceptance magnetic device consisting of a large aperture vertically focusing quadrupole and a horizontally bending dipole magnet. A hybrid Focal Plane detector (FPD) featuring a gaseous tracker and a wall of silicon pad sensors is mounted at the focal plane to detect the reaction ejectiles [25] [26]. MAGNEX was designed to investigate processes characterized by very low yields, guaranteeing the identification of heavy ions with quite high mass (ΔA/A∼1/160), angle (Δθ∼0.2°) and energy resolutions (ΔE/E∼1/1000), within a large solid angle (Ω∼50 msr) and momentum range (−14% < Δp/p < +10%). High- resolution measurements for quasi-elastic processes, characterized by differential cross-sections as low as tens of nb/sr, were already performed with this setup [27] [28]. 2 The motivation Recently, accurate cross sections for heavy-ion DCE reactions have been measured at the INFN-LNS laboratory, thanks to the use of the MAGNEX spectrometer. Neutrinoless double beta decay (0νββ) is an hypothetical process in which a “parent” nucleus is spontaneously transformed into a “daughter” isobar by the conversion of two neutron/protons into two protons/neutrons. The process is accompanied by the emission of two electrons (or positrons), which guarantee also electric charge conservation. If detected, 0νββ would provide evidence for the Majorana nature of the neutrino (i.e. the equivalence of neutrino and antineutrino) and give access to the neutrino mass [13] [14] [15] [16]. Under the hypothesis that the neutrino, electrons and nuclear degrees of freedom can be separated in 0νββ, the decay rate explicitly depends on the nuclear transition probability, accounted for in the so-called Nuclear Matrix Element (NME). The 0νββ NMEs are very sensitive to nuclear physics involved in decay, since they probe the nuclear wave functions of the initial and final states. In particular, the nuclear response to high-momentum second-order isospin operators is probed in 0νββ, similarly to what happen in DCE reactions [17]. However, there are no experimental methods to directly measure 0νββ NMEs and accurate theoretical calculations are challenging, leading to discrepancy factors larger than two, as presently reported in literature [13]. Thus experimental inputs from SCE and DCE studies are useful to help evaluate the 0νββ NMEs and to constrain the calculations. In this context, an experimental campaign has started at the INFN-Laboratori Nazionali del Sud in Catania using the MAGNEX large acceptance magnetic spectrometer [18]. It focuses on the study of heavy-ion induced DCE reactions and competing reaction channels involving the nuclei of interest for 0νββ decay. The accurate description of heavy-ion induced DCE is a demanding task also for nuclear theory. From the reaction theory side initial and final state interactions of projectile and target systems need to be under control and the reaction amplitude must be described properly. In this context direct reaction theory can provide the suitable toolbox. Nuclear structure theory is asked to provide accurate input for modelling both projectile and target [20]. Recently, new theoretical developments for the reaction mechanism involved in heavy ion induced reactions have been achieved alongside. Presently this activity is in progress within the NUMEN project [21] [22] [23] [24]. * Corresponding author: cappuzzello@lns.infn.it © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1051/epjconf/202125204001 https://doi.org/10.1051/epjconf/202125204001 EPJ Web of Conferences 252, 04001 (2021) HINPw6 EPJ Web of Conferences 252, 04001 (2021) transfer range. In addition, the rather tiny DCE cross sections (down to few nb) demand for high experimental sensitivity, which strongly depends on the rejection capability against unwanted spurious events. These latter may be generated by competing reaction processes, some of which are more likely to occur than DCE. For this reason highly selective particle identification is a necessary prerequisite for these experiments. In addition, spurious data associated to a wrong determination of detection parameters could casually be misinterpreted as valid DCE data. Characterizing the relevant background sources can be subtle and require stringent conditions for the experimental set-up as well as a careful study of the detection response [19]. reactions in a systematic approach. The main goal is to extract information on nuclear matrix elements for SCE and DCE processes and to identify the possible connections with double beta decay. Detailed information about these projects are found in recent articles, which we refer to [10] [11] [12] for both scientific and technological aspects. In the present paper, the global strategy and methodologies of the above mentioned programmes, the challenges of the experimental activities together with the status of the already performed experimental runs are briefly outlined. reactions in a systematic approach. The main goal is to extract information on nuclear matrix elements for SCE and DCE processes and to identify the possible connections with double beta decay. Detailed information about these projects are found in recent articles, which we refer to [10] [11] [12] for both scientific and technological aspects. In the present paper, the global strategy and methodologies of the above mentioned programmes, the challenges of the experimental activities together with the status of the already performed experimental runs are briefly outlined. 2 Heavy-ion induced double charge exchange reactions All of these information are provided in a consistent experimental framework and analysed in a multi-channel approach, based on quantum scattering theory of coupled channels. A unique opportunity is thus searched by NUMEN to build an over-constrained set of nuclear structure information, promoting the development of a new approach to 0νββ NMEs calculations. • (20Ne,18O) two-proton stripping reaction ( ) p pp g • (20Ne,19F) one-proton stripping reaction • (20Ne 22Ne) two-neutron pickup reaction • (20Ne,19F) one-proton stripping reaction • (20Ne,22Ne) two-neutron pickup reaction • (20Ne,21Ne) one-neutron pickup reaction. For these experiments, the incident beam (20Ne10+) has a lower magnetic rigidity compared to to the ejectiles of interest. Thus, for a fixed magnetic field setting, the beam is more bent than the ejectiles of interest. The spectrometer optical axis is typically placed at −3°, thus the covered angular range is −8° < θlab < +3°. The quadrupole and dipole magnetic fields are set in order that the 20Ne10+ beam reaches the low-Bρ region besides the FPD. The 20Ne9+ and 20Ne8+ components of the beam emerging for the target could be a source of background in these experiments. They are significantly reduced by the use of a supplementary stripper foil mounted after the target itself [38]. Investigations of the two classes of experiments have been started in the recent years with the present facility. These studies highlighted the strengths and the limiting aspects of the adopted technique, providing first valuable information on relevant nuclear structure aspects for a number of double beta decay emitter candidates [8]. This activity has also given precious information to guide the major upgrade of the facility toward much higher beam intensity, which is presently going on. The systems already experimentally explored using the (20Ne,20O) reaction at 15 AMeV are the 116Cd target (to study the 116Cd → 116Sn transition), the 130Te (for the 130Te → 130Xe) and the 76Ge (for the 76Ge → 76Se). The data reduction and analysis is in progress and the first results from some reaction channels have been recently published [39] [40] [41] [42]. 2 Heavy-ion induced double charge exchange reactions In this way, we can get supplementary information from the detailed study of the reaction network, profiting of the strong selectivity of quasi-elastic processes. The nucleus-nucleus interaction can be extracted from elastic scattering data and applied to the evaluation of the distorted waves in each direct reaction channel. Inelastic scattering data give selective access to the role of the deformation in the coupled channel reaction network equations. Single nucleon transfer reactions shed light on the role of single particle configurations in the involved nuclear many body states. Two nucleon transfer reactions probe pairing correlations, while SCE give access to 1particle-1hole excitations. All of these information are provided in a consistent experimental framework and analysed in a multi-channel approach, based on quantum scattering theory of coupled channels. A unique opportunity is thus searched by NUMEN to build an over-constrained set of nuclear structure information, promoting the development of a new approach to 0νββ NMEs calculations. In each case, the experiments are organized in order to explore together with the DCE reactions of interest, all the relevant quasi-elastic processes occurring in the projectile-target collision. In this way, we can get supplementary information from the detailed study of the reaction network, profiting of the strong selectivity of quasi-elastic processes. The nucleus-nucleus interaction can be extracted from elastic scattering data and applied to the evaluation of the distorted waves in each direct reaction channel. Inelastic scattering data give selective access to the role of the deformation in the coupled channel reaction network equations. Single nucleon transfer reactions shed light on the role of single particle configurations in the involved nuclear many body states. 3.2. Experiments with 20Ne beam (β−β−direction) In the class of experiments triggered by 20Ne10+ beams, the reaction channels we are interested are the following: • (20Ne,20Ne) elastic and inelastic scattering • (20Ne,20Ne) elastic and inelastic scattering • (20Ne,20O) DCE reaction • (20Ne,20F) SCE reaction Two nucleon transfer reactions probe pairing correlations, while SCE give access to 1particle-1hole excitations. All of these information are provided in a consistent experimental framework and analysed in a multi-channel approach, based on quantum scattering theory of coupled channels. A unique opportunity is thus searched by NUMEN to build an over-constrained set of nuclear structure information, promoting the development of a new approach to 0νββ NMEs calculations. Two nucleon transfer reactions probe pairing correlations, while SCE give access to 1particle-1hole excitations. 2 Heavy-ion induced double charge exchange reactions Challenging experimental issues must be addressed to measure heavy-ion induced DCE cross sections. Indeed one needs to detect heavy ions with good isotopic separation and energy resolution in a wide angular range, including zero-degree, in order to distinguish transitions to individual states and explore a wide momentum 2 https://doi.org/10.1051/epjconf/202125204001 EPJ Web of Conferences 252, 04001 (2021) HINPw6 EPJ Web of Conferences 252, 04001 (2021) 6 θlab < 9° is explored. The MAGNEX quadrupole and dipole magnetic fields are set in order that the incident beam, after passing through the magnets, reaches a region besides the FPD. For this class of experiments, in fact, the incident ion beam (18O8+) has higher magnetic rigidity (Bρ) than the ejectiles of interest (namely fully stripped 18Ne, 18F, 20Ne, 19F, 16O, 17O ions). The beam stops in a Faraday cup, placed in the high-Bρ region besides the FPD, which measures the collected charge for each run. The experimental activity with accelerated beams proposed and presently in progress consists of two main classes of experiments, aiming at the exploration of the two directions of isospin lowering τ− τ− and rising τ+ τ+, characteristic of β−β− and β+β+ decays, respectively. In particular, the β+β+ direction in the target excitation modes is investigated using a 18O8+ beam and measuring the (18O,18Ne) DCE transitions, together with other reaction channels involving same beam and target. Similarly, the β−β− direction is explored via the (20Ne,20O) reaction, using a 20Ne10+ beam and detecting the reaction products from the DCE channel and from the other open reaction channels. 116Sn, 76Se and 48Ti are the targets of interest for 0νββ, which have already been explored by NUMEN, via (18O,18Ne) reaction at 15 and 22 AMeV. The purpose was to study the 116Sn → 116Cd, 76Se → 76Ge and 48Ti → 48Ca transitions and the competing channels as shown in Fig. 2. A dedicated run was also performed on 12C target, in order to compare reaction models with an easier light system. The reduction and analysis of the collected data from these campaigns is presently in progress. First results have been already published in ref. [34] [35] [36] [37]. reaction channels. In each case, the experiments are organized in order to explore together with the DCE reactions of interest, all the relevant quasi-elastic processes occurring in the projectile-target collision. Acknowledgments This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No 714625). This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No 714625). References [1] Alford, W.P.; Spicer, B.M. Nucleon charge-exchange reactions at intermediate energy. Adv. Nucl. Phys. 1998, 1, 24. Fig. 2 Scheme of the reaction channels already studied within the NURE and NUMEN experiments. [2] Osterfeld, F. Nuclear spin and isospin excitations. Rev. Mod. Phys. 1992, 64, 491.. [3] Taddeucci, T.N.; et al. The (p, n) reaction as a probe of beta decay strength. Nucl. Phys. A 1987, 469, 125.. [4] Lenske, H.; Cappuzzello, F.; Cavallaro, M.; Colonna, M. Heavy ion charge exchange reactions as probes for nuclear β-decay. Prog. Part. Nucl. Phys. 2019, 109, 103716.. [5] F. Cappuzzello et al., Eur. Phys. J. A 51, 145 (2015). [6] Cappuzzello, F.; Cavallaro, M.; Agodi, C.; Bondì, M.; Carbone, D.; Cunsolo, A.;, Foti, A. Heavy-ion double charge exchange reactions: A tool toward 0νββ nuclear matrix elements. Eur. Phys. J. A 2015, 51, 145. [7] Cappuzzello, F., and Agodi, C. (2021). The NUMEN Project: Shedding Light on Neutrinoless Double Beta Decay by Heavy-Ion Nuclear Reactions. Nucl, Phys. News, in press, DOI 10.1080/10619127.2021.1881368. [8] Cappuzzello, F., Agodi, C., Cavallaro, M., Carbone, D., Tudisco, S., Lo Presti, D., et al.. (2018). The NUMEN project: NUclear Matrix Elements. Eur. Phys. J. A. 54, 72. [9] Cavallaro, M., Aciksoz, E., Acosta, L., Agodi, C., Auerbach, N., Bellone, J. I., et al., (2017). “NURE: an ERC project to study nuclear reactions,” in Proceedings, 55th International Winter Meeting on Nuclear Physics Bormio, (Bormio). doi: 10.22323/1.302.. [10] P. Finocchiaro et al., Universe 2020, 6, 129; doi:10.3390/universe6090129. [11] C. Agodi et al., Universe 2021, 7, 72. https://doi.org/10.3390/universe7030072. [12] F. Cappuzzello et al., The NUMEN Project: An Update of the Experimental Campaigns. Front. Astron. Space Sci. 8:668587. doi: 10.3389/fspas.2021.668587. [13] H. Ejiri, J. Suhonen, K. Zuber, Physics Reports 797 (2019) 1. [14] J. Engel, J. Menéndez, Rep. Progr. Phys. 60 (2017) 046301. [15] S. Dell’Oro, S. Marcocci, and F. Vissani, Phys. Rev. D 90, 033005 (2014). [16] J. Vergados, H. Ejiri, F. Šimkovic, Rep. Progr. Phys. 75 (2012) 106301. Fig. 2 Scheme of the reaction channels already studied within the NURE and NUMEN experiments. [17] Cappuzzello, F., and Cavallaro, M. (2020). Nuclear Response to Second-Order Isospin Probes in Connection to Double Beta Decay. Universe. 6, 217. 3.1. Experiments with 18O beam study the Cd → Sn transition), the Te (for the Te → 130Xe) and the 76Ge (for the 76Ge → 76Se). The data reduction and analysis is in progress and the first results from some reaction channels have been recently published [39] [40] [41] [42]. For the experiments of this class, the reaction channels of our interest are listed below: For the experiments of this class, the reaction channels of our interest are listed below: • (18O,18O) elastic and inelastic scattering 18 18 • (18O,18Ne) DCE reaction A scheme of the transitions already studied in the experimental runs is shown in Fig. 2. • (18O,18F) SCE reaction • (18O,20Ne) two-proton pickup reaction 18 19 • (18O,19F) one-proton pickup reaction • (18O,16O) two-neutron stripping reaction • (18O,17O) one-neutron stripping reaction One of the main challenges of such experiments is the measurement at very forward angles, including zero- degree. This is performed by placing the spectrometer with its optical axis at +3° with respect to the beam axis. Thanks to its large angular acceptance, the range −2° < One of the main challenges of such experiments is the measurement at very forward angles, including zero- degree. This is performed by placing the spectrometer with its optical axis at +3° with respect to the beam axis. Thanks to its large angular acceptance, the range −2° < 3 https://doi.org/10.1051/epjconf/202125204001 EPJ Web of Conferences 252, 04001 (2021) HINPw6 EPJ Web of Conferences 252, 04001 (2021) ig. 2 Scheme of the reaction channels already within the NURE and NUMEN experiments. Conclusions [18] F. Cappuzzello et al. The MAGNEX spectrometer: Results and perspectives. Eur. Phys. J. A (2015) 51:145. A systematic study of heavy-ion induced DCE reactions and of the other reaction channels characterized by the same projectile and target is in progress at INFN-LNS. The main goal is to investigate the nuclear response to DCE reactions for all the isotopes candidate for 0νββ decay towards the determination of 0νββ NMEs. [19] S. Calabrese et al. Analysis of the background on cross section measurements with the MAGNEX spectrometer: The (20Ne,20O) Double Charge Exchange case. Nuclear Inst. and Methods in Physics Research, A 980 (2020) 164500. 4 4 EPJ Web of Conferences 252, 04001 (2021) HINPw6 https://doi.org/10.1051/epjconf/202125204001 EPJ Web of Conferences 252, 04001 (2021) HINP 6 [20] N. Shimizu, J. Menendez, and K. Yako, Phys. Rev. Lett. 120, 142502 (2018).. [21] H. Lenske, J.I. Bellone, M. Colonna, J. A. Lay, Phys. Rev. C 98 (4), 044620 (2018).. [22] E. Santopinto, H. García-Tecocoatzi, RIM Vsevolodovna, J. Ferretti, Phys. Rev. C 98 (6), 061601 (2018). [22] E. Santopinto, H. García-Tecocoatzi, RIM [23] J.I. Bellone, S. Burrello, M. Colonna, J. A. Lay, H. Lenske, Phys. Lett. B 807, 135528 (2020). [24] Lenske, H.; Bellone, J.; Colonna, M.; Gambacurta, D. Nuclear Matrix Elements for Heavy Ion Sequential Double Charge Exchange Reactions. Universe 2021, 7, 98. https://doi.org/10.3390/universe7040098. [25] Torresi, D., Sgouros, O., Soukeras, V., Cavallaro, M., Cappuzzello, F., Carbone, D., et al. (2021). An upgraded focal plane detector for the MAGNEX spectrometer. Nucl. Instr. and Meth. A. 989, 164918. [26] M. Cavallaro et al. The low-pressure focal plane detector of the MAGNEX. Eur. Phys. J. A (2012) 48: 59. DOI 10.1140/epja/i2012-12059-8. [27] J.R.B. Oliveira et al., Journal of Physics G: Nuclear and Particle Physics, 40 (2013) 105101. [28] D. Pereira et al. Phys. Lett. B 710 (2012) 426. [29] A. Lazzaro et al., Nucl. Instr. and Methods A 591, 394 (2008).. [30] A. Lazzaro et al., Nucl. Instr. and Methods A 585, 136 (2008). [31] A. Lazzaro et al., Nucl. Instr. and Methods A 570, 192 (2007). [32] A. Lazzaro et al., Nucl. Instr. and Methods A 602, 494 (2009). [33] F. Cappuzzello et al., Eur. Phys. J. A 52 (2016) 169. [34] O. Sgouros et al., Phys. Rev. C, accepted. [35] D. Carbone et al., Universe 7 (2021) 58.. [36] J. L. Ferreira et al., Phys. Rev. C 103 (2021) 054604. [37] M. Conclusions Cavallaro et al., Frontiers in Astron. and Space Science 8 (2021) 659815. [38] M. Cavallaro et al., Results in Phys. 13 (2019) 102191. [39] A. Spatafora et al., Phys. Rev. C 100 (2019) 034620. [40] V. Soukeras et al., Results in Physics, (2021) submitted. [41] S. Calabrese et al., Nucl. Instr. and Meth. A, 980 (2020) 164500. [42] S. Calabrese et al., Acta Phys. Pol. B 49 (2018) 275. 5 5
https://openalex.org/W2561924782
https://scindeks-clanci.ceon.rs/data/pdf/0354-3471/2016/0354-34711602129V.pdf
Croatian
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Evaluation of brand from the perspective of consumers
Marketing
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cc-by-sa
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UVOD Globalizacija i kapitalizam kao osnovne odlike savremenog poslovnog okruženja, podigle su pojam konkurencije na jedan potpuno novi nivo. Nadmetanje na globalnom tržištu postaje sve intenzivnije i složenije. Kako bi opstale i napredovale u ovakvim uslovima, kompanije teže ka maksimalnom iskorišćavanju sopstvenih potencijala, razvijanju kom- parativnih prednosti i diferenciranju u odnosu na konkurente. Upravo brend, postaje sve značajniji instrument diferenciranja i sticanja konku- rentske prednosti kako u proizvodnom, tako i u uslužnom sektoru. Prvi korak koji kompanija mora načiniti na putu ka izgradnji snažnog brenda je identifikovanje jedinstvenih prednosti proizvoda/usluga na kojima će bazirati brend. Međutim, brendiranje kao proces, ne treba precenjivati. Napor ulo- žen u brendiranje, sam po sebi, ne može prikriti nedostatke proizvoda. S tim u vezi, uspešan proces brendiranja treba da teče u smeru: proizvod – brend. Drugim rečima, brendiranje treba posmatrati kao završni mo- menat. U domaćoj literaturi, autori su se bavili merenjem vrednosti brenda, ali isključivo sa teorijskog stanovišta u smislu objašnjavanja modela za merenje performansi. Cilj ovog rada je identifikovanje i analiza fakto- ra koji imaju uticaj na preferenciju brenda. Na ovaj način, sprovedeno empirijsko istraživanje pruža doprinos postojećoj literaturi. Pored toga, nalazi ovog istraživanja mogu poslužiti kompanijama kao koristan alat za upravljanje brendom, te alat za unapređenje efikasnosti marketing strate- gije i konkurentske pozicije preduzeća. Ključne reči:  dimenzije vrednosti brenda, merenje vrednosti brenda, prefe- rencija brenda Originalni naučni rad, UDK 003.65:339.138 Originalni naučni rad, UDK 003.65:339.138 Vrednovanje brenda iz perspektive potrošača Marija Vranješ, Dragoljub Jovičić, Dragana Drinić Marija Vranješ, Dragoljub Jovičić, Dragana Drinić JEL klasifikacija: M31, D12 JEL klasifikacija: M31, D12 JEL klasifikacija: M31, D12 Apstrakt:  Brend je izuzetno važna intelektualna svojina kompanije. Kako bi privukle nove potrošače i zadržale što više postojećih, kompanije moraju neprestano da rade na unapređenju vrednosti brenda, što u krajnjoj liniji dovodi do rasta tržišnog učešća, prihoda i profita. Cilj rada je da istraži na koji način određene dimenzije vrednosti brenda utiču na preferenciju brenda. U skladu sa navedenim ciljem, korišćene su mere deskriptivne statistike, analiza pouzdanosti, korelaciona i regresi- ona analiza. Rezultati istraživanja ukazuju da dve dimenzije vrednosti brenda: me- đuljudski odnosi i komparativna prednost brenda, imaju statistički značajan uticaj na povećanje nivoa preferencije. S druge strane, razumevanje i istorija, kao dimen- zije vrednosti brenda, nemaju statistički značajan uticaj. Nalazi sprovedene studije od koristi su menadžmentu kompanija prilikom formiranja odgovarajućih okvira za upravljanje brendom u cilju povećanja preferencije brenda, što bi u krajnjoj liniji trebalo da dovede do unapređenja konku- rentske pozicije kompanije. Ključne reči:  dimenzije vrednosti brenda, merenje vrednosti brenda, prefe- rencija brenda 1.1 Brend Savremeno poslovno okruženje karakteriše sve jači intenzitet konkuren- cije. U takvim uslovima, kompanije nastoje da se što više diferenciraju u odnosu na konkurente. S tim u vezi, kompanije mogu diferencirati svoj proizvod, fokusirajući se na bilo koje materijalno svojstvo proizvo- 129 da (npr. ukus, dizajn) ili nematerijalno svojstvo (npr. cena, brend, zemlja porekla) (Zeugner-Roth, Dia- mantopoulos & Montesinos, 2008). Upravo brend po- staje sve značajniji instrument diferenciranja i sticanja konkurentske prednosti. Postojeća literatura obiluje definicijama ovog pojma. Prema definiciji američ- kog udruženja za marketing, brend predstavlja „ime, pojam, znak, simbol, dizajn, ili pak, kombinacija na- vedenog, čime se identifikuje roba ili usluge jednog prodavca/grupe prodavaca i diferencira u odnosu na konkurentske“ (Kotler & Keller, 2006, p. 274). Vrednost brenda, moguće je posmatrati iz dve per- spektive: potrošača i kompanije. Ove dve perspektive su povezane na način da finansijska vrednost brenda počiva na vrednosti brenda za potrošače. Upravo la- nac vrednosti brenda, na odgovarajući način pred- stavlja navedeni odnos. Naime, proces kreiranja bren- da započinje investiranjem kompanije u marketing program čiji je osnovni cilj da utiče na stav kupaca o brendu. Stav velikog broja kupaca dalje utiče na tr- žišni učinak, koji definiše vrednost brenda na tržištu (Kotler & Keller, 2006). U daljem radu biće razmatra- na vrednost brenda iz perspektive potrošača. Jedna od osnovnih uloga brenda je da potrošačima stvori sliku o vrednosti proizvoda, pa je brend moguće posmatrati i kao izvestan „sertifikat o kvalitetu proi- zvoda“ (Keegan & Green, 2008, p. 328). S tim u vezi, „brendovi omogućavaju potrošačima da skrate vreme potrebno za izbor pravih alternativa dok istovreme- no predstavljaju garanciju da će proizvodi i usluge zadovoljiti njihova očekivanja“ (Veljković i Đorđević, 2010, p. 10). p p p „Brend treba da omogući funkcionalne, simbo- ličke i iskustvene koristi potrošaču“ (Helgeson & Supphellen, 2004, p. 205). To praktično znači da brend, s jedne strane, treba da omogući rešavanje problema potrošača, dok s druge strane, treba da pruži i određe- ne simboličke koristi, odnosno odgovarajuće značenje za potrošača. Kada se govori o simboličkim koristima, veoma je važno da postoji podudarnost ličnosti brenda i ličnosti potrošača. Ova podudarnost definiše se kao veza ovih koncepata i pokazuje koliko se potrošačeva predstava o sebi poklapa sa imidžom tipičnog korisni- ka brenda (Escalas & Bettman, 2005). Na kraju, brend treba da omogući i iskustvene koristi, koje se vezuju za čulna zadovoljstvo ili kognitivna uzbuđenja. 1.1 Brend S obzirom na navedene koristi, kompanije sve više pažnje posvećuju dizajniranju brenda. „Brend dizaj- niran prema zahtevima ciljnog tržišta mora imati sle- deće atribute: jedinstvenost, posebnost, originalnost i kompatibilnost sa atributima proizvoda“ (Kocić, Šapić i Kijevčanin, 2013, p. 220). Zavisno od toga kako je brend dizajniran, reakcije potrošača mogu da se kre- ću od veoma niskog nivoa na kom postoji samo svest o brendu ili upoznatost, pa sve do visokog nivoa koji podrazumeva lojalnost brendu. Međutim, snagu bren- da ne treba precenjivati. „Ključ uspeha kompanije je, pre svega, u izvrsnom proizvodu, a zatim u njegovom brendu“ (Keller, 2011, p. 38). To praktično znači da „potrošač neće biti zainteresovan za kupovinu proi- zvoda ili usluga u slučaju kada su fizičke manjkavosti prikrivene snažnom promotivnom kampanjom“ (Ko- cić i Senić, 2010, p. 90). Pored percepcija, koje direktno utiču na vrednost brenda, veoma je važno razmotriti i ponašanja potro- šača koja počivaju na percepcijama i tako indirektno definišu vrednost brenda. Upravo šestodimenzionalni model (Lehmann, Keller & Farley, 2008) ukazuje na povezanost dimenzija vrednosti brenda, te predstavlja jedan od indirektnih metoda za određivanje vrednosti brenda za potrošače. Model je ustanovljen na osnovu 3 izvora (Lehmann et al., 2008): 1. istraživanja koja su sprovodili pomenuti autori i Fournier (1998) Jednom ustanovljen brend potrebno je kontinui- rano pratiti i usavršavati. „Uspešnost brenda upravo zavisi od sposobnosti kompanije da održava njegovu vrednost, istovremeno uzimajući u obzir i aktivnosti konkurenata“ (Kocić et al., 2013, p. 220). 2. istraživanja koje je sproveo Ambler (2003), i 3. metodologije BAV, BrandZ i Research International’s Equity Engine Na osnovu navedenih izvora autori su došli do 27 stavki vezanih za vrednost brenda, koje su, nakon analize međuzavisnosti faktora, svrstali u šest grupa: razumevanje, komparativna prednost marke, među- ljudski odnosi, istorija, preferencija i vezanost potro- šača za marku. 1.3. Preferencija brenda Pored podataka o ispitanicima, upitnik je obuhva- tio 5 grupa pitanja, sa ukupno 27 konstatacija koje odražavaju različite dimenzije vrednosti brenda. Kon- statacije su odabrane na osnovu pregleda relevantne literature, pri čemu je svaka varijabla modela merena putem minimum 4 konstatacije. Teorijski osnov za iz- bor konstatacija temelji se na modelu za određivanje vrednosti brenda za potrošače, koji su kreirali Leh- mann et al., (2008). Sve konstatacije uvršćene u upit- nik izabrane su iz pomenutog modela i modifikovane od strane autora za potrebe sprovođenja ovog istaživa- nja. Ispitanici su stepen slaganja sa navedenim konsta- tacijama iskazivali na petostepenoj Likertovoj skali (1 – potpuno nesaglasan/a, 2 – delimično nesaglasan/a, 3 – neutralan/a, 4 – delimično saglasan/a i 5 – potpuno saglasan/a). Preferenciju brenda moguće je definisati na više nači- na. S tim u vezi, ovaj pojam može se posmatrati kao mera u kojoj potrošači favorizuju određeni brend u odnosu na konkurente, koji se takođe razmatraju (Hellier, Geursen, Carr & Rickard, 2003). Drugi auto- ri, preferenciju brenda vezuju za pristrasnost potroša- ča i njegovu nameru da kupi određeni brend (Cobb- Walgren, Ruble & Donthu, 1995). Osnovni preduslov stvaranja preferencija i naklonosti potrošača ka bren- du, upravo je izgradnja imovine brenda (Myers, 2003). Pojedini autori prave jasnu razliku između stavova i preferencija (Franzen & Bouwman, 2001), dok neki drugi stavljaju znak jednakosti između ova dva poj- ma, smatrajući da pozitivni stavovi doprinose jačanju preferencija (Jun, Cho & Kwon, 2008). Analiza prikupljenih podataka, izvršena je pomo- ću statističkog paketa SPSS (The Statistical Package for the Social Sciences). Od statističkih metoda analize podataka, primenjene su: deskriptivna statistička ana- liza, analiza vrednosti koeficijenta Cronbach’s alpha, korelacija, te prosta i višestruka regresiona analiza. U prvom koraku je primenjena deskriptivna statistička analiza na celom uzorku, i izračunata je aritmetička sredina i standardna devijacija za svaku konstataci- ju. Na taj način, mereni su stavovi potrošača vezani za pojedine dimenzije vrednosti brenda i utvrđena je Rasprostranjenost, poverenje i diferenciranost su osnovne snage brenda (Predović, 2007). „Snažan brend može da poboljša preferencije potrošača i sklo- nost ka određenom proizvodu, pa čak i hedonistič- ko iskustvo sa proizvodom i to van njegovih fizičkih svojstava“ (Ariely & Norton, 2009; Ariely & Berns, 2010). Upravo iz tih razloga, izgradnja jakog brenda višestruko je korisna za kompaniju. 1.3. Preferencija brenda 1.2 Vrednost brenda Jedna od najpoznatijih definicija vrednosti brenda je- ste Akerova definicija. Prema ovoj definiciji, vrednost brenda predstavlja „set sredstava i obaveza vezanih za marku, njeno ime i simbol, koji povećavaju ili uma- njuju vrednost omogućenu proizvodom ili uslugom“ (Christodoulides & Chernatony, 2010, p. 45, 46). Poznavanje osnovnih dimenzija koje čine vrednost brenda, kao i načina na koji one utiču na formiranje preferencija potrošača te donošenje odluke o kupovi- ni, predstavalja prvi korak ka uspešnom upravljanju ovom vrednom imovinom. 130      Marija Vranješ, Dragoljub Jovičić, Dragana Drinić Vrednovanje brenda iz perspektive potrošača      131 2. METODOLOGIJA ISTRAŽIVANJA Empirijsko istraživanje uticaja dimenzija vrednosti brenda na preferenciju brenda od strane potrošača, sprovedeno je metodom ankete. U pitanju je jedan od najčešće korišćenih metoda u terenskim marke- tinškim istraživanjima. U istraživanje je uključeno 140 ispitanika. Podaci su prikupljeni u periodu 02.10. - 20.10.2015. godine. Upitnik je koncipiran u on-li- ne formi i distribuiran ispitanicima putem društvene mreže Facebook. Tabela 1. Struktura uzorka (n=140) Demografski profil Broj ispitanika (n) Procenat (%) Pol   Muškarci 61 43,6   Žene 79 56,4 Starost   ≤ 25 godina 49 35,0   26 – 35 godina 74 52,9   36 – 45 godina 11 7,9   46 – 65 godina 6 4,3 Stepen stručne spreme   Niža stručna sprema 2 1,4   srednja stručna sprema 50 35,7   viša stručna sprema 9 6,4   visoka stručna sprema 79 56,4 Region   Vojvodina 110 78,6   Beograd 12 8,6   Šumadija i Zapadna Srbija 6 4,3   Južna i Istočna Srbija 12 8,6 Tabela 1. Struktura uzorka (n=140) Kako bi se ispitanicima olakšalo odgovaranje na postavljena pitanja i dobili što precizniji odgovori, istraživanje je vršeno na određenoj kategoriji proi- zvoda – zubne paste. Od ispitanika je traženo da se opredele za brend zubne paste koji najviše kupuju tj. preferiraju. Ponuđeni su sledeći odgovori: Colga- te (Colgate Palmolive), Kolynos (Colgate Palmolive), Blenda-med (Procter&Gamble), Signal (Unilever), Aqua Fresh (GlaxoSmithKline), Vademecum (Henkel – Merima), Denta fresh (Henkel – Merima), ostalo. Na ovaj način, izvršeno je istraživanje bazirano na percepcijama potrošača vezanim za tačno određen brend zubne paste koju preferiraju. Napred navedene konstatacije, mogu se grupisati u 5 faktora odnosno varijabli. Prvi faktor „Razumeva- nje“ obuhvata sledeće konstatacije: homogenost stavova potrošača. U narednom koraku, pouzdanost dimenzija vrednosti brenda i unutrašnja konzistentnost konstatacija, mereni su na osnovu vrednosti koeficijenta alfa (Cronbach’s alpha). Na- kon toga, sprovedena je korelaciona analiza kojom je utvrđen stepen zavisnosti između varijabli u modelu. Na kraju, ispitivanje pojedinačnog uticaja dimenzija vrednosti brenda tj. nezavisnih promenljivih, na pre- ferenciju potrošača, kao zavisnu promenljivu, utvrđe- no je prostom regresionom analizom. − Brend „X“ je veoma prisutan u kanalima marke- tinga i lako ga je pronaći. − O brendu „X“, prisutne su i dostupne brojne infor- macije iz promocije i drugih izvora. − Brend „X“ je jedna od prvih asocijacija na paste za zube. − Do detalja sam upoznat sa karakteristikama i načinom upotrebe brenda X“ Istraživanjem je obuhvaćena i višestruka regresi- ona analiza putem koje je utvrđena statistička zna- čajnost zajedničkog uticaja sve četiri identifikovane komponente vrednosti brenda, na nivo preferencije brenda. 2. METODOLOGIJA ISTRAŽIVANJA Na osnovu vrednosti VIF-a (Variance inflati- on factor) ispitan je problem multikolinearnosti. načinom upotrebe brenda „X“. − Koristim brend „X“. Drugi faktor „Komparativna prednost brenda“, uk- ljučuje sledeće konstatacije: Drugi faktor „Komparativna prednost brenda“, uk- ljučuje sledeće konstatacije: − Brend „X“ se izdvaja od konkurencije. j Na osnovu tabele 1 može se zaključiti da su u uzorku žene (56,4%) zastupljene u većem procentu od anketiranih muškaraca (43,6%). Ako se posmatra struktura ispitanika prema starosti, primećuje se da najmanji broj ispitanika starosti od 46 do 65 godina (4,3%), dok je najviše ispitanika pripadalo grupi oso- ba starih između 26 i 35 godina (52,9%). Analizom uzorka prema stručnoj spremi, dolazi se do zaključka da najveći broj ispitanika ima visoku stručnu spremu (56,4%), dok je najmanje onih sa nižom stručnom spremom (1,4%). Najzad, shodno regionu u kom žive, najveći broj ispitanika bilo je iz Vojvodine (78,6 %), dok je najmanje njih bilo iz Šumadije i Zapadne Srbije (4,3%). Na osnovu tabele 1 može se zaključiti da su u uzorku žene (56,4%) zastupljene u većem procentu od anketiranih muškaraca (43,6%). Ako se posmatra struktura ispitanika prema starosti, primećuje se da najmanji broj ispitanika starosti od 46 do 65 godina (4,3%), dok je najviše ispitanika pripadalo grupi oso- ba starih između 26 i 35 godina (52,9%). Analizom uzorka prema stručnoj spremi, dolazi se do zaključka da najveći broj ispitanika ima visoku stručnu spremu (56,4%), dok je najmanje onih sa nižom stručnom spremom (1,4%). Najzad, shodno regionu u kom žive, najveći broj ispitanika bilo je iz Vojvodine (78,6 %), dok je najmanje njih bilo iz Šumadije i Zapadne Srbije (4,3%). − Imam visoko mišljenje o brendu „X“. − Brend „X“ ima snažnu reputaciju. − Brend „X“ ispunjava moja očekivanja. − Smatram da ne mogu pogrešiti ako izaberem brend „X“. − Brend „X“ je prihvaćen i od strane mojih prijate- lja, rođaka, kolega. Treći faktor „Međuljudski odnosi“, obuhvata slede- će konstatacije: Treći faktor „Međuljudski odnosi“, obuhvata slede- će konstatacije: − Smatram da je brend „X“ posvećen interesu po- trošača. − Smatram da je brend „X“ prestižan. − Smatram da je brend „X“ prestižan. − Ukoliko se javi problem vezan za brend „X“, veru- jem da će ga kompanija brzo rešiti. − Brend „X“ je inovativan i stalno se unapređuje. 132      Marija Vranješ, Dragoljub Jovičić, Dragana Drinić 3. REZULTATI ISTRAŽIVANJA Četvrti faktor „Istorija“ odnosi se na sledeće kon- statacije: Primenom deskriptivne statistike izračunate su vred- nosti aritmetičke sredine i standardne devijacije za 27 definisanih konstatacija. Koristeći ceo uzorak, re- zultati deskriptivne statističke analize ukazuju da is- pitanici generalno visoko vrednuju preferirani brend. Najpovoljniji stav ispitanika je iskazan kod tvrdnje da ispitanik koristi odnosno upotrebljava preferira- ni brend (aritmetička sredina = 4,46), dok je najne- povoljniji stav dobijen u slučaju konstatacije da za preferirani brend ispitanik, odnosno potrošač vezuje radosna sećanja (aritmetička sredina = 2,63). Najveći stepen neslaganja među ispitanicima zabeležen je kod konstatacije vezane za upotrebu preferiranog brenda od strane roditelja ispitanika (standardna devijacija = 1,395), dok se najmanji stepen neslaganja među ispi- tanicima vidi kod konstatacije koja se odnosi na ge- neralno mišljenje o preferiranom brendu (standardna devijacija = 0,771) (Tabela 2). − Brend „X“ ima snažnu istoriju i dugo je prisutan na tržištu. − Brend „X“ su koristili i moji roditelji. − Sećam se brenda „X“ još iz svoje mladosti/detinj- stva. − Za brend „X“ vezujem radosna sećanja. Peti faktor „Preferencija“ sadrži sledeće konstata- cije: Peti faktor „Preferencija“ sadrži sledeće konstata- cije: − Brend „X“ je zaslužio moje poverenje. − Lojalan sam brendu „X“. − Spreman sam da platim višu cenu za brend „X“. − Brend „X“ je nezamenljiv i u slučaju da ga nema na određenom prodajnom mestu, potražiću ga na drugom. 132      Marija Vranješ, Dragoljub Jovičić, Dragana Drinić Tabela 2. Rezultati deskriptivne statističke analize Tabela 2. Rezultati deskriptivne statističke analize Konstatacije M SD Brend „X“ je veoma prisutan u kanalima marketinga i lako ga je pronaći. 4,29 1,027 O brendu „X“, prisutne su i dostupne brojne informacije iz promocije i drugih izvora. 3,75 1,080 Brend „X“ je jedna od prvih asocijacija na paste za zube. 4,09 1,035 Do detalja sam upoznat sa karakteristikama i načinom upotrebe brenda „X“. 3,66 1,044 Koristim brend „X“. 4,46 0,876 Brend „X“ se izdvaja od konkurencije. 3,61 0,941 Imam visoko mišljenje o brendu „X“. 3,81 0,905 Brend „X“ ima snažnu reputaciju. 3,92 0,930 Brend „X“ ispunjava moja očekivanja. 4,01 0,831 Smatram da ne mogu pogrešiti ako izaberem brend „X“. 3,84 0,954 Brend „X“ je prihvaćen i od strane mojih prijatelja, rođaka, kolega. 3,52 0,925 Smatram da je brend „X“ posvećen interesu potrošača. 3,64 0,858 Smatram da je brend „X“ prestižan. 3,34 0,950 Ukoliko se javi problem vezan za brend „X“, verujem da će ga kompanija brzo rešiti. 3. REZULTATI ISTRAŽIVANJA 3,71 0,884 Brend „X“ je inovativan i stalno se unapređuje. 3,88 0,971 Brend „X“ ima snažnu istoriju i dugo je prisutan na tržištu. 3,84 0,954 Brend „X“ su koristili i moji roditelji. 2,94 1,395 Sećam se brenda „X“ još iz svoje mladosti/detinjstva. 2,85 1,330 Za brend „X“ vezujem radosna sećanja. 2,63 1,183 Brend „X“ je zaslužio moje poverenje. 3,76 0,944 Lojalan sam brendu „X“. 3,57 1,074 Spreman sam da platim višu cenu za brend „X“. 3,03 1,269 Brend „X“ je nezamenljiv i u slučaju da ga nema na određenom prodajnom mestu, potražiću ga na drugom. 2,81 1,324 Kada kupujem pastu za zube, brend „X“ je moj prvi izbor. 4,06 0,976 Smatram da kupovinom brenda „X“ dobijam odličnu vrednost za utrošen novac. 3,88 0,869 Spreman sam da kupim i probam i druge proizvode brenda „X“. 3,89 0,968 Generalno, imam pozitivno mišljenje o brendu „X“. 4,16 0,771 M aritmetička sredina; SD standardna devijacija SD M – aritmetička sredina; SD – standardna devijacija Rezultati korelacione analize prikazani su u tabeli 4. Vrednosti Pirsonovog koeficijenta pokazuju stepen zavisnosti između varijabli u modelu.i − Kada kupujem pastu za zube, brend „X“ je moj prvi izbor. p − Smatram da kupovinom brenda „X“ dobijam odličnu vrednost za utrošen novac. Interkorelaciona matrica ukazuje na signifikantne vrednosti Pirsonovog koeficijenta. Između varijabli modela, javlja se statistički značajan stepen korelaci- je. Međutim, ta korelacija nije svuda jaka. Vrednost − Spreman sam da kupim i probam i druge proizvo- de brenda „X“. − Generalno, imam pozitivno mišljenje o brendu „X“. Na osnovu koeficijenta Cronbach’s alpha, utvrđe- na je pouzdanost i interna konzistentnost konstatacija u upitniku. Rezultati su prikazani u tabeli 3. Vred- nosti koeficijenta Cronbach’s alpha kreću se od 0 do 1, pri čemu se smatra da vrednosti veće od 0,7 uka- zuju na adekvatnu pouzdanost i internu konzisten- tnost tvrdnji (Nunnally, 1978). Vrednosti koeficijenta Cronbach’s alpha u istraživanju kreće se u rasponu od 0,774 (razumevanje) do 0,884 (preferencija). Dobije- ne vrednosti ukazuju na adekvatnu pouzdanost i in- ternu konzistentnost varijabli. Vrednovanje brenda iz perspektive potrošača      133 Tabela 3. Pouzdanost varijabli - vrednosti koeficijenta Cronbach’s alpha Varijable Cronbach’s alpha Razumevanje 0,774 Komparativna prednost brenda 0,857 Međuljudski odnosi 0,783 Istorija 0,799 Preferencija 0,884 Tabela 3. Pouzdanost varijabli - vrednosti koeficijenta Cronbach’s alpha Varijable Cronbach’s alpha Razumevanje 0,774 Komparativna prednost brenda 0,857 Međuljudski odnosi 0,783 Istorija 0,799 Preferencija 0,884 Tabela 3. 3. REZULTATI ISTRAŽIVANJA Pouzdanost varijabli - vrednosti koeficijenta Cronbach’s alpha Vrednovanje brenda iz perspektive potrošača      133 Tabela 4. Korelaciona analiza Varijable 1 2 3 4 5 Razumevanje 1 0,655** 0,616** 0,335** 0,583** Komparativna prednost brenda 0,655** 1 0,724** 0,257** 0,805** Međuljudski odnosi 0,616** 0,724** 1 0,277** 0,690** Istorija 0,335** 0,257** 0,277** 1 0,303** Preferencija brenda 0,583** 0,805** 0,690** 0,303** 1 Napomena:  ** Korelacija je značajna na nivou 0,01 Tabela 4. Korelaciona analiza Tabela 5. Prosta regresiona analiza (Zavisna varijabla: Preferencija brenda) Varijable β t Sig. R² F Razumevanje 0,583 8,423 0,000** 0,340 70,954 Komparativna prednost brenda 0,805 15,931 0,000** 0,648 253,802 Međuljudski odnosi 0,690 11,196 0,000** 0,476 125,342 Istorija 0,303 3,735 0,000** 0,092 13,947 Napomena:  p <0.01 (**) Tabela 5. Prosta regresiona analiza (Zavisna varijabla: Preferencija brenda) Pirsonovog koeficijenta preko 0,6 ukazuje na visok stepen korelacije između varijabli, odnosno na jaku koreleciju; vrednost 0,4 – 0,6 na umerenu korelaciju, a ispod 0,4 na slabu korelaciju. Rezultati korelacione analize pokazuju visok stepen korelacije između ra- zumevanja i komparativne prednosti brenda (0,655), razumevanja i međuljudskih odnosa (0,616), kom- parativne prednosti brenda i međuljudskih odnosa (0,724), komparativne prednosti brenda i preferen- cije (0,805) kao i međuljudskih odnosa i preferencije (0,690). Umeren stepen korelacije, zabeležen je izme- đu razumevanja i preferencije (0,583), dok je nizak stepen korelacije prisutan između istorije i razume- vanja (0,335), istorije i komparativne prednosti bren- da (0,257), istorije i međuljudskih odnosa (0,277), te istorije i preferencije (0,303). imaju signifikantan uticaj na preferenciju brenda. Pri tom, najveći uticaj na preferenciju ima komparativna prednost brenda: ß = 0,805, t = 15,931, p <0,01; dok je najmanji uticaj zabeležen kod istorije kao dimenzija vrednosti brenda: ß = 0,303, t = 3,735, p <0,01. Na kraju, sprovedena je i višestruka regresija ko- jom je analiziran zajednički uticaj nezavisnih varijabli na nivo preferencije brenda koja je zavisna varijabla. Rezultati analize prikazani su u tabeli 6. Koeficijent determinacije (R2) iznosi 0,678 što znači da je 67,8% varijabiliteta u preferenciji brenda opisano preko če- tiri nezavisne varijable (razumevanje, komparativna prednost brenda, međuljudski odnosi i istorija), dok je ostatak varijabiliteta pod uticajem drugih faktora. Rezultati istraživanja pokazali su da dva faktora ima- ju statistički značajan uticaj na preferenciju brenda: komparativna prednost brenda: ß = 0,622, t = 8,117, p <0,01; i međuljudski odnosi: ß = 0,203, t = 2,757, p <0,01; pri čemu, komparativna prednost brenda ima jači uticaj. 3. Christodoulides, G. and Chernatony, L. (2010). Consumer-based brand equitz conceptualization and measurement. International Journal of Market Research, 52 (1), 43-66. 4. Cobb-Walgren, C. J., Ruble, C. A. and Donthu, N. (1995). Brand equity, brand preference, and purchase intent. Journal of Advertising, 24 (3), 25–40. 3. REZULTATI ISTRAŽIVANJA Razumevanje i istorija kao dimenzije vred- nosti brenda, nemaju statistički značajan uticaj na preferenciju brenda. U sledećem koraku sprovedena je i regresiona analiza kako bi se utvrdilo koje varijable imaju signi- fikantan uticaj na preferenciju brenda. U tom cilju, korišćena je prosta linearna regresija (tabela 5). Testi- ran je pojedinačni uticaj nezavisnih varijabli na pre- ferenciju brenda koja je zavisna varijabla. Analiza do- bijenih rezultata pokazuje da sve nezavisne varijable Tabela 6. Višestruka regresiona analiza (Zavisna varijabla: Preferencija brenda) Tabela 6. Višestruka regresiona analiza (Zavisna varijabla: Preferencija brenda) Varijable β t Sig. VIF Razumevanje 0,023 0,337 0,737 1,973 Komparativna prednost brenda 0,622 8,117 0,000** 2,467 Međuljudski odnosi 0,203 2,757 0,007** 2,284 Istorija 0,079 1,524 0,130 1,137 Napomena:  p<0.01 (**); R2=0,678 134      Marija Vranješ, Dragoljub Jovičić, Dragana Drinić pravcu treba usmeriti napore. S tim u vezi, kompanije moraju kontinuirano da rade na izgradnji komparativ- ne prednosti kroz unapređenje reputacije brenda, pozi- tivnog mišljenja, te ispunjavanje očekivanja potrošača. Pored toga, neophodno je punu pažnju posvetiti i me- đuljudskim odnosima koji se ogledaju u inovativnosti, prestižu i posvećenosti potrošačima. Ovaj rezultat je u skladu sa stavovima autora Helgeson & Supphellen, (2004.) prema kojima brend treba da omogući funk- cionalne, iskustvene ali i simboličke koristi potrošaču. S druge strane, razumevanje i istorija nisu statistički značajne za nivo preferencije brenda. Međutim, ni ove dimenzije ne treba zanemariti, jer testiranjem poje- dinačnog uticaja ovih varijabli, potvrđena je njihova signifikantnost za nivo preferencije brenda. Na ovaj način, menadžmentu je omogućeno da razume na- čin izgradnje vrednosti brenda, da formira adekvatan okvir za upravljanje brendom što bi u krajnjoj liniji trebalo da dovede do rasta nivoa preferencije brenda i poboljšanja konkurentske pozicije kompanije. Pre sprovođenja višestruke regresione analize, utvrđen je faktor povećanja varijanse (VIF) kako bi se utvrdila eventualna multikolinearnost. Problem mul- tikolinearnosti se neće javiti, ukoliko vrednosti VIF koeficijenta budu manje od 5 (Field, 2000). S obzirom da su vrednosti navedenog koeficijenta u ovom istra- živanju bile adekvatne i kretale se u rasponu 1,137- 2,467, može se zaključiti da je bilo opravdano sprove- sti regresionu analizu. 1. Ariely, D. and Berns, G. S. (2010). Neuromarketing: the hope and hype of neuroimaging in business. Nature reviews Neuroscience, 11 (4), 284–292. 2. Ariely, D. and Norton, M. I. (2009). Conceptual consumption. Annual Review of Psychology, 60 (1), 475–499. ZAKLJUČAK Poslovno okruženje u kom funkcionišu savremene kompanije, postalo je izuzetno kompleksno. S jedne strane, kompanije se suočavaju sa sve većim troškovi- ma i sve intenzivnijom konkurencijom. S druge stra- ne, potrošači postaju sve zahtevniji, dok je broj alter- nativa koje im se nude sve veći. U takvim uslovima, kada je neophodno razviti lojalnost, koncept brenda dobija sve veći značaj. Samim tim, brendiranje sve manje postaje stvar izbora i nameće se kao neminov- nost. S ozirom na navedenu činjenicu, cilj ovog rada je bio da utvrdi uticaj određenih dimenzija vrednosti brenda na unapređenje nivoa preferencije brenda od strane potrošača. Pored praktičnog doprinosa, sprovedeno istraživanje pruža i doprinos postojećoj literaturi s obzirom na to da identifikuje najznačajnije faktore koji utiču na nivo preferencije brenda. Radovi u domaćoj literaturi bave se merenjem vrednosti brenda, ali isključivo sa teorijskog stanovišta u smislu objašnjavanja modela za merenje performansi, ali bez empirijske analize i potvrde. Rezultati istraživanja primenom proste regresione analize navode na zaključak da sve dimenzije vred- nosti brenda imaju statistički signifikantan uticaj na povećanje nivoa preferencije brenda, a to su: razume- vanje, komparativna prednost brenda, međuljudski odnosi i istorija. Međutim, primena višestruke re- gresione analize, pokazuje da u slučaju zajedničkog delovanja sve četiri nezavisne varijable, dve varijable: međuljudski odnosi i komparativna prednost brenda imaju statistički značajan uticaj na nivo preferencije, s tim da komparativna prednost brenda ima jači uticaj. S druge strane, preostale dve dimenzije: razumevanje i istorija nemaju signifikantan uticaj. Pored doprinosa, ovo istraživanje sa sobom nosi i neka ograničenja koja bi se mogla prevazići u budu- ćim istraživanjima. Jedno od ograničenja odnosi se na veličinu uzorka. S tim u vezi, u narednom istraživanju poželjno bi bilo da uzorak obuhvati veći broj ispitani- ka. Pored toga, korisno bi bilo analizom obuhvatiti i neke nove konstatacije kako bi se dobila sveobuhvat- nija analiza. Takođe, buduća istraživanja bi mogla da ispitaju dalji uticaj preferencije brenda, na nivo veza- nosti potrošača za brend posmatranu kroz doslednost potrošača pri korišćenju brenda, bez obzira na povre- meni pad kvaliteta, kao i aktivnost potrošača vezane za traganje za dodatnim informacijama o brendu i raz- menjivanje iskustava sa prijateljima. Najzad, dimenzije vrednosti brenda bi trebalo kontinuirano inovirati i adaptirati u skladu sa novonastalim okolnostima. i Sprovedeno istraživanje pruža korisne informa- cije menadžmentu kompanija. Rezultati pokazuju da međuljudski odnosi i komparativna prednost imaju snažan uticaj na nivo preferencije. Pored toga, jačina uticaja statistički značajnih dimenzija, sugeriše u kom Reference: 2. Ariely, D. and Norton, M. I. (2009). Conceptual consumption. Annual Review of Psychology, 60 (1), 475–499. 1. Ariely, D. and Berns, G. S. (2010). Neuromarketing: the hope and hype of neuroimaging in business. Nature reviews Neuroscience, 11 (4), 284–292. Reference: Vrednovanje brenda iz perspektive potrošača      135 13. Kocić, M. i Senić, V. (2010). Ponuda kao kreativna osnova direktnog marketinga. Marketing, 41 (2), 87-96. 5. Escalas, J. E. and Bettman, J. R. (2005). Self-Construal, Reference Groups, and Brand Meaning. Journal of Consumer Research, 32 (3), 378–389. g g g 14. Kocić, M., Šapić, S. i Kijevčanin V. (2013). Strategic implications of Internet marketing plan, Proceedings of the 2nd International scientistic conference: Contemporary issues in economics, business and management (p. 217-226.), Kragujevac: Ekonomski fakultet, Univerzitet u Kragujevcu. 6. Field, A. (2000). Discovering statistics using SPSS for Windows. Thousand Oaks, USA: Sage Publication.h h g 7. Franzen, G. and Bouwman, M. (2001). The Mental World of Brands: Mind, Memory and Brand Success. Oxfordshire: World Advertising Research Center. g 8. Helgeson, J. and Supphellen, M. (2004). A conceptual and measurement comparison of self-congruity and brand personality. The impact of socially desirable responding. International Journal of Market Research, 46 (2), 205-233. 15. Kotler, P. and Keller, K. (2006). Marketing Menadžment (12th edition). Beograd: Data status. g 16. Lehmann, D. R., Keller, K. L. and Farley, J. U. (2008). The Structure of Survey-Based Brand Metrics. Journal of International Marketing, 16 (4), 29–56. 9. Hellier, P. K., Geursen, G. M., Carr, R. A. and Rickard, J. A. (2003). Customer repurchase intention: A general structural equation model. European Journal of Marketing, 37 (11/12), 1762–1800.h 17. Myers, C. A. (2003). Managing brand equity: A look at the impact of attributes. Journal of Product and Brand Management, 12 (1), 39–51. g 18. Nunnally, J. C. (1978). Introduction to Psychological Measurement. New York: McGraw-Hill. f g 10. Jun, J. W., Cho, C. and Kwon, H. J. (2008). The Role of Affect and Cognition in Consumer Evaluations of Corporate Visual Identity: Perspectives from the United States and Korea. Journal of Brand Management, 15 (6), 382-398. 19. Predović, D. (2007). Vrednovanje marke, Od tržišne vrednosti marke bazirane na potrošaču do finansijskog vrednovanja. Zagreb: Mate. j g 20. Veljković, S. i Đorđević, A. (2010). Vrednost brenda za potrošače i preduzeća. Marketing, 41 (1), 3-16. g 11. Keegan, J. W. and Green, C. M. (2008). Global Marketing. London: Pearson Education Ltd. g 21. Zeugner-Roth, K. P., Diamantopoulos, A. and Montesinos, A. (2008). Home country image, country brand equity and consumers product preferences: An empirical study. Management International Review, 48 (5), 577-602. 12. Keller, L. K. (2011). How to Navigate the Future of Brand Management. Reference: Marketing Management, 20 (2), 36-43. Evaluation of Brand from the Perspective of Consumers Marija Vranješ, Dragoljub Jovičić, Dragana Drinić Brand is extremely important intellectual property of a company. In order to attract new customers and keep as many existing ones, companies have to work continuously to improve the brand value, which ultimately leads to the market share, revenues and profits growth. the level of preference - interpersonal relations and compara- tive advantage of the brand. On the other hand, understand- ing of brand and its history, doesn’t have statistically sig- nificant effect. Results of these conducted studies may help company’s management when formulating the appropriate management’s framework of the brand in order to increase brand preference, which ultimately may lead to improving the competitive position of the company. i The aim is to explore how certain aspect of brand value influences the preference of a brand. In order to fulfill these objectives, certain types of descriptive statistics measures were used, together with reliability analysis, correlation and regression analysis. The results show that statistically there are two dimensions of brand value which effect increase of Keywords: dimensions of brand value, brand value mea- surement, brand preference 136      Marija Vranješ, Dragoljub Jovičić, Dragana Drinić Kontakt: M. Sc. Marija Vranješ, asistent vranjesmarija.vps@gmail.com dr Dragoljub Jovičić, professor strukovnih studija djovicic@uns.ac.rs M.Sc. Dragana Drinić, asistent dragana.vps@gmail.com Visoka poslovna škola strukovnih studija u Novom Sadu, Vladimira Perića Valtera 4, Novi Sad M. Sc. Marija Vranješ, asistent vranjesmarija.vps@gmail.com dr Dragoljub Jovičić, professor strukovnih studija djovicic@uns.ac.rs M.Sc. Dragana Drinić, asistent dragana.vps@gmail.com Visoka poslovna škola strukovnih studija u Novom Sadu, Vladimira Perića Valtera 4, Novi Sad M. Sc. Marija Vranješ, asistent vranjesmarija.vps@gmail.com dr Dragoljub Jovičić, professor strukovnih studija djovicic@uns.ac.rs M.Sc. Dragana Drinić, asistent dragana.vps@gmail.com Visoka poslovna škola strukovnih studija u Novom Sadu, Vladimira Perića Valtera 4, Novi Sad 136      Marija Vranješ, Dragoljub Jovičić, Dragana Drinić
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The Paneth Cell: The Curator and Defender of the Immature Small Intestine
Frontiers in immunology
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The Paneth Cell: The Curator and Defender of the Immature Small Intestine Shiloh R. Lueschow 1† and Steven J. McElroy 1,2*† 1 Department of Microbiology and Immunology, University of Iowa, Iowa City, IA, United States, 2 Stead Family Department of Pediatrics, University of Iowa, Iowa City, IA, United States Paneth cells were first described in the late 19th century by Gustav Schwalbe and Josef Paneth as columnar epithelial cells possessing prominent eosinophilic granules in their cytoplasm. Decades later there is continued interest in Paneth cells as they play an integral role in maintaining intestinal homeostasis and modulating the physiology of the small intestine and its associated microbial flora. Paneth cells are highly specialized secretory epithelial cells located in the small intestinal crypts of Lieberkühn. The dense granules produced by Paneth cells contain an abundance of antimicrobial peptides and immunomodulating proteins that function to regulate the composition of the intestinal flora. This in turn plays a significant role in secondary regulation of the host microvasculature, the normal injury and repair mechanisms of the intestinal epithelial layer, and the levels of intestinal inflammation. These critical functions may have even more importance in the immature intestine of premature infants. While Paneth cells begin to develop in the middle of human gestation, they do not become immune competent or reach their adult density until closer to term gestation. This leaves preterm infants deficient in normal Paneth cell biology during the greatest window of susceptibility to develop intestinal pathology such as necrotizing enterocolitis (NEC). As 10% of infants worldwide are currently born prematurely, there is a significant population of infants contending with an inadequate cohort of Paneth cells. Infants who have developed NEC have decreased Paneth cell numbers compared to age-matched controls, and ablation of murine Paneth cells results in a NEC-like phenotype suggesting again that Paneth cell function is critical to homeostasis to the immature intestine. This review will provide an up to date and comprehensive look at Paneth cell ontogeny, the impact Paneth cells have on the host-microbial axis in the immature intestine, and the repercussions of Paneth cell dysfunction or loss on injury and repair mechanisms in the immature gut. †ORCID: Shiloh R. Lueschow orcid.org/0000-0001-6185-7612 Steven J. McElroy orcid.org/0000-0002-4321-723X Specialty section: This article was submitted to Mucosal Immunity, a section of the journal Frontiers in Immunology Received: 17 December 2019 Accepted: 13 March 2020 Published: 03 April 2020 Keywords: paneth cell, necrotizing enterocolitis, immature intestine, defensins, cathelicidin (LL37), cell death Edited by: Tobias Strunk, King Edward Memorial Hospital, Australia Reviewed by: Claudio Nicoletti, University of Florence, Italy Luisa Cervantes-Barragan, Emory University, United States *Correspondence: Steven J. McElroy steven-mcelroy@uiowa.edu REVIEW published: 03 April 2020 doi: 10.3389/fimmu.2020.00587 REVIEW published: 03 April 2020 doi: 10.3389/fimmu.2020.00587 THE ANATOMY OF THE PANETH CELL The human gastrointestinal surface is the largest surface area of the body that is in contact with the external environment (7, 8). This massive surface area is required to allow sufficient nutrient absorption to support growth and health of the host. The small intestine, where Paneth cells reside, has an estimated surface area of 950 cm2 at birth, which grows and expands to over 30 m2 by adulthood (7, 8). To achieve such a massive surface area, the intestinal surface is clad by fingerlike projections that stick out into the intestinal lumen creating an expansive folding system. This systems’ entire surface is covered by a single layer of columnar intestinal epithelial cells (IECs). The intestinal epithelium is the most rapidly-renewing tissue in the adult mammal (9) and undergoes continuous turnover that is generated from Intestinal Stem cells (ISC). The ISC reside at or near the base of the pocket-like intestinal crypts (10, 11) and continuously generate daughter cells that differentiate near the top of the crypts before migrating toward their final destinations. The differentiated cell types are generally grouped by their function as belonging to either the absorptive (enterocytes), or secretory (mucus-secreting goblet, antimicrobial-secreting Paneth, hormone-secreting enteroendocrine cells, and chemosensing/immunomodulatory cytokine-secreting tuft cells) lineage, with clear markers (e.g., hes1 expression of absorptive and sox9 for secretory) defining y Paneth cells, like all other intestinal epithelial cell types, are derived from ISCs. In the last decade, it has become clear that ISCs are quite complex. Current models suggest multiple, potentially interconvertible populations of stem cells exist. The first is the crypt-base columnar (CBC) cells (20), slender cells wedged at the very base of the crypt between the Paneth cells. CBC cells carry the specific marker LGR5 and are actively proliferating (21, 22). The second ISC population express Bmi1, mTert, and Lrig1 markers, and have been hypothesized to be quiescent stem cells until injury occurs, at which time they actively proliferate and produce daughter progeny (23). Interconversion between the two compartments and overlap between the populations has been demonstrated (24). Under normal conditions, the LGR5+ ISCs proliferate to generate daughter cells that move out of the crypt. These cells become differentiated as they migrate, and both their differentiation and the maintenance of the stem cells in their proper place is driven through gradients and juxtracrine signaling of Bmp, Wnt, Notch, and growth factor pathways (25, 26). Citation: Lueschow SR and McElroy SJ (2020) The Paneth Cell: The Curator and Defender of the Immature Small Intestine. Front. Immunol. 11:587. doi: 10.3389/fimmu.2020.00587 In the small intestine, intestinal epithelial cells form an important physical and biochemical barrier that prevents the microbial communities contained within the lumen from accessing the rest of the body and causing infection (1). One particular type of intestinal epithelial cell, the Paneth cell, was first discovered by Gustav Schwalbe in the late 19th century based on the eosinophilic April 2020 | Volume 11 | Article 587 Frontiers in Immunology | www.frontiersin.org Paneth Cell Lueschow and McElroy granules evident in their cytoplasm. A few years later, Paneth cells were described in depth by their namesake, Joseph Paneth (2, 3). They are now well-recognized as pyramidal shaped, columnar, secretory cells situated at the base of the crypts of Lieberkühn, which are small depressions in the mucosal surface along the small intestine (4). While Paneth cells have occasionally also been found patchily dispersed in the stomach and colon, this is generally associated with mucosal inflammation as opposed to homeostasis (4). commitment to one or the other arm (12). The typical pattern for these cells is to migrate upwards toward the villus tip in a conveyor-belt-type fashion until they are sloughed offthe upper villus into the lumen. However, a unique aspect of Paneth cell biology compared to the other intestinal epithelial cell types is that instead of flowing upward out of the crypt, Paneth cells move downwards further into the crypt as they mature. In addition, while most epithelial cells are rapidly turned over in a few days, Paneth cells can persist for just under 1 month (13). Paneth cell presence is an intestinal priority and their density is rapidly repopulated following their depletion (14). Following their descent into the crypts, Paneth cells are interspersed between the ISCs and can be distinguished by their columnar to pyramidal shape and by the presence of eosinophilic granules within their cytoplasm (Figure 1). Although Paneth cells were first discovered and described in humans, they are not specific to humans. Paneth cells can be found in many other vertebrates including primates, rodents, horses, sheep, certain fish, and chickens (5, 6). Citation: While Paneth cells have been found in this wide variety of other organisms aside from humans, the ontogeny and function are not well- understood for most of them aside from the well-studied and characterized rodents as well as humans. Today, Paneth cells still capture the attention of researchers as they serve an essential role in modulating the microbiome, playing a key part of the innate immune response, and aiding in the proliferation and differentiation of the intestinal epithelium. While Paneth cells have been shown to play important roles in the healthy gut of adults, the development and role of Paneth cells in the immature gut of the preterm infant remains an understudied, but crucial avenue of research that could aid in the understanding of the development of intestinal diseases such as necrotizing enterocolitis (NEC). This review sets out to unveil some of the mystery surrounding Paneth cells in the context of the preterm infant gut and how it relates to NEC. PANETH CELL ONTOGENY AND DIFFERENTIATION Paneth cells first appear in the small intestine of humans at 13.5 weeks gestational age (15, 16). Paneth cell density in the developing fetal intestine is relatively low, but gradually increases throughout gestation, with significant increases in the third trimester after 29 weeks completed gestation (17, 18). Paneth cell levels do not reach quantities similar to adult levels until term gestation or later (17). Because Paneth cells are located primarily in the distal small intestine, studies using human tissues have been challenging. Thus, much of our understanding of in vivo Paneth cell biology has been generated using animal models, predominantly in mice. It is therefore important to note that not all mammals develop Paneth cells prenatally, but instead develop them mid-way through intestinal development after villus development, but before intestinal maturity according to a normal developmental pattern. For example, the commonly used C57Bl/6 mouse strain does not develop Paneth cells until 7–10 days after birth (18, 19). Frontiers in Immunology | www.frontiersin.org THE ANATOMY OF THE PANETH CELL Furthermore, while the exact sources of ligands for these pathways are not fully April 2020 | Volume 11 | Article 587 Frontiers in Immunology | www.frontiersin.org 2 Paneth Cell Lueschow and McElroy FIGURE 1 | The intestinal epithelium. (Left) H&E stained ileum from P14 C57Bl6 mouse with vilus, crypt, and lamina propria labeled. (Right) Schematic of the intestinal epithelium, associated microbial flora, epithelial cell types (goblet cells, Paneth cells, enterocytes, and stem cells) intestinal microvasculature, and mucus layer. Corresponding labels for vilus, crypt, and lamina propria labeled are placed on the schematic to compare to the H&E stained section. FIGURE 1 | The intestinal epithelium. (Left) H&E stained ileum from P14 C57Bl6 mouse with vilus, crypt, and lamina propria labeled. (Right) Schematic of the intestinal epithelium, associated microbial flora, epithelial cell types (goblet cells, Paneth cells, enterocytes, and stem cells) intestinal microvasculature, and mucus layer. Corresponding labels for vilus, crypt, and lamina propria labeled are placed on the schematic to compare to the H&E stained section. C57Bl/6 mice normally develop Paneth cells by day 10 of life (19). It is however important to note that modifications to Atoh1 signal pathways also affect goblet cell differentiation (36), so understanding of signal pathways that distinguish goblet cell from Paneth cell differentiation downstream of Atoh1 is still incomplete. understood, it is important to note that Paneth cells produce EGF, Notch, and Wnt, which in turn promote stem cell proliferation and maintenance (27). In fact, Paneth cells can support LGR5+ cell growth and survival in vitro, and have been proposed as a key nurse cell for the actively dividing stem population (27). Several biochemical pathways have been implicated in the development of Paneth cells (Figure 2). Naïve daughter cells are driven to either an absorptive enterocyte phenotype by Notch signaling, or to a secretory phenotype through Wnt signal pathways. The Wnt/β-catenin pathway is an important stimulator of Paneth cell differentiation (28, 29). However, the Wnt signal pathway and its relationship to Paneth cell development is complex and still not completely elucidated. Genetic knockout of LGR-5, a downstream target of Wnt signaling has been shown to produce precocious Paneth cell differentiation in fetal intestine (29, 30). This contradictory data may be due to alterations in negative feedback mediators in the Wnt pathway. THE ANATOMY OF THE PANETH CELL Following differentiation into a secretory lineage, activation of the transcription factors Atoh1 (also known as Math1) induces differentiation into a combined goblet/Paneth cell precursor cell lineage (31–35), while genetic ablation of Atoh1 in transgenic mice has been shown to result in loss of Paneth cell lineages (35, 36). Atoh1 has also been shown to be affected by ErbB3, a Receptor Tyrosine Kinase also known as neuregulin (37). Genetic loss of ErbB3 in mice results in unchecked activity of the transcription factor Atoh1 and induces precocious appearance of Paneth cells (37). In addition, activation of ErbB3 can delay normal Paneth cell development. Frontiers in Immunology | www.frontiersin.org PANETH CELL ROLE IN THE SMALL INTESTINE After their migration to the crypt base and subsequent maturation, Paneth cells can be easily distinguished by their prominent acidophilic granules. The granules hold many of the proteins and peptides that Paneth cells secrete to both modulate the microbiome and mediate the inflammatory response. These include: α-defensins (cryptdins in mice), lysozyme, secretory phospholipase A2 (sPLa2), TNF, RegIII, angiogenin-4, MMP-7, CD15, CD95 ligand, xanthine oxidase, IgA, CRIP, metallothionine, adipokines, serum amyloid A, α- 1-antitrypsin IL-17A, IL-1β and lipokines (3, 38, 39). These granular components are assembled and packaged by an extensive endoplasmic reticulum (ER) and Golgi apparatus network into dense core granules. (13, 39–43) It is important to note that it is possible that some components of the granules may be produced elsewhere before being collected and added to the granules. IgA is one such component which may be produced by plasma cells in the lamina propria before April 2020 | Volume 11 | Article 587 Frontiers in Immunology | www.frontiersin.org 3 Paneth Cell Lueschow and McElroy communicates with the external environment (7, 52), protection of the host from injury or bacterial invasion from the intestinal flora (53) requires a complex system of defense mechanisms. In the small intestine, a key component of host defense is epithelial derived antimicrobial peptides (AMPs). AMPs are small peptides generally >5 kDa in length, cationic at a neutral pH, and have broad spectrum microbicidal activities at low concentrations (45). These peptides are the main product contained in Paneth cell granules. FIGURE 2 | Intestinal epithelial cell differentiation pathways. The intestinal stem cell (ISC) differentiates into absorptive (enterocyte) or secretory precursors through Wnt/Notch signaling. While enterocytes further differentiate through HES-1 signaling, secretory lineages can differentiate into different cell types depending on conditions. Wnt signal pathways drive ISC differentiation into secretory precursor cells. Secretory precursors then develop either into enteroendocrine cells through Neurog3 signaling, or into goblet and Paneth cells following activation of Atoh1. Differentiation signal pathways to separate development of goblet cells and Paneth cells are still unknown. It is also important to note that recent data has shown that activation of ErbB3 acts as a suppressor of Atoh1, while genetic deletion of ErbB3 induces precocious development of Paneth cells. In humans, there are two major classes of AMPs: cathelicidins and defensins. Cathelicidins are antimicrobial peptides with broad antibacterial (54), anti-fungal (55), and anti-viral activity (56), and are characterized by a highly conserved N-terminal domain. PANETH CELL ROLE IN THE SMALL INTESTINE Only after cleavage of the AMP does the protein exert its myriad activities (57). Humans express only one cathelicidin, LL-37 (originally hCAP-18) (58) and it is expressed in various cells of the body including those of the intestinal epithelium (59–62). However, in the small intestine, cathelicidin expression is restricted to the neonatal period (63, 64) before markedly decreasing and disappearing. The timing of this decrease is important as it coincides with the appearance of Paneth cells (19, 65) and the onset of expression of Paneth cell AMPs such as α-Defensins (18). This “switch” from one AMP to another occurs at roughly the mid-point of development of the small intestine (66). It is important to note that mid-development of the small intestine is also around the time when NEC often occurs in infants born extremely prematurely (67) (Figure 3). FIGURE 2 | Intestinal epithelial cell differentiation pathways. The intestinal stem cell (ISC) differentiates into absorptive (enterocyte) or secretory precursors through Wnt/Notch signaling. While enterocytes further differentiate through HES-1 signaling, secretory lineages can differentiate into different cell types depending on conditions. Wnt signal pathways drive ISC differentiation into secretory precursor cells. Secretory precursors then develop either into enteroendocrine cells through Neurog3 signaling, or into goblet and Paneth cells following activation of Atoh1. Differentiation signal pathways to separate development of goblet cells and Paneth cells are still unknown. It is also important to note that recent data has shown that activation of ErbB3 acts as a suppressor of Atoh1, while genetic deletion of ErbB3 induces precocious development of Paneth cells. FIGURE 2 | Intestinal epithelial cell differentiation pathways. The intestinal stem cell (ISC) differentiates into absorptive (enterocyte) or secretory precursors through Wnt/Notch signaling. While enterocytes further differentiate through HES-1 signaling, secretory lineages can differentiate into different cell types depending on conditions. Wnt signal pathways drive ISC differentiation into secretory precursor cells. Secretory precursors then develop either into enteroendocrine cells through Neurog3 signaling, or into goblet and Paneth cells following activation of Atoh1. Differentiation signal pathways to separate development of goblet cells and Paneth cells are still unknown. It is also important to note that recent data has shown that activation of ErbB3 acts as a suppressor of Atoh1, while genetic deletion of ErbB3 induces precocious development of Paneth cells. The second class of AMP found in the small intestine are defensins. PANETH CELL ROLE IN THE SMALL INTESTINE Defensins are abundant in human cells and tissues that are involved in host defense and have two main subtypes: α-defensins, which are found in granule containing cells such as neutrophils and Paneth cells (also known as cryptdins in mice), and β-defensins which are produced by epithelial cells (68–71). Human Paneth cells produce two main α-defensins known as HD-5 and HD-6 (72). In mice, loss of matrilysin (the proteolytic enzyme needed to activate cryptdins) have altered microbiomes and are more susceptible to Salmonella infections (73–75). In addition, mice that have been genetically modified to express HD-5 have enhanced resistance to bacterial invasion (74). accumulating and associating in Paneth cell granules (44). Since Paneth cells are not currently able to be cultured without other epithelial and stem cells, most of the data we have on granular contents is from immunohistochemistry techniques. The granules are then released at the apical surface of the cell into the lumen of the intestine where they serve a variety of biological functions, primarily as microbiocidal agents against bacteria, fungi, spirochetes, protozoa, and enveloped viruses (45). Paneth cell granules are secreted both constitutively and in response to pathogenic exposure, with common stimuli including cholinergic stimulation and exposure to bacterial antigens (45–47). This secretion of Paneth cell granular components is under tight regulatory control, as these mediators are vital for maintenance of intestinal homeostasis (38, 48, 49). AMPs work by inserting themselves into the bacterial membrane and forming pores, which result in the leakage of bacterial cytoplasmic content (76–78). They can also degenerate bacterial cytoplasmic structures and form extracellular net-like structures, which result in bacterial trapping (79). In animal models, AMPs have been shown to preferentially target non- commensal bacteria while sparing commensal normal flora (47, 80). In addition to killing pathogens, AMPs can also influence the immune system through white blood cell chemotaxis (81), activation of dendritic cells (82), and downregulation of immunomodulators such as cortisol (68, 71). Frontiers in Immunology | www.frontiersin.org PANETH CELLS AND MECHANISMS OF CELLULAR DEATH Paneth cell health remains a critical priority to the homeostasis of the small intestine. We and others have shown that following dithizone-induced loss, the small intestine replenishes Paneth cell populations within 72 h (14, 50, 51). Since the mammalian intestinal tract represents the largest surface area that Cells of the body undergo death for a multitude of reasons and through various mechanisms. The mechanisms of cell April 2020 | Volume 11 | Article 587 Frontiers in Immunology | www.frontiersin.org 4 Paneth Cell Lueschow and McElroy FIGURE 3 | Small intestinal AMP switch during intestinal development. During development, the immature intestine is protected by the AMP CRAMP (LL-37 in humans). However, CRAMP expression decreases around mid-development, at roughly the time that Paneth cells begin to develop. This “switch” occurs during mid-intestinal development which is around post-natal (P) day 10–21 in C57Bl6 mice and in the second trimester (between 20 and 28 weeks of gestation) in humans. In infants born prematurely, this switch is temporally similar to when extremely preterm infants are most susceptible to develop NEC (18). FIGURE 3 | Small intestinal AMP switch during intestinal development. During development, the immature intestine is protected by the AMP CRAMP (LL-37 in humans). However, CRAMP expression decreases around mid-development, at roughly the time that Paneth cells begin to develop. This “switch” occurs during mid-intestinal development which is around post-natal (P) day 10–21 in C57Bl6 mice and in the second trimester (between 20 and 28 weeks of gestation) in humans In infants born prematurely, this switch is temporally similar to when extremely preterm infants are most susceptible to develop NEC (18). has been attached to the cryptdin-2 promoter of Paneth cells (14, 65). When these mice are exposed to diphtheria toxin, all Paneth cells expressing the construct are lysed through apoptotic pathways (90). death include apoptosis, necrosis, necroptosis, pyroptosis, and autophagy. While NEC is defined by necrosis of the intestinal tissue, many of these different cellular death pathways have been implicated in the pathogenesis of NEC. Importantly, several of these pathways are also mechanistically tied to Paneth cell biology. Another form of cell death directly related to Paneth cells is autophagy, which is a self-degradative process thought to help remove cells with misfolded or aggregated proteins or other intracellular damage (91). Autophagy is characterized by creation of an intracellular vacuole known as the autophagosome (83). PANETH CELLS AND MECHANISMS OF CELLULAR DEATH The autophagosome is formed around damaged intracellular organelles or other selected targets. The autophagosome is then fused with a lysosome allowing for degradation of the components within the autophagosome followed by chromatin condensation (83). The morphologic changes that occur tend to be relatively well-regulated similar to the degree of regulation of apoptosis. Also similar to apoptosis, because the degradation of the dying cell takes place within another cell, this process tends to prevent inflammation (83). Autophagy is also an important process for Paneth cells. Because Paneth cells tend to live longer than most other cells of the gut and have many aggregated proteins that could be recycled by other neighboring cells, as damage and stressors to the cells occur, autophagy becomes activated (92). When mutations occur in the autophagy pathway such as in Atg16l1, Paneth cells can become dysfunctional and ultimately trigger intestinal inflammation, which can have implications for gut health such is suggested to be the case with Crohn’s disease (92) and NEC (93). Our laboratory has also shown that autophagy may play a role in development of Apoptosis is a normal part of intestinal health that results in disassembly of the cell and, in general, tends to avoid causing inflammation (83). During apoptosis, cells tend to retract pseudopods, condense chromatin (pyknosis), undergo nuclear fragmentation and then experience blebbing of the plasma membrane (84). This contrasts with cellular necrosis where cells experience organelle swelling, extensive vacuole formation, condensation of nuclei, and release of inflammatory cytokines in a passive or accidental manner (83, 84). One type of apoptosis seen in the intestinal epithelial layer is when the epithelial cells move upward from the crypt toward the tip of the villus. Once they reach the tip, cells are sloughed into the intestinal lumen in a process called anoikis, which is a form of apoptosis (84). There is evidence to show that apoptosis is also involved in the cell death experienced by cells in the stem cell region within the small intestinal crypts although the regulation of the process is not well-understood (84). Apoptosis has been shown by multiple investigators to be important in development of NEC (85–89). Frontiers in Immunology | www.frontiersin.org Frontiers in Immunology | www.frontiersin.org PANETH CELLS AND MECHANISMS OF CELLULAR DEATH Additionally, apoptosis is directly relevant to Paneth cell biology and NEC as our lab has shown that NEC- like injury can be induced in mice by delivering diphtheria toxin to PC-DTR mice where a human diphtheria toxin receptor April 2020 | Volume 11 | Article 587 Frontiers in Immunology | www.frontiersin.org 5 Paneth Cell Lueschow and McElroy FIGURE 4 | Proposed role of the Paneth cell in development of NEC. As the immature intestine (A) is exposed to inflammation (B), oxygen radicals are produced creating a selective advantage for Proteobacteria sp. over obligate anaerobes such as the Firmicutes. This creates a feedback loop for sustaining and increasing the pro-inflammatory state in the immature intestine. Previous work from our lab has shown that intestinal inflammation can reduce intestinal mucus production and cause loss of Paneth cells (112, 129). (C) Loss of these important chemical and physical aspects of innate immunity allows bacteria to move from the mucus layer of the intestinal lumen and gain closer proximity to the epithelial surface, (D) followed eventually by attachment and invasion of the epithelium. (E) Once bacteria invade the intestinal tissue, further inflammation occurs including recruitment of leukocytes including neutrophils, macrophages, and monocytes (133–135) which lead to eventual FIGURE 4 | Proposed role of the Paneth cell in development of NEC. As the immature intestine (A) is exposed to inflammation (B), oxygen radicals are produced creating a selective advantage for Proteobacteria sp. over obligate anaerobes such as the Firmicutes. This creates a feedback loop for sustaining and increasing the pro-inflammatory state in the immature intestine. Previous work from our lab has shown that intestinal inflammation can reduce intestinal mucus production and cause loss of Paneth cells (112, 129). (C) Loss of these important chemical and physical aspects of innate immunity allows bacteria to move from the mucus layer of the intestinal lumen and gain closer proximity to the epithelial surface, (D) followed eventually by attachment and invasion of the epithelium. (E) Once bacteria invade the intestinal tissue, further inflammation occurs including recruitment of leukocytes including neutrophils, macrophages, and monocytes (133–135) which lead to eventual death of the tissues. FIGURE 4 | Proposed role of the Paneth cell in development of NEC. As the immature intestine (A) is exposed to inflammation (B), oxygen radicals are produced creating a selective advantage for Proteobacteria sp. over obligate anaerobes such as the Firmicutes. PANETH CELLS AND MECHANISMS OF CELLULAR DEATH This creates a feedback loop for sustaining and increasing the pro-inflammatory state in the immature intestine. Previous work from our lab has shown that intestinal inflammation can reduce intestinal mucus production and cause loss of Paneth cells (112, 129). (C) Loss of these important chemical and physical aspects of innate immunity allows bacteria to move from the mucus layer of the intestinal lumen and gain closer proximity to the epithelial surface, (D) followed eventually by attachment and invasion of the epithelium. (E) Once bacteria invade the intestinal tissue, further inflammation occurs including recruitment of leukocytes including neutrophils, macrophages, and monocytes (133–135) which lead to eventual death of the tissues. NEC. Lueschow et al. (14) showed that dithizone-induced Paneth cell loss in an experimental murine NEC model resulted in upregulation of autophagy pathways in Paneth cells (14). as induce necroptosis when RIP1 and RIP3 are recruited and deubiquitinated (84). RIP1 and RIP3 are generally under the control of caspase-8, but when an inactivation of the caspase- 8 gene occurs, induction of necroptotic cell death ensues although the mechanism by which this occurs is not completely understood (84). Necroptosis is an increasingly important mechanism of cellular death in the intestinal epithelium. Studies have shown that necroptosis of intestinal epithelial cells can result in intestinal inflammation and ultimately produce pathophysiology similar to inflammatory bowel disease (IBD). This was done by creating conditional knockout mice with deletion of FADD or caspase-8, the regulator of necroptosis, in intestinal epithelial cells (54, 95, 96). Interestingly, in addition to induction of necroptosis, this knockout also resulted in Lastly, a more newly described type of cellular death is necroptosis which acts as an intermediate between necrosis and apoptosis. Cells undergoing necroptotic death show features more morphologically similar to necrosis and the immune system creates a highly inflammatory response, but in contrast to necrosis, necroptosis is a well-regulated process, similar to apoptosis (84, 94). Along with this relationship, necroptosis, and apoptosis have a great deal of overlap in their regulation. Apoptosis is promoted by TNFα binding and conversion of the TNFR complex I to the TNFR complex II/alternative TNFR complex (84). PANETH CELLS AND NECROTIZING ENTEROCOLITIS (NEC) For preterm infants, one of the leading causes of morbidity and mortality, and the most devastating intestinal complication, is development of NEC (97). The incidence of NEC varies widely among developed countries, ranging from 5 to 22% in infants with birth weight <1,000 g (98), and in the US is around 7% (97). Risk factors associated with development of NEC in the preterm infant include degree of prematurity, low birth weight, formula feeding, intestinal ischemia, prolonged antibiotic use, and anemia (99–102). However, the exact etiologic mechanisms and pathophysiology of NEC is still incomplete. In addition, the NEC phenotype may actually be the result of a final common pathway starting from multiple inciting events that results in an imbalance between mucosal injury and epithelial defense and repair, with activation of an unchecked pro-inflammatory cascade (103). As a disease process, NEC is unique in the Neonatal Intensive Care Unit (NICU) population. While the incidence of NEC is directly correlated to the degree of prematurity (the more premature, the more likely to develop NEC), the onset of NEC doesn’t happen at birth, but rather weeks after and this delay is longer in the more premature infants. The result is that the incidence of NEC begins to increase at 28 weeks corrected gestational age, peaks at 32 weeks corrected gestational age, and steadily decreases at older corrected gestational ages (67). Theories have been suggested to explain this delay including feeding practices, development of microbial dysbiosis, or the accumulation of mesenteric hypoxic events (99–102). However, there is currently no universally accepted mechanistic explanation. We propose that another plausible reason may be a disruption in the function or quantity of Paneth cells (17, 67, 104). p g p g Studying Paneth cell mechanistic biology in the immature intestine is challenging in humans due to the difficulty of obtaining tissue specimens for preterm infants (115, 116). To help understand the potential role of Paneth cell biology in NEC, several laboratories have instead utilized animal models (100). Interestingly, when Paneth cells are disrupted in neonatal rats followed by enteral exposure to E. coli, there is not only an increase in bacterial translocation, but also a development of NEC-like injury to the small intestinal tract (105). PANETH CELLS AND MECHANISMS OF CELLULAR DEATH Necroptosis has also been recently shown to play a role in development of NEC (94). In preterm infants who develop NEC, there is a higher degree of expression in genes related to necroptosis such as RIPK1, RIPK2, and MLKL compared to preterm infants who do not develop NEC (94). Moreover, increased expression of these three necroptosis related genes was correlated with a greater degree of NEC severity (94). This trend was also observed in murine experimental NEC conditions (94). Overall, these studies highlight the importance of necroptosis as well as Paneth cells in NEC. PANETH CELLS AND MECHANISMS OF CELLULAR DEATH Also, the TNFR complex II can regulate as well April 2020 | Volume 11 | Article 587 Frontiers in Immunology | www.frontiersin.org 6 Paneth Cell Lueschow and McElroy until approximately halfway through intestinal development and maturation (22–24 weeks of human gestation and P7-10 or mouse age—normal intestinal development in the mouse occurs following birth while in the human it occurs in utero) (19, 65, 110). It is also important to note that these early Paneth cells do not possess all the constituents contained in mature granules (65), and it takes weeks in mice and months in humans before the Paneth cell cohort reaches its optimal density and before it becomes fully functional (17). Because of this developmental pattern, premature infants are thus born before they can develop a full complement of functional Paneth cells. As Paneth cells help regulate the intestinal bacterial flora, and NEC requires bacteria to induce intestinal injury, disruption of normal Paneth cell function, especially in the immature intestine could very well be involved in development of the NEC phenotype. Supporting this theory, decreased numbers of lysozyme positive Paneth cells were documented in infants with surgical NEC compared to similar aged surgical controls in two separate studies (111, 112). These data would suggest that Paneth cells are either lost or degranulated during or prior to development of NEC. However, not all studies have shown decreases in Paneth cell function or biology. A study looking at mRNA levels of Human defensin 5 and 6 found that they were increased in infants who developed NEC compared to controls (113). This discrepancy may be explained by timing of surgical resection following the initial Paneth cell disruption. In mouse models, when Paneth cells are disrupted using the heavy metal chelator dithizone, there is an initial decrease in defensin expression followed by a significant increase starting 72 h after treatment (14). In addition, a recent article that examined presence of HD-6 showed a significant decrease following development of NEC (114). Thus, timing of the surgical collection may play a critical role in determining Paneth cell-specific gene expression following NEC. spontaneous inflammation and an absence of Paneth cells (84, 95, 96). On further examination, the authors discovered that Paneth cells were uniquely sensitive to necroptosis. This is now thought to be due to the high expression of RIP3, a key modulator of necroptosis, in Paneth cells of humans and mice (84, 95, 96). Frontiers in Immunology | www.frontiersin.org PANETH CELLS AND NECROTIZING ENTEROCOLITIS (NEC) Lastly, it is important to remember that Paneth cell loss may also impact the stem cell niche. A healthy stem cell cohort is critical to induce epithelial restitution following injury as Paneth cells support the stem cell niche through the production of EGF, Notch, and Wnt (27, 88, 104, 138). instead is a general chelator of heavy metals. To help resolve this issue, we developed the PC-DTR mouse (14, 119, 120). The PC-DTR mouse has a human diphtheria toxin receptor (DTR) inserted into mouse Paneth cells targeting the cryptdin- 2 promotor (65). Treatment with diphtheria toxin induces apoptosis of any cells possessing DTR while sparing all other cells. In this model, treatment with diphtheria toxin followed by Klebsiella pneumoniae exposure also produces intestinal injury that is equivalent to human NEC (14, 119). These data provide further evidence that it is a disruption of, and not an absence of Paneth cells that contributes to development of NEC-like injury in the immature small intestine. While these studies show a strong association for Paneth cell disfunction or loss with human NEC as well as a mechanistic relationship in mice, questions about how Paneth cell dysfunction may result in NEC remain (104, 121). It is well-established that prior to the development of NEC there is a dysbiotic change to the microbiome that is marked by a bloom of Proteobacteria, more specifically Enterobacteriaceae species (122–124). This phenomenon has also been replicated in our Paneth cell disruption model of NEC (14). In the normal homeostatic state, the microbiome acts to suppress inflammation through several mechanisms including induction of anti-inflammatory mediators such as IL- 10, suppression of pro-inflammatory mediators such as IL-17, and by breaking down and fermenting complex, non-digestible complex polysaccharides into short-chain fatty acids, which possess anti-inflammatory properties (125–127). However, a result of inflammation is increased production of nitric oxide (NO) and superoxide radicals (O2−), which can then react to form nitrates (NO3−). These nitrates can be fermented by facultative anaerobic bacteria such as Enterobacteriaceae sp. that belong to the Proteobacteria phyla by utilizing anaerobic respiration with host-derived nitrates as alternative electron acceptors. Since obligate anaerobes cannot use nitrates as a growth substrate, Proteobacteria are able to use this selective pressure to out-compete the obligate anaerobic Firmicutes and Bacteroidetes that rely on fermentation for growth (128). PANETH CELLS AND NECROTIZING ENTEROCOLITIS (NEC) In adapting this model to mice, our laboratory and others have shown that selective ablation of Paneth cells followed by enteric gavage of Klebsiella pneumoniae in 14-days old mice results in grossly necrotic intestines (89, 117–119), an increase in serum inflammatory markers (119), and alterations in the microbiome (14) that are consistent with human NEC. The use of 2-weeks old mice in this model is potentially advantageous as well as they possess a gene expression profile of epithelial cell genes that matches the expression profile seen in preterm human infants during the window when they are most susceptible to develop NEC (18, 67). Interestingly, disruption of Paneth cell biology via administration of the heavy metal chelator dithizone prior to normal Paneth cell development (5 days old mice) does not result in a NEC-like phenotype (117). One critique of this methodology is that dithizone is not specific to Paneth cells but As discussed above, Paneth cells play a key role in the homeostasis of the small intestinal epithelial lining, and loss or disruption of these cells has been shown to have significant adverse consequences including a reduction in clearance of bacterial pathogens (105, 106), disruption of normal stem cell function (3, 107), and the development of inflammatory bowel disease (108, 109). Paneth cells do not appear in the intestine April 2020 | Volume 11 | Article 587 Frontiers in Immunology | www.frontiersin.org 7 Paneth Cell Lueschow and McElroy This creates a more aerobic state leading to a competitive advantage for Proteobacteria, such as Enterobacteriaceae species. As the microbiome becomes more dysbiotic, it suppresses anti-inflammatory mechanisms creating a cycle of increasing intestinal inflammation (136). This increasing inflammation can then impact Paneth cell biology leading to a loss in Paneth cells (14, 129, 137). In an already dysbiotic environment, this combination is exactly the milieu that is modeled in our animal model and predisposes to development of injury. This is further compounded because the Paneth cells present in the immature intestine are not fully mature or functional at a baseline (18). This limited Paneth cell cohort also means that there is a limited capacity for protection via AMPs (40). As Paneth cells are lost, AMP levels will further fall, likely reaching a critical threshold under which bacterial invasion of the epithelial tissue can begin to occur (105). FUNDING Funding was provided by the Stead Family Department of Pediatrics and the Carver College of Medicine at the University of Iowa. Thus, we think that as the premature infant is exposed to foreign antigens such as formula feedings (132), there is an increase in production of inflammatory cytokines (Figure 4). PANETH CELLS AND NECROTIZING ENTEROCOLITIS (NEC) As the proportion of commensal bacteria such as Firmicutes and Bacteroidetes decrease, the production of anti-inflammatory mediators also decreases which further facilitates increased inflammation and dysbiosis. Our laboratory has previously shown that in the immature murine small intestine, exposure to inflammation can significantly decrease the density and function of Paneth cells (129–131). In summary, the Paneth cell plays a critical role in many facets of intestinal homeostasis, from regulating the microbiota that closely associate with the epithelium, to maintaining the health of the stem cell niche, to helping to regulate levels of inflammation. Disruption of these secretory cells can have an important effect on the ability of the intestinal epithelium to not only protect itself from foreign invaders, but to promote growth and development of the intestine. These functions are especially critical in the immature intestine of premature infants who have a developing intestinal tract associated with a dysbiotic microbiome. Thus, it is reasonable that Paneth cell disruption has been linked mechanistically to development of NEC-like injury. As mortality rates for NEC remain static, a greater understanding of Paneth cell biology may provide a critical novel pathway to understand the development of NEC. AUTHOR CONTRIBUTIONS SM and SL contributed equally to the drafting and editing of the work. 4. Bevins CL, Salzman NH. Paneth cells, antimicrobial peptides and maintenance of intestinal homeostasis. Nat Rev Microbiol. (2011) 9:356–68. doi: 10.1038/nrmicro2546 REFERENCES 3. Clevers HC, Bevins CL. 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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. 134. Egan CE, Sodhi CP, Good M, Lin J, Jia H, Yamaguchi Y, et al. Toll- like receptor 4-mediated lymphocyte influx induces neonatal necrotizing enterocolitis. J Clin Invest. (2016) 126:495–508. doi: 10.1172/JCI83356 April 2020 | Volume 11 | Article 587 Frontiers in Immunology | www.frontiersin.org 12
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The ecological footprint of the French-German border (1871-1914)
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L’empreinte écologique de la frontière franco-allemande (1871-1914) Benoît Vaillot Benoît Vaillot To cite this version: Benoît Vaillot. L’empreinte écologique de la frontière franco-allemande (1871-1914). Revue du Rhin Supérieur, 2021, 3, pp.21-40. ￿10.57086/rrs.183￿. ￿hal-03979335￿ Benoît Vaillot. L’empreinte écologique de la frontière franco-allemande (1871-1914). Revue du Rhin Supérieur, 2021, 3, pp.21-40. ￿10.57086/rrs.183￿. ￿hal-03979335￿ HAL Id: hal-03979335 https://hal.science/hal-03979335v1 Submitted on 8 Feb 2023 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 BENOIT VAILLOT L’empreinte écologique de la frontière franco-allemande (1871-1914) Benoit Vaillot est docteur en histoire de l’Institut Universitaire Européen, et chercheur associé à l’Université de Strasbourg et au Centre Marc-Bloch. Ses recherches portent sur la frontière franco-allemande entre 1871 et 1914, lorsque la frontière a été déplacée après l’annexion de l’Alsace-Lorraine par l’Empire allemand. Il s’intéresse à la construction de la souveraineté et des identités nationales par en bas, dans une perspective transnationale. Il a enseigné l’histoire contemporaine et la culture générale à l’université, ainsi que l’his- toire-géographie en lycée et collège. La frontière constitue un poste d’observation privilégié de l’empreinte éco- logique des sociétés humaines, dans la mesure où s’y confrontent deux conceptions différentes de la gestion de l’environnement. Les espaces forestiers sont particulièrement intéressants, parce que l’on peut y analyser finement les relations de pouvoir écologiques. Il s’agit dans cet article d’analyser sur une période relativement courte, entre 1871 et 1914, comment la frontière franco-al- lemande transforme l’environnement, et l’influence que ce dernier peut avoir sur la construction de la souveraineté et des identités nationales. 21 The border is a privileged observation post for the ecological footprint of human societes, insofar as two different conceptions of environemental management are confronted. Forest areas are particularly interesting because they allow an analysis of ecological power relations. This article analyses, over a relatively short period of time, between 1871 and 1914, how the French-German border transformed the environment, and the influence that the latter could have on the construction of sovereignty and national identities. 22 À ll À l’issue de la guerre de 1870, la France se voit contrainte de céder à l’Empire allemand les territoires qui formeront l’Alsace-Lorraine, par le traité de Francfort du 10 mai 1871. La nouvelle frontière fran- co-allemande suit alors en grande partie la ligne de crête des Vosges, et traverse principalement des espaces forestiers en altitude. Les forêts ont même été au cœur des négociations relatives à son tracé, car elles sont non seulement essentielles pour la défense du territoire, mais aussi utiles à bien des égards : on y exploite le bois, on y chasse les animaux et l’on commence à s’y rendre à des fins récréatives en cette fin de xixe siècle. Fig. 1. Profil altimétrique de la frontière franco-allemande (1871-1914) Fig. 1. 1.  Sophie Houdart et Olivier Thiery (dir.), Humains, non-humains  : comment repeupler les sciences sociales, Paris, La Découverte, 2011 ; Bruno Latour, Politiques de la nature. Comment faire entrer les sciences en démocratie, Paris, Éditions la Découverte, 1999. q faire entrer les sciences en démocratie, Paris, Éditions la Découverte, 1999. 7.  Conrad J. Bahre et Charles F. Hutchinson, « Historic Vegetation Change in La Frontera West of the Rio Grande », in G. L. Webster et C. J. Bahre (dir.), Changing Plant Life of La Frontera : Observations on Vegetation in the U.S./Mexico Borderlands, Albuquerque, University of New Mexico Press, 2001, p. 67-83. 6.  A. R. Graybill, « Boundless Nature. Borders and the Environment in North America and Beyond », art. cit. 2.  Anne-Laure Amilhat Szary, « “Montagnes mobiles ?” », Journal of Alpine Research | Revue de géographie alpine, 101-2 (2013) [En ligne : https://journals.openedition.org/rga/2135]. 5.  Jean-Pierre Husson, La forêt lorraine. Étude de géographie humaine, thèse d’État, Université de Metz, 1987. 3.  Sylvain Guyot, « The Eco-Frontier Paradigm : Rethinking the Links between Space, Nature and Politics », Geopolitics, 16-3 (2011), p. 675-706. 4.  Andrew R. Graybill, « Boundless Nature. Borders and the Environment in North America and Beyond », in A. C. Isenberg et A. R. Graybill (dir.), The Oxford Handbook of Environmental History, Oxford / New York, Oxford University Press, 2014, p. 668-687. Mexico Press, 2001, p. 67-83. 2.  Anne-Laure Amilhat Szary, « “Montagnes mobiles ?” », Journal of Alpine Research | Revue de géographie alpine, 101-2 (2013) [En ligne : https://journals.openedition.org/rga/2135]. 3.  Sylvain Guyot, « The Eco-Frontier Paradigm : Rethinking the Links between Space, Nature and Politics », Geopolitics, 16-3 (2011), p. 675-706. 4.  Andrew R. Graybill, « Boundless Nature. Borders and the Environment in North America and Beyond », in A. C. Isenberg et A. R. Graybill (dir.), The Oxford Handbook of Environmental History, Oxford / New York, Oxford University Press, 2014, p. 668-687. 5.  Jean-Pierre Husson, La forêt lorraine. Étude de géographie humaine, thèse d’État, Université de Metz, 1987. 6.  A. R. Graybill, « Boundless Nature. Borders and the Environment in North America and Beyond », art. cit. 7.  Conrad J. Bahre et Charles F. Hutchinson, « Historic Vegetation Change in La Frontera West of the Rio Grande », in G. L. Webster et C. J. Bahre (dir.), Changing Plant Life of La Frontera : Observations on Vegetation in the U.S./Mexico Borderlands, Albuquerque, University of New Mexico Press, 2001, p. 67-83. BENOIT VAILLOT L’empreinte écologique de la frontière franco-allemande (1871-1914) Profil altimétrique de la frontière franco-allemande (1871-1914) Une approche anthropocentrée nous fait oublier que les forêts sont avant tout le cadre de vie de multiples acteurs « non humains », comme les arbres, les plantes ou même les animaux1. Ces acteurs vivants « non humains » ne sont pas des données immuables, mais le produit de forces historiques. Si l’on prête suffisamment attention à l’environnement, l’étude des frontières révèle l’empreinte des sociétés humaines sur leurs écosys- tèmes. Les forêts reflètent les sociétés humaines dans leurs actions et dans leurs perceptions, et c’est pourquoi elles intéressent l’historien ; elles per- mettent de penser les conditions de production des relations sociales dans des espaces caractérisés par des spécificités environnementales, qu’elles soient climatiques, topographiques ou culturelles2. Il s’agit de proposer une approche environnementale des rapports de domination au sein des sociétés dans leur relation aux acteurs vivants « non humains »3. La question environnementale reste un angle mort de l’historiogra- phie des frontières. Il faut principalement se tourner vers la géographie, la géohistoire en particulier, si l’on veut s’appuyer sur des études articu- lant environnement et frontières4. L’œuvre de Jean-Pierre Husson, auteur d’une histoire de la forêt lorraine dont la grande spécificité est précisément d’avoir été séparée par la frontière franco-allemande entre 1871 et 1918, fait exception5. Certains historiens nord-américains ont par ailleurs précoce- ment étudié l’impact des sociétés humaines sur les écosystèmes en mettant en avant la question des frontières6. Leurs études ont mis en évidence les divergences environnementales qui se sont développées au fur et à mesure de la formation des États. Conrad J. Bahre et Charles F. Hutchinson, par exemple, ont montré en quoi les modifications intervenues dans la flore au xxe siècle le long de la frontière mexicano-étatsunienne, ne sont pas dues aux conditions climatiques, mais bel et bien aux sociétés humaines7. À partir d’une série de photographies des obélisques démarquant la frontière à l’ouest du Rio Grande prises dans les années 1890 et de clichés réalisés entre les années 1960 et 1990, ils retracent l’évolution de la végétation. Frontières et espaces transfrontaliers, une approche environnementale 24 24 Il n’est malheureusement pas possible d’adopter la même méthode, ne disposant pas de photographies ou de descriptions suffisantes de la frontière franco-allemande au début des années 1870. 8.  La Revue des Eaux et Forêts rattachée à l’École nationale des Eaux et Forêts de Nancy, la Forstwissenschaftliches Centralblatt et le Journal für das Forst-, Jagd- und Fischereiwesen qui dépendent de la Höhere Forstlehranstalt d’Eberswalde, sont des revues scientifiques centrali- sant les débats et l’actualité de la science forestière en France et en Allemagne, mais qui ne se limitent pas au cadre national. 9.  Henri Gerdolle, « Die Wälder Deutsch-Lothringen », Deutsche Geographische Blätter, 18 (1877), p. 19-36 ; Id., L’administration forestière allemande en Alsace-Lorraine, Metz, Charles Thomas, 1877. 10.  August Kahl, Der Buchenhochwald auf dem Vogesensandstein der Oberförsterei Bannstein (Canton Bitsch, Lothringen) nach Standort und Art der Verjüngung, Munich, Johannes Falk, 1883 ; Id., Forstgeschichtliche Skizzen aus den Staats- und Gemeindewaldungen von Rappoltsweiler und Reichenweier aus der Zeit vom Ausgange des Mittelalters bis zu Anfang des xix. Jahrhunderts, Strasbourg, Heitz J. H. E, 1894 ; Id., Handbuch für den Elsass-Lothringischen Förster, enthaltend eine Zusammenstellung und Erlaüterung aller wichtigen Gesetze, Verordnungen, Vorschriften fuer die Forstschutzbeamten in den Staats- und ungeteilten Waldungen und in den Gemeinde- und Anstalts-Waldungen, Melsungen, Neudamm, 1900. 8.  La Revue des Eaux et Forêts rattachée à l’École nationale des Eaux et Forêts de Nancy, la Forstwissenschaftliches Centralblatt et le Journal für das Forst-, Jagd- und Fischereiwesen qui dépendent de la Höhere Forstlehranstalt d’Eberswalde, sont des revues scientifiques centrali- sant les débats et l’actualité de la science forestière en France et en Allemagne, mais qui ne se limitent pas au cadre national. 9.  Henri Gerdolle, « Die Wälder Deutsch-Lothringen », Deutsche Geographische Blätter, 18 (1877), p. 19-36 ; Id., L’administration forestière allemande en Alsace-Lorraine, Metz, Charles Thomas, 1877. 10.  August Kahl, Der Buchenhochwald auf dem Vogesensandstein der Oberförsterei Bannstein (Canton Bitsch, Lothringen) nach Standort und Art der Verjüngung, Munich, Johannes Falk, 1883 ; Id., Forstgeschichtliche Skizzen aus den Staats- und Gemeindewaldungen von Rappoltsweiler und Reichenweier aus der Zeit vom Ausgange des Mittelalters bis zu Anfang des xix. Jahrhunderts, Strasbourg, Heitz J. H. E, 1894 ; Id., Handbuch für den Elsass-Lothringischen Förster, enthaltend eine Zusammenstellung und Erlaüterung aller wichtigen Gesetze, Verordnungen, Vorschriften fuer die Forstschutzbeamten in den Staats- und ungeteilten Waldungen und in den Gemeinde- und Anstalts-Waldungen, Melsungen, Neudamm, 1900. 11.  Gustave Huffel, « Les forêts d’Alsace-Lorraine », Revue des Eaux et Forêts, 6 (1918), p. 265-280. 12.  C. Zwilling, « La conversion des taillis sous futaie du département de la Moselle en haute futaie », Revue des Eaux et Forêts, 1921, p. 33-39. 13.  Robin Degron et Jean-Pierre Husson, « Les forêts d’Alsace-Lorraine (1871-1918)  : les contrastes de l’héritage allemand, reflets de l’annexion », Histoire, économie & société, 18-3 (1999), p. 593-611. 13.  Robin Degron et Jean-Pierre Husson, « Les forêts d’Alsace-Lorraine (1871-1918)  : les contrastes de l’héritage allemand, reflets de l’annexion », Histoire, économie & société, 18-3 (1999), p. 593-611. 11.  Gustave Huffel, « Les forêts d’Alsace-Lorraine », Revue des Eaux et Forêts, 6 (1918), p. 265-280. 12.  C. Zwilling, « La conversion des taillis sous futaie du département de la Moselle en haute futaie », Revue des Eaux et Forêts, 1921, p. 33-39. 11.  Gustave Huffel, « Les forêts d’Alsace-Lorraine », Revue des Eaux et Forêts, 6 (1918), p. 265-280. 12.  C. Zwilling, « La conversion des taillis sous futaie du département de la Moselle en haute futaie », Revue des Eaux et Forêts, 1921, p. 33-39. 13.  Robin Degron et Jean-Pierre Husson, « Les forêts d’Alsace-Lorraine (1871-1918)  : les d l’hé i ll d fl d l’ i Hi i é i ié é 1877. 10.  August Kahl, Der Buchenhochwald auf dem Vogesensandstein der Oberförsterei Bannstein (Canton Bitsch, Lothringen) nach Standort und Art der Verjüngung, Munich, Johannes Falk, 1883 ; Id., Forstgeschichtliche Skizzen aus den Staats- und Gemeindewaldungen von Rappoltsweiler und Reichenweier aus der Zeit vom Ausgange des Mittelalters bis zu Anfang des xix. Jahrhunderts, Strasbourg, Heitz J. H. E, 1894 ; Id., Handbuch für den Elsass-Lothringischen Förster, enthaltend eine Zusammenstellung und Erlaüterung aller wichtigen Gesetze, Verordnungen, Vorschriften fuer die Forstschutzbeamten in den Staats- und ungeteilten Waldungen und in den Gemeinde- und Anstalts-Waldungen, Melsungen, Neudamm, 1900. 11.  Gustave Huffel, « Les forêts d’Alsace-Lorraine », Revue des Eaux et Forêts, 6 (1918), p. 265-280. 12.  C. Zwilling, « La conversion des taillis sous futaie du département de la Moselle en haute futaie », Revue des Eaux et Forêts, 1921, p. 33-39. 13.  Robin Degron et Jean-Pierre Husson, « Les forêts d’Alsace-Lorraine (1871-1918)  : les contrastes de l’héritage allemand, reflets de l’annexion », Histoire, économie & société, 18-3 (1999), p. 593-611. BENOIT VAILLOT L’empreinte écologique de la frontière franco-allemande (1871-1914) Même les nombreuses cartes postales réalisées entre les années 1880 et 1914 ne permettent pas de pallier ce manque, car elles sont prises sous des angles trop différents pour établir des comparaisons, et ne sont pas datées avec précision. C’est pourquoi une grande attention a été prêtée à tous ceux qui ont été sensibles à la transformation de l’environnement. À ce titre, les écrits de forestiers publiés dans des revues spécialisées constituent des sources exception- nelles8. Les témoignages des gardes forestiers restés en Alsace-Lorraine après 1871, comme Henri Gerdolle9, et de ceux, formés en Allemagne, qui s’y installent après l’annexion, comme August Kahl10, livrent quantité d’informations sur les changements écologiques qu’introduisent les admi- nistrations forestières. Les rapports de Gustave Huffel11 et C. Zwilling12, qui établissent un bilan de l’administration forestière allemande lors du retour de l’Alsace-Lorraine à la France après la Première Guerre mondiale, sont aussi très précieux, bien qu’ils souffrent d’un parti-pris qu’il faut mettre à distance13. La frontière franco-allemande tracée par le traité de Francfort constitue un poste d’observation privilégié de l’empreinte écologique des sociétés humaines, dans la mesure où s’y confrontent deux conceptions différentes de la gestion de l’environnement entre 1871 et 1914. Les espaces forestiers sont particulièrement intéressants, parce qu’adaptés à l’analyse du « schéma des relations de pouvoirs écologiques »14. Il s’agit d’analyser sur une période très courte comment une frontière transforme l’environ- nement, et l’influence que ce dernier peut avoir sur la construction de la souveraineté et des identités nationales. Cette approche transnationale permet par ailleurs d’éviter l’écueil d’une histoire environnementale dans un cadre national15. 14.  Richard Grove, Ecology, Climate and Empire. Colonialism and Global Environmental History, 1400-1940, Cambridge, White Horse Press, 1997, p. 183. 15.  Richard White, « The Nationalization of Nature », The Journal of American History, 86-3 (1999), p. 976-986. 16.  François Reitel, « Le rôle de l’armée dans la conservation des forêts en France », Bulletin de l’Association des Géographes Français, 501 (1984), p. 143-154. 17.  Jean-Paul Amat, « Le rôle stratégique de la forêt (1871-1914). Exemples dans les forêts lor- raines. », Revue historique des armées, 1 (1993), p. 62-69. 18.  Michaël Séramour, Frontières d’acier. Histoire de la fortification permanente en Lorraine et en Alsace 1871-1945, Saint-Avertin, Éditions Sutton, 2015. 14.  Richard Grove, Ecology, Climate and Empire. Colonialism and Global Environmental History, 1400-1940, Cambridge, White Horse Press, 1997, p. 183. 15.  Richard White, « The Nationalization of Nature », The Journal of American History, 86-3 (1999), p. 976-986. L’AMÉNAGEMENT DE L’ENVIRONNEMENT PAR LES AUTORITÉS MILITAIRES La mise en défense de la frontière franco-allemande a eu des consé- quences majeures sur les forêts, car les autorités militaires assignent à ces dernières un rôle stratégique majeur. Pour reprendre François Reitel, « [l]es forêts permettent de couper les routes d’invasion, de couvrir les places fortes, de canaliser un ennemi vers les forteresses, d’économiser des hommes »16. Après la guerre de 1870, les états-majors des deux pays ont dû repenser les défenses nationales pour s’adapter aux nouvelles réalités physiques de la frontière17. La situation est très préoccupante pour la France : si la citadelle de Belfort a finalement été conservée, Paris n’est plus qu’à 300 kilomètres de l’Allemagne. Inversement, le contrôle de la majeure partie du massif vosgien et la possession des places fortes de Metz et Strasbourg per- mettent à l’Empire allemand d’exclure toute menace d’offensive française à court et moyen terme. Dès le début des années 1870, plusieurs chantiers de fortification débutent de part et d’autre de la frontière, afin de consti- tuer des obstacles partout où les hauteurs et les forêts peuvent manquer. Les forts allemands de type Biehler sont construits autour de Strasbourg entre 1872 et 188518. L’extension des forts autour de Metz, débutée par les autorités militaires françaises, est achevée au profit de l’Empire allemand. L’état-major français déploie de son côté le programme Séré de Rivières Frontières et espaces transfrontaliers, une approche environnementale 26 26 à partir de 1874, qui fait sortir de terre toute une série de forts autour de Verdun, Toul et Épinal afin de former des camps retranchés, ceinturés de forts détachés. La place forte de Belfort est renforcée afin de verrouiller la trouée du même nom. Ces différents môles de résistance ont pour but de contraindre l’ennemi à s’engager dans les trouées de Stenay et des Charmes, densément boisées. Après 1885, l’état-major français se borne principalement à moderniser les ouvrages principaux et à aménager les abords. Tout au contraire, la place de Strasbourg est renforcée. L’état-major allemand entreprend, à partir des années 1890, un programme de fortification de Metz qui en fait le pivot de sa défense face à la France. Une ligne défensive allant de Thionville à Metz – appelée Moselstellung – est édifiée à partir de 1899 et devient très vite le plus grand ensemble fortifié au monde19. La trouée de Lorraine, quant à elle, est défendue par les garnisons allemandes. 19.  Clayton Donnell et Brian Delf, The German Fortress of Metz 1870-1944, Oxford/New York, Osprey Publishing, 2008. 20.  François Roth, « La frontière franco-allemande, 1871-1918 », Annales de l’Est, 1 (1992), p. 35-52. 21.  Archives du Territoire de Belfort [désormais AD90], 1 M 388, Courrier du chef de Génie de Belfort à l’inspecteur adjoint des Eaux et Forêts de Belfort, 29 mars 1893. 22.  AD90, 1  M  385, Courrier du Conservateur des Forêts de Besançon à l’Inspecteur adjoint des Forêts de Belfort, 10 avril 1877 ; Archives départementales du Haut-Rhin [désormais AD68], 8 AL 1/7152, Construction de nouveaux chemins, routes et voies forestières près de la frontière, 1892-1914. 21.  Archives du Territoire de Belfort [désormais AD90], 1 M 388, Courrier du chef de Génie de Belfort à l’inspecteur adjoint des Eaux et Forêts de Belfort, 29 mars 1893. 22.  AD90, 1  M  385, Courrier du Conservateur des Forêts de Besançon à l’Inspecteur adjoint des Forêts de Belfort, 10 avril 1877 ; Archives départementales du Haut-Rhin [désormais AD68], 8 AL 1/7152, Construction de nouveaux chemins, routes et voies forestières près de la frontière, 1892-1914. L’AMÉNAGEMENT DE L’ENVIRONNEMENT PAR LES AUTORITÉS MILITAIRES À la veille de la Première Guerre mondiale, les départements frontaliers forment la première ligne de défense française, tandis que l’Alsace-Lorraine remplit le rôle de glacis militaire de l’Empire allemand. Les ouvrages militaires ne constituent pas de lignes de défense continue comme le seront plus tard les lignes Maginot ou le Westwall, ce qui explique pourquoi l’expérience sensible des habitants de la frontière est plus marquée par le paysage forestier que par les forts et forteresses : « À la frontière on ne voyait ni blockhaus ni barbelés mais le calme étendu des champs et des forêts »20. Fig. 2. L’intégration des espaces forestiers frontaliers à la défense Fig. 2. L’intégration des espaces forestiers frontaliers à la défense L’absence ou la présence d’espaces forestiers à la frontière est fortement corrélée aux nécessités de défense nationale. Les défrichements et aména- gements forestiers sont soumis à l’autorisation d’une commission mixte des travaux publics, au sein de laquelle le génie militaire a voix prépondérante21. Les travaux de voies de communication compris non loin des ouvrages de défense doivent recevoir l’adhésion des autorités militaires22. Même le dégagement de la frontière et la construction de chemins de schlittage pour l’exploitation forestière en haute montagne ne sauraient s’en passer. En 1897, le colonel commandant du 152e régiment d’infanterie demande à ce que la section vosgienne (Nancy) du Club alpin français renonce à baliser Frontières et espaces transfrontaliers, une approche environnementale 28 28 les sentiers qui mènent à la frontière23. L’autorité militaire empêche parti- culièrement la construction de routes empierrées ou bitumées de plus de quatre mètres de large, car les voitures militaires pourraient l’emprunter. q g p p Les autorités militaires ont donc non seulement assumé de fait un rôle de conservation du couvert forestier, en raison du rôle défensif qu’elles lui attribuent, mais elles participent aussi au reboisement. Les bois de la rive droite de la Moselle, par exemple, sont propriété de l’armée allemande et protégés en raison de leur rôle stratégique et ce malgré la forte pression urbaine de l’agglomération de Metz à la fin du xixe siècle. En France, planter des arbres à la frontière devient, après 1871, un acte patriotique, car c’est préparer une « barrière » contre une éventuelle invasion allemande. 23.  Archives départementales de Meurthe-et-Moselle [désormais AD54], 4 M 179, Courrier du général commandant le 6e Corps d’armée au préfet de Meurthe-et-Moselle, 23 juillet 1897. 24.  Jean-Pierre Husson, « Les paysages anciens de la forêt domaniale de Gérardmer : contribu- tion patrimoniale à la révision d’aménagement », Revue Forestière Française, 5 (1997), p. 469-476. 25.  J.-P. Husson, La forêt lorraine, op. cit, p. 314. 26.  F. Reitel, « Le rôle de l’armée dans la conservation des forêts en France », art. cit. 27.  Jean-Paul Amat, « Guerre et milieux naturels : les forêts meurtries de l’Est de la France, 70 ans après Verdun », L’Espace géographique, 16-3 (1987), p. 217-233. 27.  Jean-Paul Amat, « Guerre et milieux naturels : les forêts meurtries de l’Est de la France, 70 ans après Verdun », L’Espace géographique, 16-3 (1987), p. 217-233. 26.  F. Reitel, « Le rôle de l’armée dans la conservation des forêts en France », art. cit. L’AMÉNAGEMENT DE L’ENVIRONNEMENT PAR LES AUTORITÉS MILITAIRES La tempête de 1902, qui provoque de nombreux chablis (chutes d’arbres) sur le versant français des Vosges, en particulier dans la forêt de Gérardmer, est vécue comme un traumatisme par les populations et l’administration des Eaux et Forêts, car elle découvre la frontière24. Le gouvernement d’Al- sace-Lorraine ne dépense pas moins de 50 000 marks par an pour l’achat de friches, afin de constituer des boisements de protection25. Jusqu’en 1914, les surfaces forestières progressent tout le long de la frontière, à l’exception du pays messin et thionvillois, en raison de la construction de la Moselstellung qui nécessite le défrichement de plusieurs bois. L’empreinte militaire sur l’environnement est rendue visible à travers les aménagements effectués dans les forêts26. Les peuplements forestiers frontaliers sont percés de trouées qui n’ont pas pour objet de régénérer les bois, mais de les rendre praticables par des hommes en armes27. Inversement, des rideaux d’arbres sont plantés pour constituer des barrières naturelles censées empêcher toute progression ennemie. Les forêts à l’est de Verdun, entre les forts de Douaumont et de Vaux, sont ainsi aménagées pour servir d’écran de protection. L’édification d’ouvrages militaires nécessite parfois le déboisement afin de dégager des vues, d’ouvrir des axes de tir, de construire des redoutes, de creuser des retranchements, de percer des lignes de chemin de fer, etc. Les forêts domaniales, communales et particulières qui entourent Épinal en 1883 sont ainsi déboisées28. À la frontière, l’expropria- tion des bois pour raison d’utilité publique est courante29. La commune de Chavelot, par exemple, est contrainte de se dessaisir de ses forêts en 1887 pour permettre la modernisation des forts d’Uxegney et de Grande-Haye. La progression des surfaces forestières à la frontière sur la période est en grande partie due à l’intervention des états-majors. La qualité et la diversité des forêts sont quant à elles profondément modifiées par les admi- nistrations forestières françaises et allemandes, qui adoptent des méthodes sylvicoles différentes. entourent Épinal en 1883 sont ainsi déboisées28. À la frontière, l’expropria- tion des bois pour raison d’utilité publique est courante29. La commune de Chavelot, par exemple, est contrainte de se dessaisir de ses forêts en 1887 pour permettre la modernisation des forts d’Uxegney et de Grande-Haye. La progression des surfaces forestières à la frontière sur la période est en grande partie due à l’intervention des états-majors. L’AMÉNAGEMENT DE L’ENVIRONNEMENT PAR LES AUTORITÉS MILITAIRES La qualité et la diversité des forêts sont quant à elles profondément modifiées par les admi- nistrations forestières françaises et allemandes, qui adoptent des méthodes sylvicoles différentes. 33.  Thaddeus Sunseri, « Reinterpreting a Colonial Rebellion  : Forestry and Social Control in German East Africa, 1874-1915 », Environmental History, 8-3 (2003), p. 430-451 : Id., « Exploiting the Urwald : German Post-Colonial Forestry in Poland and Central Africa, 1900–1960 », Past & Present, 214-1 (2012), p. 305-342 ; Jawad Daheur, « La forêt : espace de légitimation du projet colonial allemand en Chine (1898-1914) », Recherches germaniques, 43 (2013), p. 95-110. 31.  Michael Imort, « Wilhelmine Forestry and the Forest as a Symbol of Germandom », in T. Zelle et T. Lekan (dir.), Germany’s Nature: Cultural Landscapes and Environmental History, New Brunswick, Rutgers University Press, 2005, p. 55-60. 28.  Archives départementales des Vosges [désormais AD88], 38 R 12, Arrêté préfectoral concer- nant les travaux de défense, 12 avril 1883. 29.  F. Reitel, « Le rôle de l’armée dans la conservation des forêts en France », art. cit. 30.  Heinrich Rubner, « Geistige Strömungen in der Forstpolitik und ihre Auswirkungen auf die Forstwirtschaft des neuzeitlichen Frankreich », Allgemeine Forst und Jagdzeitung, 1 (1993), p. 12-17. 31.  Michael Imort, « Wilhelmine Forestry and the Forest as a Symbol of Germandom », in T. Zeller et T. Lekan (dir.), Germany’s Nature: Cultural Landscapes and Environmental History, New Brunswick, Rutgers University Press, 2005, p. 55-60. 32.  Jawad Daheur, Le Parc à bois de l’Allemagne. Course aux ressources et hégémonie commer- ciale dans les bassins de la Vistule et de la Warta (1840-1914), thèse de doctorat, Université de Strasbourg, 2016 ; Jeffrey K. Wilson, « Environmental Chauvinism in the Prussian East : Forestry as a Civilizing Mission on the Ethnic Frontier, 1871-1914 », Central European History, 41-1 (2008), p. 27-70. 33.  Thaddeus Sunseri, « Reinterpreting a Colonial Rebellion  : Forestry and Social Control in German East Africa, 1874-1915 », Environmental History, 8-3 (2003), p. 430-451 : Id., « Exploiting the Urwald : German Post-Colonial Forestry in Poland and Central Africa, 1900–1960 », Past & Present, 214-1 (2012), p. 305-342 ; Jawad Daheur, « La forêt : espace de légitimation du projet colonial allemand en Chine (1898-1914) », Recherches germaniques, 43 (2013), p. 95-110. 28.  Archives départementales des Vosges [désormais AD88], 38 R 12, Arrêté préfectoral concer- nant les travaux de défense, 12 avril 1883. 30.  Heinrich Rubner, « Geistige Strömungen in der Forstpolitik und ihre Auswirkungen auf die Forstwirtschaft des neuzeitlichen Frankreich », Allgemeine Forst und Jagdzeitung, 1 (1993), p. 12-17. 29.  F. Reitel, « Le rôle de l’armée dans la conservation des forêts en France », art. cit. 32.  Jawad Daheur, Le Parc à bois de l’Allemagne. Course aux ressources et hégémonie commer- ciale dans les bassins de la Vistule et de la Warta (1840-1914), thèse de doctorat, Université de Strasbourg, 2016 ; Jeffrey K. Wilson, « Environmental Chauvinism in the Prussian East : Forestry as a Civilizing Mission on the Ethnic Frontier, 1871-1914 », Central European History, 41-1 (2008), p. 27-70. LA CONFRONTATION DE DEUX SYLVICULTURES L’historien doit maintenant tâcher de pénétrer à l’intérieur de ces forêts, traversées par la frontière du traité de Francfort, soumises à deux sylvicultures différentes30. L’analyse des rapports de pouvoir liés à l’ex- ploitation des forêts en Alsace-Lorraine, mis en perspective avec ce qui est pratiqué dans l’Est de la France à la même époque, révèle un « impé- rialisme écologique » allemand31. À travers l’introduction de nouvelles méthodes sylvicoles, les autorités allemandes exercent leur domination sur les populations d’Alsace-Lorraine, d’une manière comparable à celle rencontrée dans les provinces polonaises de Prusse32 et dans les territoires coloniaux allemands33. 33. Thaddeus Sunseri, « Reinterpreting a Colonial Rebellion  : Forestry and Social Control in German East Africa, 1874-1915 », Environmental History, 8-3 (2003), p. 430-451 : Id., « Exploiting the Urwald : German Post-Colonial Forestry in Poland and Central Africa, 1900–1960 », Past & Present, 214-1 (2012), p. 305-342 ; Jawad Daheur, « La forêt : espace de légitimation du projet colonial allemand en Chine (1898-1914) », Recherches germaniques, 43 (2013), p. 95-110. Frontières et espaces transfrontaliers, une approche environnementale 30 30 La science forestière allemande au xixe siècle est pionnière à bien des égards et jouit d’une grande renommée internationale34. Le pivot de cette expertise repose sur la Höhere Forstlehranstalt (établissement supérieur de foresterie) d’Eberswalde, où sont formés les gardes forestiers de Prusse depuis 183035. La création concomitante de l’École nationale des Eaux et Forêts à Nancy n’a pas d’autre origine que la volonté de rivaliser avec l’Alle- magne dans ce domaine. La rivalité franco-allemande s’exprime avec éclat à la frontière, où des expériences sylvicoles sont effectuées pour démontrer sa prétendue supériorité. Ainsi, Hermann Bertog, professeur à la Höhere Forstlehranstalt, assiste en 1897 aux expériences sylvicoles effectuées dans la forêt de Nancy36. Les sciences forestières française et prussienne ne se distinguent pas tant par leurs objectifs sylvicoles – le reboisement, la conversion des taillis en futaies, l’enrésinement, etc. – que par les méthodes mises en œuvre pour y parvenir, méthodes censées refléter le génie national37. L’annexion de quelque 135 000 hectares de forêts doma- niales et de 200 000 hectares de forêts communales ou appartenant à des établissements publics offrent aux autorités allemandes l’occasion d’appli- quer à l’échelle de l’Alsace-Lorraine, les méthodes de la science forestière prussienne et de mettre fin à une gestion française jugée calamiteuse38. 34.  Henry E. Lowood, « The Calculating Forester: Quantification, Cameral Science, and the Emergence of Scientific Forestry Management in Germany », in T. Frängsmyr, J. L. Heilbron et R. E. Rider (dir.), The quantifying spirit in the 18th century, Berkeley, University of California Press, 1990, p. 315-342. 35.  H. Rubner, « Geistige Strömungen in der Forstpolitik und ihre Auswirkungen auf die Forstwirtschaft des neuzeitlichen Frankreich », art. cit. 38.  F. von Etzel, Aus dem Reichslande, Berlin, Julius Springer, 1876 ; et plus tard, Karl Eduard Ney, Zur xix. Versammlung des Elsaß-Lothringischen Forstvereins, 1897. 36.  Archives diplomatiques [désormais Arch. Dipl.], 131 CPCOM 131, Demande de Mr. Bertog, Visite de l’école forestière de Nancy. 37.  Jean-Pierre Husson, Les forêts françaises, Nancy, Presses universitaires de Nancy, 1995, p. 156. 39.  H. Gerdolle, L’administration forestière allemande en Alsace-Lorraine, op. cit. 40.  Archives départementales du Bas-Rhin [désormais AD67], 27 AL 1064 et 1065, Fonctionnaires de l’administration forestière, 1879-1916. 41.  Alexandre Barte de Sainte-Fare, « Le régime forestier en Alsace-Lorraine », Revue des Eaux et Forêts, 1879, p. 5-13 et p. 97-115. 38.  F. von Etzel, Aus dem Reichslande, Berlin, Julius Springer, 1876 ; e Zur xix. Versammlung des Elsaß-Lothringischen Forstvereins, 1897. LA CONFRONTATION DE DEUX SYLVICULTURES Avant même la signature du traité de Francfort, un Oberforstbeamter en charge de l’administration forestière est détaché auprès du commis- saire civil pour l’Alsace-Lorraine39. Très vite, l’administration forestière est réorganisée sur le modèle prussien40. Les Oberförstereien ont une superficie de moyenne de 5 600 hectares contre 6 000 à 7 000 pour les cantonne- ments français41. En revanche, les triages sont plus vastes et s’étendent en moyenne sur 600 hectares. Si les prérogatives de police des espaces naturels et des forêts des gardes forestiers français et allemands sont similaires, leur rôle de gestion et de protection des forêts est extrêmement différent. La ­hiérarchie­ de la nouvelle administration forestière d’Alsace-Lorraine est simplifiée, et ses agents disposent de plus de responsabilités. Les gardes forestiers allemands disposent d’une plus grande autonomie que leurs homologues français ; ils sont chargés de réviser les limites parcellaires, de dresser les états d’assiette et de travail d’amélioration, de marteler les coupes, de les exploiter, de les recevoir et de les vendre. L’exploitation des forêts communales et domaniales du massif des Vosges diffère ainsi profondément selon le côté de la frontière où l’on se situe42. Sur le versant français, le bois est vendu sur pied après estimation du volume et c’est à l’acheteur d’en organiser la coupe. La montagne est parsemée de scieries communales ou domaniales, attribuées par adjudi- cation. Les ouvriers qui y travaillent, appelés sagards, logent sur place et sont rémunérés par l’adjudicataire du marché public43. C’est un tout autre système qui prévaut sur le versant allemand, où l’exploitation se fait en régie. L’administration des forêts prend à sa charge l’abattage des arbres et vend le bois coupé en petits lots. Ce mode d’exploitation est plus coûteux en personnel, mais permet d’augmenter considérablement les revenus de la gestion forestière44. Les scieries communales et domaniales n’ont ainsi plus aucune utilité, et sont donc vendues ou louées, et parfois transformées en maisons forestières. Sur toute la période, les gardes forestiers allemands améliorent les dessertes forestières dans les Vosges et n’hésitent pas à créer 100 kilomètres de nouvelles pistes alors qu’en France, le génie militaire s’oppose à tous travaux d’envergure45. Au tournant du siècle, un petit réseau de chemins de fer à usage forestier voit même le jour, et permet notamment l’exploi- tation du massif du Donon. 42.  H. Gerdolle, L’administration forestière allemande en Alsace-Lorraine, op. cit. 43.  Marc Brignon et Jean-Louis Boithias, Les scieries et les anciens sagards des Vosges. Bûcherons, schlitteurs, voituriers et voileurs, Nonette, Créer, 1985. 44.  Charles Grad, Considérations sur les finances et l’administration de l’Alsace-Lorraine sous la régime allemand, Paris, Germer-Baillère, 1877. 45.  R. Degron et J.-P. Husson, « Les forêts d’Alsace-Lorraine (1871-1918) », art. cit. LA CONFRONTATION DE DEUX SYLVICULTURES Sur le versant allemand des Vosges, les coûts d’exploitation sont sensiblement plus bas que sur le versant français, où l’on débarde encore le bois par schlittage, flottage et traction animale. Les travaux d’aménagement de l’administration des forêts d’Alsace-Lorraine favorisent l’augmentation des investissements forestiers au moment même où s’accroît la demande de l’industrie locale. Frontières et espaces transfrontaliers, une approche environnementale 32 Frontières et espaces transfrontaliers, une approche environnementale 32 Les forêts du versant allemand des Vosges ne sont pas seulement exploitées avec plus d’efficacité que celles du versant français, elles sont aussi aménagées pour correspondre à la représentation que l’imaginaire national allemand s’en fait au xixe siècle46. L’administration des forêts d’Alsace-Lorraine accorde très tôt une place aux activités récréatives dans l’aménagement des forêts, particulièrement près du Donon, ce qui est inédit de l’autre côté de la frontière. Les Oberförster sont d’ailleurs souvent à la tête des sections locales du Vogesenclub, association de montagne fondée par les autorités allemandes47. L’exploitation des forêts communales et domaniales d’Alsace-Lorraine est tellement efficace qu’elle représente 11 % des revenus du Reichsland en 1913, contre seulement 0,7 % en France48. La principale question qui occupe les forestiers français et allemands au xixe siècle est celle de la conversion des taillis en futaie, c’est-à-dire la transformation de parcelles forestières combinant plusieurs niveaux de végétations différents (taillis-sous-futaie) en forêts de grands arbres aux fûts dégagés (futaie)49. Cette dernière est entreprise de façon radicalement différente de part et d’autre de la frontière. La surexploitation des forêts du versant allemand a conduit l’administration des forêts d’Alsace-Lor- raine à recourir à la régénération artificielle des parcelles forestières, chaque fois que la conversion en essences nobles et délicates était lente ou difficile. Cette régénération consiste à réaliser des coupes rases suivies de plantations, et s’oppose à la régénération naturelle privilégiée sur le versant français. En effet, les gardes forestiers français opèrent progressi- vement et prudemment la conversion des taillis en futaie. Les forêts sont régénérées par la réalisation d’éclaircies successives – c’est-à-dire par des coupes d’amélioration – destinées à favoriser la croissance du peuplement. Les arbres porte-graines sont ensuite dégagés par des coupes de régénéra- tion qui laissent aux jeunes semis la lumière dont ils ont besoin. Sur un même massif forestier, coexistent deux conceptions différentes de la forêt : peu aménagée et « naturelle » sur le versant français ; ordonnée et « artifi- cielle » sur le versant allemand. 46.  Jeffrey K. Wilson, The German Forest: Nature, Identity, and the Contestation of a National Symbol, 1871-1914, Toronto/Buffalo/Londres, University of Toronto Press, 2012. 47.  L’association prend le nom de Club Vosgien lors de la réunion de l’Alsace-Lorraine à la France en 1918. 48.  G. Huffel, « Les forêts d’Alsace-Lorraine », art. cit. 49.  L. Badre, « Évolution des aménagements des futaies résineuses dans l’Est de la France et plus spécialement dans le Haut-Rhin », Revue Forestière Française, 7-8 (1952), p. 500-506. 49.  L. Badre, « Évolution des aménagements des futaies résineuses dans l’Est de la France et plus spécialement dans le Haut-Rhin », Revue Forestière Française, 7-8 (1952), p. 500-506. 47.  L’association prend le nom de Club Vosgien lors de la réunion de l’Alsace-Lorraine à la France en 1918.f 50.  Jeffrey K. Wilson, « Environmental Chauvinism in the Prussian East : Forestry as a Civilizing Mission on the Ethnic Frontier, 1871-1914 », Central European History, 41-1 (2008), p. 27-70. 51.  La forêt de Dabo est l’exemple le plus connu d’échec d’extinction des droits ruraux. Jean- Pierre Husson, « L’héritage forestier prussien en Moselle et son évolution récente », Bulletin de l’Association de Géographes Français, 65-3 (1988), p. 217-225. 52.  G. Huffel, « Les forêts d’Alsace-Lorraine », art. cit. 53.  Michel Devèze, « Forêts françaises et forêts allemandes. Étude historique comparée », Revue historique, 235 (1966), p. 347-380 et 236 (1966), p. 47-68. 54.  Simon Schama, Le paysage et la mémoire, Paris, Seuil, 1999. LA CONFRONTATION DE DEUX SYLVICULTURES La conversion des taillis en futaie met en miroir deux sociétés dif- férentes, et deux méthodes de gestion dont les habitants ont pleinement conscience. La mise en place d’une politique sylvicole moderne et rationnelle sur le modèle prussien en Alsace-Lorraine est animée par un chauvinisme environnemental qui ne laisse pas de place aux acteurs locaux50. L’abandon brutal des taillis-sous-futaie, qui procuraient du bois de chauffage aux populations rurales, par exemple, n’est rendu possible que par l’existence d’un pouvoir autoritaire. Cette politique a par ailleurs rencontré des échecs dans les forêts domaniales51. Inversement, en France, le conservatisme de l’administration des Eaux et Forêts, qui prône une conversion progressive des taillis en futaie, correspond à une plus grande prise en compte des populations rurales. Sur les deux versants des Vosges, les États cherchent à éteindre les droits ruraux comme l’affouage, qui réserve une partie des bois des forêts communales à l’usage domestique des habitants, mais les consé- quences ne sont pas les mêmes. Sous la iiie République, les habitants des campagnes sont des citoyens qui ont des moyens juridiques et politiques pour défendre leurs droits, là où il est difficile d’aller contre la puissance publique dans l’Empire allemand. La conversion des taillis en futaie et l’extinction des droits ruraux sont donc loin d’être achevées sur le versant français des Vosges à la veille de la Première guerre mondiale, et quasiment réalisée sur le versant allemand52. Dans un même massif forestier, deux types de sylviculture coexistent et s’affrontent : intensive du côté allemand, extensive du côté français. Elles reflètent des rapports à l’environnement différents53. La frontière est ainsi productrice de différentiation spatiale jusque dans les espaces forestiers les plus reculés, ce qui n’est pas sans conséquence sur les paysages. 55.  Louis Rudault, « Comparaison entre les méthodes forestières françaises et allemandes appli- quées en Alsace », Revue des Eaux et Forêts, 1935, p. 831-844 et p. 908-921. 57.  F. von Etzel, Aus dem Reichslande, op. cit. 58.  Adolphe Tétreau, Commentaire de la loi du 4 avril 1882 sur la restauration et la conservation des terrains en montagne, Paul Dupont, 1883. 59.  C. Zwilling, « La conversion des taillis sous futaie du département de la Moselle en haute futaie », art. cit. 55.  Louis Rudault, « Comparaison entre les méthodes forestières françaises et allemandes appli- quées en Alsace », Revue des Eaux et Forêts, 1935, p. 831-844 et p. 908-921. 56.  Ibid. 57.  F. von Etzel, Aus dem Reichslande, op. cit. 58.  Adolphe Tétreau, Commentaire de la loi du 4 avril 1882 sur la restauration et la conservation des terrains en montagne, Paul Dupont, 1883. 59.  C. Zwilling, « La conversion des taillis sous futaie du département de la Moselle en haute futaie », art. cit. 58.  Adolphe Tétreau, Commentaire de la loi du 4 avril 1882 sur la restauration et la conservation des terrains en montagne, Paul Dupont, 1883. 59.  C. Zwilling, « La conversion des taillis sous futaie du département de la Moselle en haute f i i 8.  Adolphe Tétreau, Commentaire de la loi du es terrains en montagne, Paul Dupont, 1883. 60.  Vincent Moriniaux, « Les Français face à l’enrésinement, xvie-xxe siècles », Annales de géo- graphie, 609-610 (1999), p. 660-663. 61.  R. Degron et J.-P. Husson, « Les forêts d’Alsace-Lorraine (1871-1918) », art. cit. 62.  C. Zwilling, « La conversion des taillis sous futaie du département de la Moselle en haute futaie », art. cit. 63.  Gérard Leser, « Le sapin dans les rites, les croyances populaires et l’imaginaire alsacien », in A. Corvol (dir.), Le sapin. Enjeux anciens, enjeux actuels, Paris, L’Harmattan, 2001, p. 349-370. 64.  Philippe Guinier, « Trois conifères de la flore vosgienne », Bulletin de la Société Botanique de France, 106 (1959), p. 168-183. 65.  Jean-Pierre Husson, « Forêts sommitales et chaumes vosgiennes confrontées à l’essor touris- tique », Bulletin de l’Association de Géographes Français, 74-3 (1997), p. 320-328. LA TRANSFORMATION DES PAYSAGES FORESTIERS L’administration des forêts allemande a particulièrement modifié le paysage en introduisant un grand nombre d’essences d’arbres. Les paysages que nous croyons les plus épargnés par l’intervention humaine se révèlent parfois, à y regarder de plus près, ceux qui en sont le plus pétris54. À la veille de la Première Guerre mondiale, le garde forestier Louis Rudault Frontières et espaces transfrontaliers, une approche environnementale 34 34 est frappé de constater qu’en n’importe quel point, l’observateur même le moins averti est frappé par l’aspect différent des sapinières selon le côté de la frontière55. Alors que sur le côté français, les futaies régulières sont quasi absentes, sur le versant allemand des Vosges, on aperçoit des futaies régu- lières avec des peuplements équiennes (composés d’arbres ayant le même âge). C’est également vrai ailleurs, par exemple dans les bois entre Réchésy et Pfetterhouse qui jouxtent la frontière de la Suisse, où un « médiocre tail- lis-sous-futaie » en territoire français rencontre un « magnifique perchis de hêtre et de chêne » en territoire allemand56.f Les différences des cycles de coupe et de régénération des forêts l’ex- pliquent en grande partie57. En France, les pins sont abattus à l’âge de 80 ans, les sapins et les hêtres lorsqu’ils sont centenaires, et les chênes seulement après 120 ans. L’administration des forêts d’Alsace-Lorraine, pour sa part, ne coupe les sapins, les pins et les hêtres qu’une fois âgés de 120 ans et les chênes après 160 ans. À mesure que l’on avance dans la période, les classes d’âge et les essences des arbres cohabitant sur une même parcelle boisée tendent à être plus homogènes sur le territoire allemand. Les gardes forestiers ont beau, par exemple, planter le même chêne rouge de part et d’autre de la frontière, les forêts françaises ont une plus grande diversité de classes d’âge et d’essences d’arbres en raison de la méthode de régénération naturelle employée par l’administration des Eaux et Forêts. En territoire français, les coupes d’arbres sont nombreuses mais sélectives, ce qui favorise une certaine permanence du paysage, d’autant plus que la loi du 4 avril 1882 élève la forêt au rang de patrimoine national à aménager avec prudence58. Les agents forestiers allemands réalisent quant à eux des coupes rases pour régénérer les parcelles, ce qui modifie radicalement le paysage59. Fig. 3. LA TRANSFORMATION DES PAYSAGES FORESTIERS Les différentes formes de reboisement du massif vosgien (début xxe siècle) Benoit Vaillot | L’empreinte écologique de la frontière franco-allemande 35 Fig. 3. Les différentes formes de reboisement du massif vosgien (début xxe siècle) La transformation des paysages forestiers au xixe siècle est princi- palement due au reboisement qui s’est réalisé à travers l’enrésinement60. Après 1871, la substitution des feuillus par des résineux à la croissance plus rapide est mise en œuvre de façon tout à fait différente de part et d’autre de la frontière. L’enrésinement devient systématique en Alsace-Lorraine à mesure que le taillis-sous-futaie est abandonné, même si, dans la partie la plus occidentale de la frontière, la rupture avec l’administration française est moins nette61. Les gardes forestiers allemands privilégient les pins et les épicéas pour réaliser rapidement une futaie et combler les vides62. Il faut tout de même mentionner l’introduction du sapin de Nordmann dans le massif du Champ du Feu à partir de 189063. L’administration des Eaux et Forêts fait preuve, quant à elle, d’un conservatisme affirmé en favo- risant le sapin blanc, que l’on trouve à l’état naturel dans les Vosges, et le pin sylvestre64. L’administration des forêts d’Alsace-Lorraine favorise également le pin noir et le pin blanc, dont l’introduction dans les Vosges remonte au début du xixe siècle, et tente d’acclimater de nouvelles espèces, en parti- culier le pin gris65. Le pin de montagne est la manifestation la plus nette de l’impérialisme écologique allemand. En effet, il est systématiquement Frontières et espaces transfrontaliers, une approche environnementale 36 36 planté du Lac noir jusqu’à la Tête des Faux, dominant le col du Bonhomme, entre 800 et 1 200 mètres d’altitude66. Les gardes forestiers allemands pri- vilégient une unité paysagère avec la Forêt-Noire, où cette essence est très présente ; ce faisant, ils recomposent un paysage familier et homogène de part et d’autre du Rhin67. Même lorsque l’épicéa – quasiment absent des Vosges avant 1871 – est utilisé des deux côtés de la frontière, l’enrésinement diffère profondément. Les gardes forestiers allemands emploient l’épicéa commun de façon systé- matique pour le reboisement après les violentes tempêtes de 1898 et 1902, et introduisent l’épinette blanche et l’épinette de Sitka. Plus de 1 000 hectares d’épicéas sont plantés pour maintenir le potentiel productif du versant allemand des Vosges, ce qui modifie le paysage de la vallée de la Bruche, par exemple68. 69.  A. Bérard, « Les reboisements artificiels dans les forêts vosgiennes au xixe siècle », Revue Forestière Française, 5 (1959), p. 371-375. 70.  Andrée Corvol, Le sapin. Enjeux anciens, enjeux actuels, Paris, L’Harmattan, 2001 ; Bernard Debarbieux, « Imaginaires nationaux et post-nationaux du lieu », Communications, 87 (2012), p. 27-41. 66.  P. Guinier, « Trois conifères de la flore vosgienne », art. cit. 67.  D. Doll, « L’évolution des essences forestières dans le canton de Munster (1850-1950) », Revue Forestière Française, 6 (1985), p. 491-500. 68.  Le pays welche s’étend sur la vallée de la Bruche, le val d’Argent et le val d’Orbey, et situé non loin de la frontière. Éric Maire et Sylvie Guillerme, « L’évolution d’un paysage pastoral identitaire : la moyenne montagne du Pays Welche, xviiie-xxe siècle », in A. Corvol, C. Dereix, P. Gresser et F. Lormant (dir.), Forêt et montagne, Paris, L’Harmattan, 2015, p. 143-166. 69.  A. Bérard, « Les reboisements artificiels dans les forêts vosgiennes au xixe siècle », Revue Forestière Française, 5 (1959), p. 371-375. 70.  Andrée Corvol, Le sapin. Enjeux anciens, enjeux actuels, Paris, L’Harmattan, 2001 ; Bernard Debarbieux, « Imaginaires nationaux et post-nationaux du lieu », Communications, 87 (2012), p. 27-41. 71.  AD88, 109 P 3, Courrier du préfet des Vosges au directeur des douanes d’Épinal, 3 mars 1903. 68.  Le pays welche s’étend sur la vallée de la Bruche, le val d’Argent et le val d’Orbey, et situé non loin de la frontière. Éric Maire et Sylvie Guillerme, « L’évolution d’un paysage pastoral dentitaire : la moyenne montagne du Pays Welche, xviiie-xxe siècle », in A. Corvol, C. Dereix, P. Gresser et F. Lormant (dir.), Forêt et montagne, Paris, L’Harmattan, 2015, p. 143-166.i 71.  AD88, 109 P 3, Courrier du préfet des Vosges au directeur des douanes d’Épinal, 3 mars 1903. 67.  D. Doll, « L’évolution des essences forestières dans le canton de Munster (1850-1950) », Revue Forestière Française, 6 (1985), p. 491-500. 66.  P. Guinier, « Trois conifères de la flore vosgienne », art. cit. LA TRANSFORMATION DES PAYSAGES FORESTIERS Leurs collègues français ne jurent que par le sapin blanc et n’utilisent l’épicéa commun qu’à défaut de mieux pour reconstituer le sol forestier lors de la reconquête d’espaces agricoles69. Le sapin des Vosges est devenu, dans la France de la fin du xixe siècle, un véritable géo-sym- bole70. Les propriétaires privés importent néanmoins des milliers de plants d’épicéa au début du xxe siècle pour reboiser les communes fran- çaises voisines de la frontière. Pas moins de 5 000 plants d’épicéa sont ainsi repiqués à Laveline en 190371. Les gardes forestiers d’Alsace-Lor- raine formés à Eberswalde introduisent des espèces de pin et d’épicéa qui composent près des deux tiers des forêts de Prusse ; a contrario, le sapin est absent d’Allemagne du Nord. Fig. 4. Les différences de paysage dans le massif vosgien (début xxe siècle) Benoit Vaillot | L’empreinte écologique de la frontière franco-allemande 37 Fig. 4. Les différences de paysage dans le massif vosgien (début xxe siècle) À la veille de la Première Guerre mondiale, l’introduction de plusieurs espèces de pins a tellement modifié le paysage forestier du côté allemand que les espèces de sapins indigènes reculent et deviennent minoritaires en certains endroits. L’impérialisme écologique allemand a aussi eu des conséquences indirectes sur l’environnement. Les conditions de gestion et d’exploitation des forêts ont profondément modifié les milieux. Partout où les landes et forêts clairières, composées de pins et de bouleaux, ont été brutalement remplacées par des plantations denses de pins et de hêtres, l’anémone de printemps a disparu et d’autres plantes, comme le daphné camélée, le lycopode en massue et le lycopode petit-cyprès, sont devenues de plus en plus rares72. Les aménagements forestiers sur le versant alsacien, exposé au soleil levant, ont provoqué un recul des tourbières et donc la raréfaction de la lycopodiella inundata et du sedum. Les gardes forestiers allemands plantent aussi, de leur propre initia- tive, des arbres exotiques à partir des années 1880, au contraire de leurs collègues français. Ils cherchent à diversifier les essences en introduisant de nouvelles espèces. C’est grâce à leurs initiatives que l’on trouve dans les Vosges, à la veille de la Première Guerre mondiale, quelques plants de cèdre blanc, de genévrier de Virginie, de mélèze du Japon, de thuya géant, de thuya occidental, de pruche du Canada, de carya tomentosa, de noyer cendré, de noyer noir et de chêne chevelu73. 72.  Émile Walter, « Modifications survenues dans la flore d’Alsace et de Lorraine depuis 1870 », Bulletin de la Société Botanique de France, 73-6 (1926), p. 5-61. 73.  Ibid. 75.  Franz von  Oppenau, Die Pflege und Verbesserung der Hochweiden und Bergwiesen. Vortrag, gehalten den 15.  Januar 1893 in der Generalversammlung des landwirtschaftlichen Kantonalvereins zu Münster, 1893 ; Id., Die Hochweiden und Bergwiesen der Vogesen, op. cit. 76.  Albrecht Lehmann et Klaus Schriewer, Der Wald, ein deutscher Mythos ? Perspektiven eines Kulturthemas, Berlin, Dietrich Reimer, 2000. 74.  Franz von Oppenau, Die Hochweiden und Bergwiesen der Vogesen, ihr dermaliger Zustand, sowie Vorschläge zur Verbesserung des Pflanzenbestandes auf denselben. Vortrag, gehalten zu Strasburg am 12. Oktober 1899 in der Sitzung der Gesellschaft zur Förderung der Wissenschaften, des Ackerbaues und der Künste im Unter-Elsaß, 1899. 79.  François Walter, Les figures paysagères de la nation. Territoire et paysage en Europe (16e-20e siècle), Paris, Éditions de l’EHESS, 2004, p. 147. 78.  Albrecht Lehmann, « Waldbewußtsein. Zur Analyse eines Kulturthemas in der Gegenwart », Forstwissenschaftliches Centralblatt, 120 (2001), p. 38-49.i 77.  J. K. Wilson, The German Forest, op. cit. 78.  Albrecht Lehmann, « Waldbewußtsein. Zur Analyse eines Kulturthemas in der Gegenwart », Forstwissenschaftliches Centralblatt, 120 (2001), p. 38-49. 79.  François Walter, Les figures paysagères de la nation. Territoire et paysage en Europe (16e-20e siècle), Paris, Éditions de l’EHESS, 2004, p. 147. LA TRANSFORMATION DES PAYSAGES FORESTIERS Enfin, des acteurs indépendants de l’État ont aussi participé activement à modifier le paysage par l’introduction de nouvelles espèces botaniques. L’ingénieur agronome Franz von Oppenau acclimate par exemple des plantes alpines tout le long de la frontière, afin d’améliorer les herbages, pâturages et prairies. En 1894, il établit ses Frontières et espaces transfrontaliers, une approche environnementale 38 38 premières cultures à Munster, puis crée sa pépinière à Metzeral74. Oppenau distribue les graines de sa pépinière aux fermiers locaux et les encourage à les cultiver, afin de diversifier le fourrage des animaux et améliorer la qualité du lait. Il est ainsi à l’origine directe de la présence du pâturin des Alpes, de la fléole des Alpes, de méons, de la renouée vivipare, du trèfle alpin, du plantain des Alpes et de la crépide dorée sur le versant alsacien des Vosges75. Tous ces changements paysagers sont perceptibles par les populations. Lorsque l’on traverse la frontière à la veille de la Première Guerre mondiale, l’on franchit aussi une frontière sensible. Sur le versant allemand, les essences ont tendance à être moins mélangées et les arbres sont en moyenne plus petits que sur le versant français, en raison des choix sylvi- coles effectués par l’administration des forêts d’Alsace-Lorraine. Les cônes et les aiguilles en territoire français tendent à joncher plus couramment le sol et dégagent une forte odeur de résine qui indique déjà que l’on entre dans un autre territoire. Néanmoins, les fleurs semblent plus nombreuses et diverses en territoire allemand. Les autorités allemandes investissent aussi le paysage par un discours « esthético-patriotique » qui articule Heimat et Vaterland76. Les forêts ne sont pas seulement un lieu d’exploitation de ressources, mais aussi un lieu central de la construction de l’identité nationale allemande77. Dans l’imagi- naire national allemand, les forêts représentant plus que tout autre paysage la « nature », même si elles sont très aménagées78. Les individus expéri- mentent ainsi le sentiment national par le paysage forestier modelé par l’administration des forêts d’Alsace-Lorraine79. C’est la raison pour laquelle la science forestière allemande accorde une grande importance aux activités récréatives. Les lieux de randonnée sont d’ailleurs agrémentés d’arbres appréciés des touristes, comme le cerisier noir introduit à Kaysersberg à la fin du xixe siècle80. On ne retrouve pas en France le même investissement de l’imaginaire national dans les forêts. 80.  É. Walter, « Modifications survenues dans la flore d’Alsace et de Lorraine depuis 1870 », art. cit. 81.  AD88, 7 T 20, Situation des sociétés scolaires forestières de l’arrondissement de Saint-Dié, 1901-1902. 82.  Émile Cardot, Manuel de l’arbre : pour l’enseignement sylvo-pastoral dans les écoles. L’arbre, la forêt et les pâturages de montagne, Paris, Touring-Club de France, 1907. Cet ouvrage a été un grand succès de librairie. 83.  Vincent Moriniaux, « Le mythe de la ligne bleue des Vosges et la vision nationaliste de la sapinière », in A. Corvol (dir.), Le sapin. Enjeux anciens, enjeux actuels, op. cit., p. 371-383. 84.  Michel Pastoureau, Bleu. Histoire d’une couleur, Paris, Seuil, 2000. 85.  AD88, 40 J 127 : « Je désire reposer dans la même tombe que mon père et ma sœur, en face de cette ligne bleue des Vosges, d’où monte jusqu’à mon cœur fidèle la plainte touchante des vaincus » ; Aude Dontenwille-Gerbaud, « La ligne bleue des Vosges », Mots. Les langages du poli- tique, 105 (2014), p. 13-26. LA TRANSFORMATION DES PAYSAGES FORESTIERS Cependant, la connaissance des arbres et de la sylviculture entre dans la pédagogie des écoles primaires françaises au tournant du siècle. Les instituteurs soutiennent l’administration des Eaux et Forêts en assumant le rôle de vulgarisateurs sylvicoles. Comme l’écra- sante majorité des communes situées non loin de la frontière disposent de bois communaux, les enseignants sont à l’origine de « sociétés scolaires forestières »81. Leurs réalisations concrètes sont limitées ; elles ont principa- lement aidé au reboisement des terrains en friche. Mais elles ont mobilisé de nombreux acteurs locaux du monde associatif et de l’enseignement dans l’Est de la France. En 1902, on en compte au moins une à Arnould, Ban de Sapt, Denipaire, Frapelle, Le Beulay, Lesseux, Petite-Fosse et Saulcy. Les instituteurs réalisent l’apprentissage de la nature aux enfants, car « aimer les arbres c’est aimer la patrie »82. Le paysage associé à la frontière franco-allemande est néanmoins une forêt dans l’imaginaire national français83. Pour les Français, cette frontière est nécessairement bleue, en raison de la réverbération du soleil sur les arbres qui se détachent sur le ciel, mais aussi – et peut-être surtout – parce que cette couleur est associée à la France depuis le xiiie siècle84. Maurice Barrès évoque régulièrement les « montagnes bleues des Vosges » ou encore la « dentelure bleue des crêtes, la nouvelle frontière, si distincte les veilles de pluie ». Dans son testament, Jules Ferry forge une expression vouée à une grande postérité en France : celle de « ligne bleue des Vosges »85. Les surfaces forestières à la frontière franco-allemande entre 1871 et 1914 progressent, en grande partie grâce aux états-majors qui les intègrent aux systèmes défensifs. La souveraineté que les États exercent sur les Frontières et espaces transfrontaliers, une approche environnementale 40 40 populations vivant à la frontière concerne aussi les arbres, les plantes et les fleurs qui les environnent. L’administration forestière allemande fait montre d’un véritable impérialisme écologique en rompant radicalement avec les méthodes sylvicoles françaises employées jusqu’en 1871. Ainsi, deux sylvicultures obéissant à des imaginaires nationaux différents s’opposent dans le massif des Vosges, avec la ligne de crête pour front écologique ; comme une préfiguration des terribles combats que connaitra la région pendant la Première Guerre mondiale. 86.  Béatrice von  Hirschhausen, « Leçon des frontières fantômes  : les traces du passé nous viennent (aussi) du futur », L’Espace géographique, 46-2 (2017), p. 97-105. LA TRANSFORMATION DES PAYSAGES FORESTIERS Les paysages en ressortent profon- dément modifiés, si bien qu’encore aujourd’hui, après plus d’un siècle la composition et le peuplement des forêts selon le versant des Vosges restent différents : on peut parler de « frontière écologique fantôme »86.
https://openalex.org/W4285253522
https://scindeks-clanci.ceon.rs/data/pdf/0040-2389/2022/0040-23892201004M.pdf
Bosnian
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Efficiency analysis of textile companies in Serbia
Tekstilna industrija
2,022
cc-by
5,912
TEKSTILNA INDUSTRIJA · Vol. 70, No 1, 2022 TEKSTILNA INDUSTRIJA · Vol. 70, No 1, 2022 EFFICIENCY ANALYSIS OF TEXTILE COMPANIES IN SERBIA Abstract: The purpose of the research is to use the ratio analysis to determine the level of effi ciency of companies engaged in the production of textiles in the Republic of Serbia in the period from 2017 to 2019. The research was conducted on a sample of three companies engaged in the production of textiles, whose shares are listed on the Belgrade Stock Exchange. The paper observes their management effi ciency: total assets, fi xed assets, current as- sets, inventories, trade receivables and trade payables. The research determined that fl uctuations in the values of the used effi ciency indicators were present during the observed period. Further, it was determined that there is a signifi cant space for improving the effi ciency of the observed textile companies. The results of the research can be important for the management of the company, owners (shareholders) of the company, investors and creditors. eywords: textile industry, effi ciency, ratio indicators, Belgrade Stock Exchange, Republic of Serbia Marko Milašinović1, Marko Špiler2, Snežana Knežević2*, Aleksandra Mitrović1 1 Univerzitet u Kragujevcu, Fakultet za hotelijerstvo i turizam u Vrnjačkoj Banji 2 Univerzitet u Beogradu, Fakultet organizacionih nauka * e-mail: snezana.knezevic@fon.bg.ac.rs Professional paper UDC: 657: 658:005.0:677:338.4 DOI: 10.5937/TEKSTIND2201004M Professional paper UDC: 657: 658:005.0:677:338.4 DOI: 10.5937/TEKSTIND2201004M Apstrakt: Svrha istraživanja u ovom radu je da se primenom racio analize utvrdi nivo efi kasnosti preduzeća koja se bave proizvodnjom tekstila u Republici Srbiji u periodu od 2017. do 2019. godine. Istraživanje je spro- vedeno na uzorku od tri preduzeća koja se bave proizvodnjom tekstila, a čije su akcije listirane na Beogradskoj berzi. U radu je posmatrana njihova efi kasnost upravljanja: ukupnom imovinom, stalnom imovinom, obrtnom imovinom, zalihama, potraživanjima od kupaca i obavezama prema dobavljačima. Istraživanjem je utvrđeno da su tokom posmatranog perioda prisutne fl uktuacije vrednosti korišćenih pokazatelja efi kasnosti. Takođe, utvrđeno je da postoji značajan prostor za unapređenje efi kasnosti posmatranih tekstilnih preduzeća. Rezul- tati istraživanja mogu biti od značaja za menadžment preduzeća, vlasnike (akcionare) preduzeća, investitore i poverioce. 1. UVOD Naglašava se da ima dovoljno uslova i prostora za podsticanje oživljavanja tekstilne industrije u Srbiji uprkos sporom oporavku nastalom usled zastoja u privredi i nestašice kapitala [6, p. 138]. Imajući u vidu da je tekstilna industrija i dalje važna u smislu zapošljavanja i shodno tome uravnoteženog regionalnog razvoja, ističe se da postoji potreba za re- strukturisanjem ove grane iz radno intenzivne u kapi- talno intenzivnu [7, p. 7]. Ključni proizvod računovodstvenih informacija i sistema izveštavanja su fi nansijski izveštaji, a među njima kao bazični izdvajaju se bilans stanja i bilans uspeha, a pored njih, radi integrisanog pogleda na fi nansijske performanse preduzeć a, važno je pažljivo sagledati i izveštaj o novčanim tokovima za ocenu kvaliteta zarade preduzeć a [15, p. 14]. Kao značajan izvor fi nansijskih informacija o poslovanju preduzeća javljaju se fi nansijski izveštaji, koji su namenjeni kako eksternim, tako i internim korisnicima [16], [17]. Kako isti sadrže veliki broj podataka, potrebno je istaći ne- ophodnost primene neke od tehnika fi nansijske anali- ze (horizontalne, vertikalne i racio analize) radi sticanja korisnih informacija za fi nansijske odluke [18]. Kvalitet računovodstvenih informacija preduzeć a može uticati na efi kasnost njegovog odlučivanja po pitanju investi- cija [19]. Prevarno fi nansijsko izveštavanje je već dugo prisutno u računovodstvenoj praksi [20, p. 262], i takva vrsta izveštavanja ograničava upotrebljivost fi nansij- skih informacija za poslovno odlučivanje [21]. Finansij- ska racija se koriste za komparaciju fi nansijskih perfor- mansi preduzeć a za posmatrani period, komparaciju između preduzeć a, fokusirajuć i se na sličnosti, razlike i trendove [22], a široko ih koriste menadžeri, kreditori, regulatori i investitori. Ovi pokazatelji mogu da budu koristan analitički alat za razne interesne grupe [23, p. 54]. Finansijska racija su se dugo analizirala kao mere fi nansijskog stanja preduzeć a za predviđanje korpora- tivnog neuspeha [24, p. 306]. Glavni razlog za merenje poslovnih performansi je podrška donosiocima odluka na različitim hijerar- hijskim nivoima [8]. Evaluacija performansi preduze- ć a sastavni je deo upravljanja svakom kompanijom, što omoguć ava utvrđivanje uticaja odluka poslovnog upravljanja na ostvarene performanse, kao i smer re- zultata i odluka koji su potrebni da bi ih poboljšali. Analiza fi nansijskih izveštaja primenjuje analitičke ala- te i tehnike koristeći podatke iz fi nansijskih izveštaja radi izvođenja merenja i racija koji su značajni i kori- sni za donošenje odluka [9]. 1. UVOD ni sektori industrije tekstila i odeć e specifi čni po tome što zahtevaju obilje radne snage sa vrlo malo kapital- nih investicija, a to je vrsta industrije koja je potrebna zemljama u razvoju i ekonomijama [3, p. 302]. U pro- izvodnji odeć e pružaoci usluga s niskim platama (ce- nom rada) imaju apsolutnu prednost. Strani konku- renti u takozvanim „zemljama s niskim platama“ imaju niže troškove rada sa sličnim kvalitetom snadbevanja uslugama a, pored toga, imaju i apsolutne i relativne Tekstilna industrija kao grana prerađivačke indu- strije zapošljava veliki broj jeftine i niskokvalifi kovane radne snage, pri čemu nju najviše čine žene [1, p. 98]. Odevna industrija je radno intenzivna i nudi zanima- nja na početnom nivou za nekvalifi kovane radnike u razvijenim zemljama i zemljama u razvoju [2, p. 292]. Zemlje koje su u procesu razvoja svojih ekonomija, fo- kusirane su na industriju tekstila i odeće jer su određe- 4 TEKSTILNA INDUSTRIJA · Vol. 70, No 1, 2022 ekonomske prednosti [3, p. 302]. Globalna konkuren- cija je poveć ala pritis ak na proizvodne organizacije da se poboljšaju njihove aktivnosti [4]. Preduzeća koja pripadaju tekstilnoj i odevnoj industriji usko su među- sobno povezana, ali svaka industrija pokazuje razlike u poslovnom okruženju, proizvodima, proizvodnji i sklonosti kupaca [5, p. 132]. koji se odnosi na kapacitet preduzeć a da obezbedi adekvatan novčani tok koji je namenjen ažurnom iz- mirenju njegovih tekuć ih (kratkoročnih) i dugoročnih obaveza [11, p. 117]. Evaluacija performansi kompani- je je primarni cilj analize fi nansijskih izveštaja [12, p. 252]. Merenje performansi može biti defi nisano kao proces kvantifi kovanja poslovnih aktivnosti [13]. Racio analiza je verovatno jedna od najpoznatijih i najšire korišćenih alata u analizi fi nansijskih izveštaja i ima važnu ulogu u proceni učinka preduzeća i njegovog položaja [14, p. 20]. Tekstilna i odevna industrija bile su među vodećim izvoznim industrijama u Srbiji duži vremenski period i zapošljavale su veliki broj radnika. Tekstilna industrija Srbije ima dugu tradiciju i bila je jedna od najuspeš- nijih grana prerađivačke industrije, nezavisno od toga što nije imala status prioritetne privredne grane [1, p. 102]. Danas je situacija mnogo drugačija, primarna proizvodnja još nije revitalizovana, veliki deo sirovina se uvozi, što sve zajedno ima negativne implikacije na konkurentnost domaćih preduzeća u okviru tekstil- ne i odevne industrije. 1. UVOD Finansijska analiza koja obuhvata horizontalnu, vertikalnu i racio analizu, pre svega služi u dve svrhe: (a) za upoređivanje sa stan- dardom, i (b) da predvidi buduć e perspektive posma- tranog preduzeća, čime bi se zadovoljili informacioni zahtevi raznih adresata. Tradicionalno, performanse preduzeć a se ocenjuju primenom fi nansijske racio analize, ali se primeć uje da je u današnjem dinamič- nom i veoma konkurentnom poslovnom okruženju pravovremena kontrola performansi od ključne važ- nosti za održivo poslovanje, te stoga postoji sve već a pažnja posveć ena pokazateljima nefi nansijskog učin- ka [10, pp. 125–160]. Predmet ovog rada je efi kasnost proizvođača tek- stila u Republici Srbiji. Cilj rada je da se sagleda da li su posmatrana tekstilna preduzeća efi kasno upravljalala svojim resursima u periodu od 2017. do 2019. godi- ne. Efi kasnost je merena koefi cijentom obrta ukupne imovine, koefi cijentom obrta stalne imovine, koefi ci- jentom obrta obrtne imovine, koefi cijentom obrta ku- paca, koefi cijentom obrta zaliha i koefi cijentom obrta dobavljača. Akcionari, poverioci i druge zainteresovane strane evaluiraju fi nansijski položaj preduzeć a radi procene njegove sposobnosti za efi kasno obavljanje poslovne aktivnosti. Povezan s tim, važan deo poslovanja je onaj Razvijeno je nekoliko pitanja koja se odnose na tretiranu problematiku. Istraživačka pitanja su: 5 TEKSTILNA INDUSTRIJA · Vol. 70, No 1, 2022 IP1. Kako bi pokazatelji efi kasnosti mogli uticati na fi nansijske performanse kompanija? IP1. Kako bi pokazatelji efi kasnosti mogli uticati na fi nansijske performanse kompanija? Koefi cijent obrta stalne imovine meri efi kasnost korišćenja stalne imovine preduzeća u generisanju prihoda od prodaje [30]. Usled činjenice da su stalna sredstva skupa za kupovinu i održavanje, neophodno je da budu što efi kasnija u generisanju prodaje [26]. Vrednost ovog pokazatelja u velikoj meri zavisi i od in- dustrije kojoj preduzeće pripada [31]. Niska vrednost ovog pokazatelja ukazuje da preduzeće nedovoljno koristi svoje kapacitete ili da su oni predimenzionirani. IP2. Koji su ključni aspekti koji se odnose na efi ka- snost a ukazuju na negativne tendencije kompanija? IP2. Koji su ključni aspekti koji se odnose na efi ka- snost a ukazuju na negativne tendencije kompanija? Rad je podeljen u pet delova. Nakon uvodnog dela sledi prikaz najčešće korišćenih pokazatelja efi ka- snosti (fi nansijskih racija) koji se javljaju u akademskoj i stručnoj literaturi. Slede podaci o uzorku u trećem delu. Rezultati istraživanja prikazani su u četvrtom delu. 1. UVOD U poslednjem delu izneta su zaključna razmatra- nja, kao i ograničenja u sprovedenom istraživanju iz kojih proističu dalji pravci istraživanja. Koefi cijent obrta obrtne imovine, kao odnos pri- hoda od prodaje i prosečne obrtne imovine, pokazu- je koliko se puta, u proseku, obrtna imovine obrne u toku godine kroz prihode od prodaje [26]. Drugim re- čima, ovaj pokazatelj meri sposobnost preduzeća da generiše prihode od prodaje kroz svoja obrtna sred- stva. Preduzeće treba nastojati da ovaj pokazatelj ima višu vrednost, uz trend rasta, jer to znači da preduzeće radi na doslednom poboljšanju svojih politika u obla- sti zaliha, potraživanja, gotovine i drugih obrtnih sred- stava [26]. 2. MERE EFIKASNOSTI POSLOVANJA PREDUZEĆA Pokazatelji efi kasnosti mere uspešnost upravlja- nja različitim poslovnim resursima preduzeća [25], odnosno mere sposobnost preduzeća da konvertu- je sopstvena sredstva u gotovinu ili prihode [26]. Za ovu grupu pokazatelja nisu zainteresovani samo me- nadžeri preduzeća, kao lica kojima je povereno uprav- ljanje sredstvima, već i poverioci (kreditori, dobavljači i dr.); [27]. Ova grupa racija omogućava sagledavanje broja obrta pojedinih kategorija (sredstava) u poslov- nom ciklusu, odnosno podataka o vremenu njihovog vezivanja [28]. Pokazatelji efi kasnosti povezani su sa pokazateljima profi tabilnosti, odnosno ukoliko je pre- duzeće efi kasnije u korišćenju sredstava, ono postaje profi tabilno [26]. Prilikom utvrđivanja racio pokaza- telja efi kasnosti, koriste se podaci iz bilansa stanja i bilansa uspeha. Kao najčešće korišćeni pokazatelji efi - kasnosti javljaju se: Koefi cijent obrta potraživanja od kupaca, kao od- nos prihoda od prodaje i prosečnih potraživanja od kupaca, meri efi kasnost kojom preduzeće naplaćuje svoja potraživanja od kupaca [32]. Ovaj pokazatelj ukazuje koliko je puta preduzeće bilo u mogućnosti da pretvori potraživanja od kupaca u gotovinu [32]. Za preduzeće je bolje da vrednost ovog pokazatelja bude viša, jer to ukazuje da brže naplaćuje svoja po- traživanja a, samim tim, brže dolazi do gotovine (kao najlikvidnijeg oblika sredstava preduzeća). Adekvatna naplata potraživanja je, pored profi tabilnosti, druga najznačajnija mera uspešnosti poslovanja preduzeća [33]. U situacija kada preduzeće vodi izuzetno strogu kreditnu politiku, vrednost ovog pokazatelja će biti izuzetno visoka [34]. Preterana prodaja na kredit, ne- mogućnost kupaca da plate obaveze ili slaba naplata potraživanja imaće za posledicu nisku vrednost koe- fi cijenta. • Koefi cijent obrta ukupne imovine. • Koefi cijent obrta stalne imovine. • Koefi cijent obrta obrtne imovine. • Koefi cijent obrta kupaca. Koefi cijent obrta zaliha ukazuje koliko efi kasno preduzeće upravlja zalihama, odnosno koliko se puta u toku godine, u proseku, zalihe obrnu [32]. Za razliku od prethodno korišćenih pokazatelja aktivnosti, kod kojih se u brojiocu nalaze prihodi od prodaje, kod ko- efi cijenta obrta zaliha u brojiocu se nalazi cena košta- nja prodatih proizvoda. Konkretno, ovaj pokazatelj se utvrđuje stavljanjem u odnos cene koštanja prodatih proizvoda i prosečne vrednosti zaliha [32]. Zbog ne- mogućnosti da se iz bilansa uspeha utvrdi cena košta- nja prodatih proizvoda, u brojiocu se nalaze troškovi proizvodnje uvećani za povećanje vrednosti zaliha, odnosno umanjeni za smanjenje vrednosti zaliha [35]. Viša vrednost koefi cijenta znači efi kasnije upravljanje zalihama u preduzeću, odnosno zalihe se kraće zadr- • Koefi cijent obrta zaliha. 2. MERE EFIKASNOSTI POSLOVANJA PREDUZEĆA • Koefi cijent obrta dobavljača. Koefi cijent obrta ukupne poslovne imovine pred- stavlja najuopšteniji pokazatelj aktivnosti preduzeća, budući da predstavlja odnos prihoda od prodaje i prosečnog stanja ukupnih poslovnih sredstava [29]. On pokazuje koliko se puta u toku godine vrednost ukupno angažovane poslovne imovine preduzeća ge- nerisala u prodaji. Na njegovu visinu se može uticati povećanjem koefi cijenta obrta pojedinih sredstava (npr. stalnih sredstava, obrtnih sredstva i njenih obli- ka). Što je vrednost ovog koefi cijneta viša, to je i veća sposobnost preduzeća da koristi svoju ukupnu imovi- nu u stvaranju prihoda od prodaje [26]. 6 TEKSTILNA INDUSTRIJA · Vol. 70, No 1, 2022 žavaju u skladištu. To može ukazivati da preduzeće manje ulaže u zalihe, kao i na postojanje ubrzanog poslovnog ciklusa pretvaranja zaliha u gotovinu i po- stojanje manje verovatnoće nestanka zaliha [36]. Kako Mitrović & Knežević [37, p. 64] ističu, niska vrednost koefi cijenta obrta zaliha može se javiti kao rezultat niskog kvaliteta proizvodnje, vrednovanja zaliha po visokim cenama, uključivanja bezvrednih i starih proi- zvoda u zalihe i drugo. žavaju u skladištu. To može ukazivati da preduzeće manje ulaže u zalihe, kao i na postojanje ubrzanog poslovnog ciklusa pretvaranja zaliha u gotovinu i po- stojanje manje verovatnoće nestanka zaliha [36]. Kako Mitrović & Knežević [37, p. 64] ističu, niska vrednost koefi cijenta obrta zaliha može se javiti kao rezultat niskog kvaliteta proizvodnje, vrednovanja zaliha po visokim cenama, uključivanja bezvrednih i starih proi- zvoda u zalihe i drugo. 13 – Proizvodnja tekstila), a analizom je obuhvaćeno njihovo poslovanje od 2017. do 2019. godine na osno- vu javno dostupnih fi nansijskih izveštaja [40]. Od pet preduzeća, koja pripadaju pomenutoj oblasti a čije su akcije na dan 14. 07. 2021. godine listirane na Beo- gradskoj berzi, iz daljeg istraživanja isključena su dva preduzeća [39]. Razlog njihovog isključivanja nalazi se u činjenici da se jedno preduzeće nalazi u stečaju, dok drugo preduzeće nema javno dostupne fi nansijske izveštaje [40]. Na problem javne nedostupnosti fi nan- sijskih izveštaja preduzeća koja su listirana na Beo- gradskoj berzi ukazuju i [41], [42]. Tri posmatrana pre- duzeća u daljem tekstu su označena kao preduzeće A (Dunav A.D. Grocka), B (Kluz Padobrani A.D. Beograd) i C (Yumco, A.D. Vranje). Koefi cijent obrta dobavljača predstavlja odnos nabavki na kredit i prosečnih obaveza prema dobav- ljačima [34]. 4. REZULTATI ISTRAŽIVANJA Radi odgovora na defi nisana istraživačka pitanja, primenjena su selektovana fi nansijska racija za testira- nje efi kasnosti preduzeća. U nastavku su predstavljeni dobijeni rezultati. Vrednost navedenih pokazatelja efi - kasnosti posmatranih tekstilnih preduzeća u periodu od 2017. do 2019. godine prikazana je u sledećoj ta- beli. Tokom posmatranog perioda prisutne su fl uktu- acije vrednosti svih šest korišćenih pokazatelja efi ka- snosti. Tako je kod preduzeća A, tokom posmatranog perioda, došlo do pada vrednosti koefi cijenta obrta ukupne imovine. U prvoj posmatranoj godini, predu- zeće A je na svaki dinar prosečno angažovane imovi- ne ostvarilo 0,55 dinara prihoda od prodaje, da bi u poslednoj posmatranoj godini ostvarivalo 0,45 dinara. Pad vrednosti ovog pokazatelja efi kasnosti kod pre- duzeća A javlja se usled većeg pada vrednosti prihoda od prodaje, u odnosu na zabeleženo smanjenje vred- nosti ukupne imovine. Međutim, i pored navedenih oscilacija, preduzeće A je u sve tri posmatrane godine 2. MERE EFIKASNOSTI POSLOVANJA PREDUZEĆA Usled nedostatka podataka o nabavkama na kredit u fi nansijskim izveštajima, prilikom utvrđi- vanja ovog pokazatelja vrši se aproksimacija nabavki na kredit sa vrednošću cene koštanja prodatih goto- vih proizvoda, uvećane za povećanje vrednosti zaliha, odnosno umanjene za smanjenje zaliha [34]. Pokazuje koliko se puta prosečno u toku godine izvrši isplata obaveza prema dobavljačima. Niža vrednost pokaza- telja ukazuje na postojanje dužeg perioda između dve isplate dobavljačima. Stavljajući u odnos broj dana u godini i koefi cijent obrta dobavljača dobija se pro- sečno vreme plaćanja obaveza prema dobavljačima. Pokazuje koliko u proseku protekne dana od trenutka nabavke sirovina, materijala i usluga od dobavljača na kredit do trenutka isplate tih obaveza. U slučaju da se upoređivanjem utvrdi viša vrednost pokazatelja u od- nosu na ugovoreni rok plaćanja prema dobavljačima, to može ukazivati da preduzeće ima problem s nedo- statkom gotovine [38]. 3. PODACI O UZORKU Istraživanje u radu je sprovedeno na uzorku pre- duzeća čije su akcije listirane na Beogradskoj berzi u okviru Sektora C-Prerađivačka industrija [39], koja se bave proizvodnjom tekstila (odnosno preduzeća čija je šifra oblasti prema Uredbi o sektorskoj klasifi kaciji Tabela: Efi kasnost tekstilnih preduzeća u periodu od 2017. do 2019. godine   A B C 2017 2018 2019 2017 2018 2019 2017 2018 2019 Koefi cijent obrta ukupne imovine 0,5538 0,4938 0,4503 0,0085 0,0027 0,0000 0,2263 0,3697 0,3366 Koefi cijent obrta stalne imovine 0,8227 0,7177 0,6723 0,0113 0,0036 0,0000 0,3776 0,4831 0,4654 Koefi cijent obrta obrtne imovine 16,942 15,823 13,639 0,0350 0,0107 0,0000 0,5657 15,745 12,166 Koefi cijent obrta kupaca 132,906 171,546 160,754 0,4326 0,1376 0,0000 26,212 239,944 60,827 Koefi cijent obrta zaliha 14,721 13,748 11,090 0,0381 0,0117 0,0003 11,247 24,651 22,895 Koefi cijent obrta dobavljača 26,648 37,247 44,919 0,2078 0,2538 0,2807 0,4396 0,9316 10,210 Tabela: Efi kasnost tekstilnih preduzeća u periodu od 2017. do 2019. godine 7 TEKSTILNA INDUSTRIJA · Vol. 70, No 1, 2022 zabeležilo najvišu vrednost ovog pokazatelja. Sma- njenje vrednosti koefi cijenta obrta ukupne imovine zabeleženo je i kod preduzeća B, da bi u poslednjoj posmatranoj godini usled neostvarivanja prihoda od prodaje, vrednost ovog pokazatelja bila 0. Preduzeće C je u 2018. godini, usled rasta prihoda od prodaje i smanjenja vrednosti prosečne imovine u odnosu na 2017. godinu, ostvarilo rast koefi cijenta ukupne imovine, odnosno na svaki dinar prosečno uložene imovine ostvarilo je 0,37 dinara prihoda od prodaje. Međutim, u 2019. godini je usledio pad vrednosti koe- fi cijenta obrta ukupne imovine u odnosu na 2018. go- dinu. Tako je preduzeće C u 2019. godini ostvarilo 0,34 dinara prihoda od prodaje na svaki dinar prosečno angažovane ukupne imovine. Razlog za smanjenje se nalazi u činjenici da je zabeleženo povećanje vredno- sti ukupne imovine, ali i smanjenje vrednosti prihoda od prodaje. U sve tri posmatrane godine, preduzeće A je ostvarilo najvišu vrednost efi kasnosti ukupne imo- vine. dinara prihoda od prodaje. U 2018. godini ta vrednost iznosi 1,58, odnosno 1,36 u 2019. godini. Preduzeće B je u 2017. godini na svaki dinar prosečno uložene obrtne imovine ostvarilo 0,03 dinara prihoda od pro- daje, da bi u 2018. godini, ostvarilo 0,01 dinar. Usled već navedenih činjenica (da preduzeće B u 2019. go- dini nije imalo prihode od prodaje), u poslednjoj po- smatranoj godini vrednost koefi cijenta obrta obrtne imovine iznosio je 0. 3. PODACI O UZORKU Usled rasta prihoda od prodaje i smanjenja vrednosti prosečnih potraživanja, preduze- će C je u 2018. godini zabeležilo rast koefi cijenta obrta obrtne imovine u odnosu na 2017. godinu. Međutim, u 2019. godini zabeleženo je smanjenje efi kasnosti obrtne imovine. Kao i u slučaju prethodna dva kori- šćena pokazatelja, preduzeće A je u sve tri posmatra- ne godine ostvarilo najviše vrednosti koefi cijenta obr- ta obrtne imovine. Iz tabele se može videti da je tokom posmatranog perioda vrednost koefi cijenta obrta kupaca zabeležila fl uktuacije. Preduzeća A i C su u 2018. godini zabele- žila rast vrednosti ovog pokazatelja efi kasnosti u od- nosu na 2017. godinu, da bi u 2019. godini usledilo smanjenje vrednosti (pri čemu je ta vrednost viša u odnosu na 2017. godinu). Dakle, u 2018. godini pre- duzeća A i C su skratila period naplate potraživanja od kupaca u odnosu na 2017. godinu, da bi u 2019. godini došlo do dužeg perioda naplate potraživanja od kupaca. Preduzeće A je u 2017. godini 13,29 puta naplaćivalo prosečna potraživanja od kupaca, u 2018. godini 17,15 puta, a 16,07 puta u 2019. godini. Predu- zeće B beleži trend konstantnog smanjenja vrednosti koefi cijenta obrta kupaca, da bi u poslednjoj posma- tranoj godini ta vrednost bila 0 (usled odsustva priho- da od prodaje). To ukazuje da preduzeće ima velikih problema sa prodajom i naplatom svojih potraživanja. Kao što je navedeno, preduzeće C je u 2018. godini (vrednost koefi cijent obrta kupaca iznosi 23,99) pove- ćalo svoju efi kasnost naplate potraživanja od kupaca u odnosu na 2017. godinu (kada je vrednost koefi ci- jenta iznosila 2,62), da bi u 2019. godini usledilo sma- njenje efi kasnosti. U 2017. i 2019. godini, preduzeće A je zabeležilo najvišu vrednost koefi cijenta obrta kupa- ca, dok je u 2018. godini to bilo preduzeće C. Preduzeće A usled smanjena vrednosti prihoda od prodaje i smanjenja vrednosti prosečne stalne imovine beleži smanjenje vrednosti koefi cijenta obrta stalne imovine tokom posmatranog perioda. Tako je preduzeće A u 2017. godini na svaki dinar prosečno angažovane stalne imovine stvorilo 0,82 dinara priho- da od prodaje, da bi u 2019. godini, stvorilo 0,67 di- nara. Preduzeće B takođe beleži trend smanjenja efi - kasnosti stalne imovine u periodu od 2017. do 2019. godine. Ovo preduzeće je u 2017. godini na svaki dinar prosečno angažovane stalne imovine ostvarilo 0,01 dinara prihoda od prodaje. 3. PODACI O UZORKU U poslednjoj posma- tranoj godini, usled nepostojanja prihoda od prodaje, vrednost koefi cijenta obrta stalne imovine iznosi 0. Usled rasta vrednosti prihoda od prodaje i smanjenja prosečne vrednosti stalne imovine, preduzeće C je u 2018. godini ostvarilo višu vrednost efi kasnosti stalne imovine u odnosu na 2017. godinu. Preduzeće C je u 2018. godini na svaki dinar prosečno uložene stalne imovine ostvarilo 0,48 dinara, dok je u 2017. godini to bilo 0,38 dinara. U 2019. godini je usledio pad efi ka- snosti stalne imovine preduzeća C. Tako je, na kraju poslednje posmatrane godine, preduzeće C na svaki dinar prosečno angažovane stalne imovine ostvarilo 0,46 dinara prihoda od prodaje. Kako je preduzeće A u sve tri posmatrane godine zabeležilo najvišu vrednost koefi cijenta stalne imovine, može se reći da je najefi - kasnije koristilo stalnu imovinu u generisanju prihoda od prodaje. U posmatranom periodu preduzeća A i B su sma- njila efi kasnost upravljanja zalihama, odnosno prisu- tan je trend smanjenja vrednosti koefi cijenta obrta zaliha. U preduzeću A, zalihe se obrnu 1,47 puta u 2017. godini, 1,37 puta u 2018. i 1,11 puta u 2019. Pre- duzeće C je u 2018. godini povećalo efi kasnost zaliha u odnosu na 2017. godinu, da bi u 2019. godini došlo do pada efi kasnosti zaliha. U 2017. godini preduzeće A je najefi kasnije upravljalo zalihama, dok je u 2018. i 2019. godini to činilo preduzeće C. Preduzeća A i B su zabeležila trend smanjenja efi kasnosti obrtne imovine u generisanju prihoda od prodaje. Na svaki dinar prosečno uložene obrtne imovine, preduzeće A je u 2017. godini ostvarilo 1,69 8 TEKSTILNA INDUSTRIJA · Vol. 70, No 1, 2022 Koefi cijent obrta dobavljača preduzeća A u 2017. godini iznosio je 2,66, što znači da se 2,66 puta godiš- nje isplate prosečne obaveze prema dobavljačima. U 2018. godini vrednost ovog pokazatelja je porasla na 3,72, da bi trend rasta bio nastavljen i 2019. godine (kada je koefi cijent obrta dobavljača 4,49). Vrednost koefi cijenta obrta dobavljača kod preduzeća B u sve tri posmatrane godine, kao i kod preduzeća C u prve dve posmatrane godine, niža je od 1. To ukazuje da iz- među dve isplate obaveza prema dobavljačima prođe više od godinu dana, što može biti jedan od signala da je sposobnost preduzeća za izvršenje obaveza do- vedena u pitanje. U sve tri posmatrane godine, predu- zeće A je ostvarilo najvišu vrednost koefi cijenta obrta dobavljača. 3. PODACI O UZORKU njiće se troškovi skladištenja istih. Takođe, preduzeća na taj način slobodna novčana sredstva (koja bi bila “zarobljena” u zalihama) mogu uložiti u druge aktiv- nosti koje će doneti određenu zaradu. Kroz adekvatnu analizu poslovanja (postojećih i potencijalnih) kupa- ca, preduzeća treba da sagledaju realne mogućnosti za naplatu svojih potraživanja kako bi uskladila prilive (po osnovu naplate potraživanja) sa odlivima gotovi- ne (plaćanja prema dobavljačima, kreditorima i dr.). U radu se javlja nekoliko ograničenja. Kako su po- smatrana samo akcionarska tekstilna preduzeća sa Beogradske berze, naredna istraživanja treba usme- riti i na tekstilna preduzeća koja nisu registrovana kao akcionarska društva. Takođe, treba posmatrati poslovanje tekstilnih preduzeća u dužem vremen- skom periodu. Povećanjem veličine uzorka i vremena posmatranja može se steći preciznija i potpunija sli- ka o poslovanju preduzeća. Kako istraživanjem nije obuhvaćeno poslovanje tekstilnih preduzeća u 2020. godini, godini pandemije COVID-19, u narednim istra- živanjima treba sagledati i uticaj pandemije na efi ka- snost tekstilnih preduzeća. Da bi se stekla potpuna slika o poslovanju preduzeća, pored pokazatelja efi - kasnosti, budućim istraživanjima bi trebalo obuhvatiti i pokazatelje likvidnosti, solventnosti i profi tabilnosti [43]. Kako se zadnjih godina značajna pažnja posve- ćuje zaštiti životne sredine [44], [45], [46], naredna istraživanja treba usmeriti i na ispitivanje efi kasnosti tekstilnih preduzeća upravljanjem otpadom, kao ne- fi nansijskom performansom u poslovanju preduzeća. 5. ZAKLJUČAK Kako se efi kasnost pozitivno odražava na profi ta- bilnost preduzeća, neophodno je Praćenje efi kasnosti preduzeća predstavlja važan zadatak koji se stavlja pred menadžment preduzeća, posebno kada se uzme u obzir činjenica da efi kasno korišćenje sredstava do- vodi i do profi tabilnog poslovanja preduzeća. Spro- vedenim istraživanjem je utvrđeno da postoji znača- jan prostor za unapređenje efi kasnosti posmatranih tekstilnih preduzeća, što je potvrđeno rezultatima istraživanja da određena preduzeća nisu ostvarila pri- hode od prodaje, ili su ih ostvarila na veoma niskom nivou, čime nastavak poslovanja može biti doveden u pitanje. Takođe, utvrđeno je da određena preduzeća nemaju javno dostupne fi nansijske izveštaje, čime je blagovremeno donošenja poslovnih odluka dovede- no u pitanje. LITERATURA [1] Urošević, S., Đorđević, D., Cvijanović, J. M. (2009). Značaj doradnih poslova za razvoj tekstilne i odev- ne industrija Srbije u procesu tranzicije, Industrija, 2, 97–125. Koristeć i informacije koje su predstavljene kao rezultat primenjene fi nansijske analiize, mogu se dati preporuke koje ć e omoguć iti preduzećima da poprave (ili maksimizuju) svoje performanse u segmentu efi ka- snosti. 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This article is an open access article distributed under the terms and condi- tions of the Creative Commons Attribution 4.0 Inter- national license (CC BY) (https://creativecommons. org/licenses/by/4.0/) ______________________________ © 2021 Authors. Published by Union of Textile Engi- neers and Technicians of Serbia. This article is an open access article distributed under the terms and condi- tions of the Creative Commons Attribution 4.0 Inter- national license (CC BY) (https://creativecommons. org/licenses/by/4.0/) [38] Weetman, P. (2011). ___________________ Primljeno/Received on: 05.08.2021. Revidirano/ Revised on: 07.02.2022. Prihvaćeno/Accepted on: 08.02.2022. ______________________________ © 2021 Authors. Published by Union of Textile Engi- neers and Technicians of Serbia. This article is an open access article distributed under the terms and condi- tions of the Creative Commons Attribution 4.0 Inter- national license (CC BY) (https://creativecommons. org/licenses/by/4.0/) Primljeno/Received on: 05.08.2021. LITERATURA Financial Accounting: An Intro- duction, 5th edition, Pearson Education, Harlow. [39] Sektor C, Beogradska berza, pristupljeno preko https://www.belex.rs/trzista_i_hartije/sektori/C, da- tum pristupa 14. 07. 2021. godine. 11
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English
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New bio-polyol based on white mustard seed oil for rigid PUR-PIR foams
Polish Journal of Chemical Technology
2,018
cc-by
5,759
INTRODUCTION polyols based on citric acid (2-hydroxy-1,2,3-propanetri- carboxylic acid) and various glycols to obtained a rigid polyurethane-polyisocyanurate foams. These composites were characterized by good thermal insulation properties, high aging resistance and low fl ammability. Polyurethanes are the important group of thermosets because of their wide application in everyday life. The use of these materials is directly related to their properties: physical (density), mechanical (brittleness, compressive strength), thermal (thermal conductivity) and fl amma- bility. Environmental aspects (e.g reuse of waste from this materials) and economic aspects (e.g. price of the fi nished product) are also signifi cant. Often, the desire to obtain specifi c properties of polyurethanes is related to the incorporation into the technological formulation of additives1–4. Many chemical companies strives to receive green product solutions and keep sustainable develop- ment strategies. Raw materials of plant origin are recently more and more used for the production of polyurethane materials, e.g. bio-polyols based on vegetable oils1, 5–7. They are usually produced by pressing oil plants. The unrefi ned vegetable oil obtained is additionally subjected to a fi ltration process to remove solid seed residues8. In recent years there has been a huge interest in these raw materials because of their green nature and an alterna- tive to shrinking oil and gas reserves1. The aim of the study was to obtain a new bio-polyol based on crude mustard oil and 2,2’-mercaptodiethanol and to investigate its physicochemical properties. The effect of this bio-polyol on the properties of rigid poly- urethane-polyisocyanurate foams was also investigated. New bio-polyol based on white mustard seed oil for rigid PUR-PIR foams Kazimierz Wielki University, J.K. Chodkiewicza 30, 85-064, Bydgoszcz, Poland *Corresponding author: e-mail: m.borowicz@ukw.edu.pl Kazimierz Wielki University, J.K. Chodkiewicza 30, 85-064, Bydgoszcz, Poland *Corresponding author: e-mail: m.borowicz@ukw.edu.pl A new bio-polyol based on white mustard oil (Synapis alba) and 2,2’-mercaptodiethanol (2,2’-MDE) was obtained. The synthesis was carried out by two-step method. In the fi rst stage, the double bond of the unsaturated fatty acid residues was oxidized, and in the second step the epoxy rings were opened with 2,2’-MDE. The properties of the obtained bio-polyol for application as raw material in polyurethane-polyisocyanurate foams (PUR-PIR) - hydroxyl number, acid number, density, viscosity, pH, water content, FTIR, 1H NMR and 13C NMR were investigated. Based on the obtained results, foaming formulations containing 0 to 0.6 R of the new bio-polyol were prepared. Signifi cant impact of bio-polyol on apparent density, compressive strength, brittleness, fl ammability, water absorption and thermal conductivity of polyurethane composites were noted. Modifi ed foam had better functional properties than reference foam e.g. lower brittleness, better thermal insulation properties and better fi re resistance. Keywords: polyurethane foam, white mustard seed oil, 2,2’-mercaptodiethanol, bio-polyol. 24 Pol. J. Chem. Tech., Vo 2, 2018 Polish Journal of Chemical Technology, 20, 2, 24—31, 10.2478/pjct-2018-0019 Properties of bio-polyol based on mustard oil (Sigma-Aldrich, USA). Anhydrous calcium chloride (POCh, Poland) was used to neutralize the catalyst and remove residual water. (Sigma-Aldrich, USA). Anhydrous calcium chloride (POCh, Poland) was used to neutralize the catalyst and remove residual water. New bio-polyol was subjected to physicochemical, analytical and spectroscopic studies to determine its suitability for the synthesis of rigid PUR-PIR foams. The properties are determined in accordance with the standards applicable to polyol raw materials: Polyether polyol- Rokopol RF 551 – sorbitol oxypro- pylene product with HN = 420 mgKOH/g (PCC Rokita, Poland), was used for the synthesis of PUR-PIR foams. Purocyn B, a technical polyisocyanate (purchased in Pu- rinova, Poland) was used as a isocyanate raw material. Main ingredient was 4,4’-diphenylmethane diisocyanate. NCO content was 31%. – Hydroxyl number (HN), was determined in accor- dance with the factory standard of Purinova Chemical Plant No. WT/06/07/PURINOVA. – Acid value (AV) was determined in accordance with PN-ISO 660 standard. The catalyst system for the preparation of PUR-PIR rigid foams was anhydrous potassium acetate (Chempur, Gliwice) used as a 33% solution in diethylene glycol (catalyst 12) and DABCO – 1,4-diazabicyclo [2.2.2] octane (Hülls, Germany) used as a 33% solution in diethylene glycol. The foam structure stabilizer was Sili- cone L-6900 – poly-siloxane poly-oxy-alkylene surfactant (Witco, Sweden). The foaming agent was Solkane HFC 365/227 (Solvay, Belgium) – a mixture of 1,1,1,3,3-pen- tafl uorobutane and 1,1,1,2,3,3,3-heptafl uoropropane in a weight ratio of 87:13. The fl ame retardant was Antiblaze TCMP - tri[2-chloro-1-methylethyl phosphate] (Albright and Wilson, UK). – Iodine value (IV) was determined in accordance with PN-ISO 3961:2013-10 standard. – Epoxy value (EV) was determined in accordance with PN-ISO 3001:1999 standard. – Viscosity of bio-polyol determined by Fungilab digital rheometer at 20°C (293 K). Measurements were made using a standard DIN-87 spindle co-operating with the ULA-DIN-87 bushing. Keeping the constant tempera- ture is ensured by a thermostat connected to the water jacket of the sleeve. j – Density was measured at 25oC (298 K) in an adiabatic pycnometer in accordance with PN-92/C-04504. – Water content was determined by Carl-Fisher me- thod using Titraqual branded reagent in accordance with PN-81/C-04959. Material and methods For the synthesis of a new bio-polyol, the two-stage method used unrefi ned white mustard seed (Sinapis alba) oil, as a light yellow liquid with a density of 0.880 g/cm3 and a viscosity of 160 mPa · s, produced by the company jedzpij.pl (Poland). The iodine value was 0.426 mol I2/100g of oil and the unsaturated fatty acid content was 95.88 wt% of total fatty acid in the oil. The fatty acid profi le of the oil used is shown in Table 1. A number of studies on the synthesis of bio-polyols based on oils: soybean9–11, castor12–15 and rapeseed16–19 can be found in the literature. Studies on the use of polyols based on soya oil for rigid foam were conducted by Tu et al.20. The obtained materials were characterized by a low coeffi cient of thermal conductivity. Veronese et al.21 used a mixture of petrochemical polyols and soybean-based polyol to obtain the resultant transesterifi cation of the hydroxylated oil with tri-ethanolamine to produce rigid polyurethane foams. The resulting bio-composites exhi- bited better mechanical properties than their equivalents obtained from conventional raw materials. Prociak et al.22–25 developed composites with bio-polyol based on rapeseed oil, which was characterized by higher content of closed cells and lower thermal conductivity. Horak and Benes26 have used fi sh oil waste to chemically recycle fl exible polyurethane foams. As a result of the process, the authors obtained liquid recycled polyols that were used to obtain new semi-rigid foams. The properties of these materials were, however, worse than these of pure raw materials. Liszkowska et al.27–28 with the team used Table 1. Fatty acid profi le of white mustard oil Table 1. Fatty acid profi le of white mustard oil In the fi rst stage of the synthesis, an oxidizing system was used: 99.5% acetic acid (Chempur, Poland) and 30% solution of hydrogen peroxide (Chempur, Poland). 96% sulfuric acid (VI) (POCh, Poland). Anhydrous magnesium sulphate (VI) (Chempur, Poland) was used to dry the purifi ed epoxidized white mustard oil. p p 98% of 2,2’-mercaptodiethanol in presence of the acid catalyst (as above) was used to open the oxirane rings 25 Pol. J. Chem. Tech., Vol. 20, No. 2, 2018 Synthesis of new bio-polyol 500 g of crude mustard oil (MO) with an iodine value of 0.426 mol I2/100g, 128.55 g of 99.5% acetic acid (AC) and 4.35g of 96% sulfuric acid (VI) (SA) were trans- ferred into a 1000 cm3 reactor equipped with a refl ux condenser, thermometer, addition funnel and mechanical stirrer. The mixture was heated to 40°C at constant stirrer speed (700 rpm) and 362.10g of 30% hydrogen peroxide (HP) was added via dropping funnel. The molar ratio of the reactants was 1:1:1.5:0.02 for MO:AC:HP:SA. After adding all hydrogen peroxide solution, the mixture was heated to 60oC. Hydrogen peroxide in the presence of acetic acid produced peracetic acid in situ, which oxidized the double bonds contained in the oil to the epoxy groups. The reaction was carried out for 3 hours. During synthesis, the epoxy number of the mixture was determined all the time. Then the reaction system was cooled and allowed to stand for 24 hours to separate the oil phase from the aqueous phase. The oil phase was washed with distilled water to remove residual acetic acid and catalyst, and the remaining water was removed with anhydrous magnesium sulphate (VI). The obtained intermediate (epoxidized oil) was subjected to analytical tests and was used to further synthesis. – The pH was measured by Hanna Instruments mi- croprocessor laboratory pH-meter (ORP/ISO/oC) with RS 22 C connector. – Elemental analysis with the Elementar Vario EL III CHNSO. – Elemental analysis with the Elementar Vario EL III CHNSO. – Spectroscopic analysis of the chemical structure of crude mustard oil, epoxidized oil and bio-polyol was also performed: – Spectroscopic analysis of the chemical structure of crude mustard oil, epoxidized oil and bio-polyol was also performed: – In infrared spectroscopy was performed using the Brücker Vector Spectrophotometer with the KBr tech- nique in the range of 400 to 4000 cm–1. – In infrared spectroscopy was performed using the Brücker Vector Spectrophotometer with the KBr tech- nique in the range of 400 to 4000 cm–1. q g – 1H NMR and 13C NMR were performed using the Brücker Ascend III NMR spectrometer at 400 MHz in deuterated chloroform. q g – 1H NMR and 13C NMR were performed using the Brücker Ascend III NMR spectrometer at 400 MHz in deuterated chloroform. Preparation of PUR-PIR foams The preparation of rigid PUR-PIR foams with white mustard oil based bio-polyol required experimental studies to determine the optimal composition of addi- tives (catalysts, surfactants, fl ame retardants and blow- ing agents). The basis for determining the amount of polyols raw materials was the hydroxyl number. Polyol formulation was developed to keep a constant isocyanate index. The addition of isocyanate was selected taking into account the equivalent (R) ratio of the NCO to OH groups in the reaction mixture, which for PUR-PIR rigid foams was set to 3:1. It is essential for the reaction of the NCO groups with OH to produce the urethane bond (Scheme 1) and the trimerization of the three NCO groups to the isocyanurate ring (Scheme 2). In the next step, 500 g of epoxidized white mustard oil (EMO), 234.24 g of 98% 2,2’-mercaptodiethanol (MDE) and 1.96 g of 96% sulfuric acid (VI) were transferred into a 1000 cm3 reactor. The molar ratio of the reagents was 1:1:0.01 for EMO:MDE:SA. The mixture was heated to 120°C under continuous stirring (700 rpm). The synthesis was performed for 4 hours to open all epoxy rings (EV = 0). After synthesis, the obtained polyol was neutralized and dried with solid anhydrous calcium chloride. The content of additives, i.e. catalyst of reaction be- tween OH and NCO (1 wt%), catalyst of trimerization reaction (2.5 wt%), physical blowing agent (10 wt%), fl ame retardant (17 wt%) and surfactant (1.7 wt% ). The properties of the new bio-polyol (PG3) based on white mustard oil were studied. The reaction product was then used for the synthesis of rigid polyurethane- -polyisocyanurate foams. The foams were prepared on a laboratory scale from two-component system by using one-stage method. Component A was obtained by precisely mixing the Pol. J. Chem. Tech., Vol. 20, No. 2, 2018 26 (stability of linear dimensions, change of geometrical volume) and PN-ISO 4590:1994 (mass loss). Scheme 1. Reaction of urethane bond formation. ) ( ) – The thermal conductivity of the foams was determi- ned by Lasercomp FOX 200. ) ( ) – The thermal conductivity of the foams was determi- ned by Lasercomp FOX 200. p – Absorbability and water absorption were carried out in accordance with DIN 53433. – Absorbability and water absorption were carried out in accordance with DIN 53433. Scheme 1. Reaction of urethane bond formation. Preparation of PUR-PIR foams – Retention (Bütler’s vertical test) was determined in accordance with ASTM D3014-73. Scheme 2. The trimerization of isocyanate to isocyanurate ring. – Test of horizontal burning was determined in accor- dance with PN-78 C-05012. – Oxygen index (OI) was determined by Concept Equ- ipment apparatus in accordance with ASTM D2863-1970. RESULTS AND DISCUSSION Scheme 2. The trimerization of isocyanate to isocyanurate ring. Synthesis and characterisation of polyols appropriate amounts of polyols, catalysts, surfactant, blowing agent and fl ame retardant. Component B was Purocyn B. Components A and B were mixed for 10 s by using a mechanical stirrer (1800 rpm) at a suitable mass ratio. Then mixture was poured into a rectangular opened mould with an internal dimension of 25 x 25 x 30 cm, where was free rise of foam. Milk-white epoxidized oil (EMO) with an iodine va- lue of 0.039 mol I2/100g of oil and an epoxy number of 0.384 mol/100g of oil was obtained by reaction of white mustard oil (MO) with peracetic acid produced in situ. A simplifi ed reaction on the example of an oleic acid triglyceride, was shown in Scheme 3. A series of P3 foams (P3.0-P3.6) was added with an increase in the equivalent of bio-polyol from 0 to 0.6 (0.1R increments) at the expense of the petrochemical polyol (Table 2). The synthesis of PUR-PIR foams was repeated twice. The resulting foam was cured for 4 hours at 120°C. Scheme 3. Epoxidation of unsaturated bonds in fatty acids Assessing the properties of rigid PUR-PIR foams The selected performance characteristics of the PUR- -PIR foams have been determined according to the accepted standards for rigid polyurethane foam: – Direction of the foaming process was analysed by measuring of typical foaming times: start, growth and gelation by electronic stopwatch29–30. Scheme 3. Epoxidation of unsaturated bonds in fatty acids – Apparent density foam is marked in accordance with ISO 845-1988. The effi ciency of oxidation the double bonds was de- termined as the ratio of the epoxy number of EMO to the initial iodine number of MO. It was equal 89.67% 31. – Compressive strength was achieved on Instron’s universal 5544 machine in accordance with PN 93/C- 89071 (ISO 844). q The epoxidized mustard oil was reacted to open the epoxy rings with the 2,2’-mercaptodiethanol according to Scheme 4. – The brittleness of the obtained foams was determined in accordance with ASTM C-421-61. – Aging tests of foam were determined after 48h thermostat at 120oC in accordance with ISO 1923:1981 The physicochemical properties of obtained light yellow bio-polyol (PG3) were shown in Table 3. Table 2. Formulation of rigid PUR-PIR foams with bio-polyol Table 2. Formulation of rigid PUR-PIR foams with bio-polyol Pol. J. Chem. Tech., Vol. 20, No. 2, 2018 27 Scheme 4. Reaction of epoxy ring opening by 2,2’-mercapto- diethanol Scheme 4. Reaction of epoxy ring opening by 2,2’-mercapto- diethanol group (–OH) stretching, (2) 2950 and 2925 cm–1 of C-H bond stretching in -CH2- and -CH3, (3) 1740 cm–1 of carbonyl group (C=O) stretching, (4) 1465 and 1380 cm–1 of C-H bond deformation in -CH2- and -CH3, (5) 1240, The effi ciency of the epoxy ring opening process was equal 100% because the epoxy value of the bio-polyol was 0 mol/100g of oil. The results of the elemental analysis of the obtained bio-polyol (PG3) are presented in Table 4. Table 3. Properties of bio-polyol based on white mustard oil Table 4. Results of elemental analysis of oil and bio-polyol Table 3. Properties of bio-polyol based on white mustard oil 1160 and 1089 cm–1 of torsional vibration of the ester group and (6) 725 cm–1 of oscillation of -CH2- group. There was a signifi cant increase in oxygen and sulphur content, at the expense of carbon and hydrogen. The increase in oxygen was due to the oxidation of double bonds and the opening of the rings with 2,2’-mercap- todiethanol. Assessing the properties of rigid PUR-PIR foams As a result, two -OH groups (one at the fatty acid carbon, the other at the end of the glycol chain) were obtained per a C=C bond. The increase in sulphur content is attributable to the attachment of the 2,2’-mercaptodiethanol molecule. Analysis of 1H NMR spectrum (Figure 2) showed characteristic chemical shift for: (1) protons of oleic groups of fatty acids -CH=CH-; (2) methane protons of glyceryl -CH2-CH-CH2-; (3) methylene protons of glyceryl -CH2-CH-CH2-; (4) protons of hydroxyl groups at the end of the chain -OH; (5) protons of α-CH2 groups to the hydroxyl group -CH2-OH; (6) protons of the β-CH2 group to the hydroxyl group -CH2-CH2-OH; (7) protons of α-CH2 groups to the sulphide group -CH2-S-CH2; (8) protons of the α-CH2 group to the carbonyl group -CH2- CO-; (9) protons of hydroxyl groups inside the chain -OH; (10) protons of α-CH2 groups with respect to olefi n group -CH2-CH=CH-; (11) protons of the β-CH2 group to the carbonyl group -CH2-CH2-CO-; (12) protons of CH2 groups in the fatty acid chain; (13) protons of the end groups -CH3. Analysis of 1H NMR spectrum (Figure 2) showed characteristic chemical shift for: (1) protons of oleic groups of fatty acids -CH=CH-; (2) methane protons of glyceryl -CH2-CH-CH2-; (3) methylene protons of glyceryl -CH2-CH-CH2-; (4) protons of hydroxyl groups at the end of the chain -OH; (5) protons of α-CH2 groups to the hydroxyl group -CH2-OH; (6) protons of the β-CH2 group to the hydroxyl group -CH2-CH2-OH; (7) protons of α-CH2 groups to the sulphide group -CH2-S-CH2; (8) protons of the α-CH2 group to the carbonyl group -CH2- CO-; (9) protons of hydroxyl groups inside the chain -OH; (10) protons of α-CH2 groups with respect to olefi n group -CH2-CH=CH-; (11) protons of the β-CH2 group to the carbonyl group -CH2-CH2-CO-; (12) protons of CH2 groups in the fatty acid chain; (13) protons of the end groups -CH3. The presence of sulphur in the bio-polyol molecule also has other important implications. This element belongs to the so-called group fl ame retardant. This suggests that it can play a dual role in the polyurethane preparation process as a polyol component and as an internal fl ame retardant. The chemical structure of synthesized bio-polyol was confi rmed by spectroscopic analyses: FTIR, 1H NMR and 13C NMR. The FTIR method (Figure 1) showed the presence of band intensity at: (1) 3450 cm–1 of hydroxyl Figure 1. Assessing the properties of rigid PUR-PIR foams FTIR spectrum of bio-polyol based on mustard oil Figure 1. FTIR spectrum of bio-polyol based on mustard oil Pol. J. Chem. Tech., Vol. 20, No. 2, 2018 28 the fatty acid chain; (12) carbons of penultimate groups -CH2-CH3; (13) carbons of the terminal groups -CH3. FTIR spectroscopy, 1H NMR and 13C NMR confi r- med the assumed chemical structure of the synthesized bio-polyol. Analysis of the 13C NMR spectrum (Figure 3) showed characteristic chemical shift for: (1) carbons of car- bonyl groups >C=O; (2) carbons of olefi nic fatty acids -CH=CH-; (3) carbons of α-CH2 groups to the alkoxyl group -CH2-O-CH2-; (4) carbons of α-CH groups linked to a hydroxyl group within the chain >CH-OH; (5) Figure 2. 1H NMR spectrum of bio-polyol based on mustard oil Figure 2 1H NMR spectrum of bio polyol based on mustard oil Figure 2. 1H NMR spectrum of bio-polyol based on mustard oil Analysis of the 13C NMR spectrum (Figure 3) showed characteristic chemical shift for: (1) carbons of car- bonyl groups >C=O; (2) carbons of olefi nic fatty acids -CH=CH-; (3) carbons of α-CH2 groups to the alkoxyl group -CH2-O-CH2-; (4) carbons of α-CH groups linked to a hydroxyl group within the chain >CH-OH; (5) methane carbons of glyceryl -CH2-CH-CH2-; (6) meth- ylene carbons of glyceryl -CH2-CH-CH2-; (7) carbons of α-CH2 groups to the hydroxyl group at the end of the chain -CH2-OH; (8) carbons of the α-CH2 group to the sulphide group -CH2-S-CH2-; (9) carbons of β-CH2 groups to the hydroxyl group at the end of the chain -CH2- CH2-OH; (10) carbons of α-CH2 groups to the carbonyl group -CH2-OOC-CH2-; (11) carbons of CH2 groups in the fatty acid chain; (12) carbons of penultimate groups -CH2-CH3; (13) carbons of the terminal groups -CH3. the fatty acid chain; (12) carbons of penultimate groups -CH2-CH3; (13) carbons of the terminal groups -CH3. the fatty acid chain; (12) carbons of penultimate groups -CH2-CH3; (13) carbons of the terminal groups -CH3. FTIR spectroscopy, 1H NMR and 13C NMR confi r- med the assumed chemical structure of the synthesized bio-polyol. 2 3 ( ) g p 3 FTIR spectroscopy, 1H NMR and 13C NMR confi r- med the assumed chemical structure of the synthesized bio-polyol. FTIR spectroscopy, 1H NMR and 13C NMR confi r- med the assumed chemical structure of the synthesized bio-polyol. Pol. J. Chem. Tech., Vol. 20, No. 2, 2018 In both cases absorbability and water absorption was due to the introduction of hydrophobic groups derived from vegetable oil into the polyurethane macromolecule. An important parameter for PUR-PIR rigid foams is the apparent density. It indirectly affects compression strength or brittleness. The increase in the share of the new polyol raw material resulted in a decrease in appa- rent density from 50.19 kg/m3 for the reference foam to 33.07 kg/m3 for the foam with 0.6 R. The incorporation to polyurethane, a component containing long linear chains results in a decrease in packing of the polymer macromolecules. It follows that fatty acid residues are fl exible segments with low crosslinking. Also water con- tent in the bio-polyol had an impact on apparent density. Free water molecule could react with NCO group and produce a chemical blowing agent – CO2. More blowing agent in the polyurethane formulation caused apparent density decrease. This phenomenon also signifi cantly affected on the compressive strength of rigid foams. With the increase in the number of elastic segments and the apparent density decrease, this parameter decreased from 377.43 kPa (PG3.0) to 293.31 kPa (PG3.6). In spite of a marked decrease in compressive strength, the value of this parameter was at a satisfactory level. Another consequence of the decrease in degree of packing was slight decrease in stiffness. This leads to a reduction in the brittleness of the obtained PUR-PIR rigid foams. In case of application of bio-polyol based on white mustard oil, the parameter was reduced from 40.17% for P3.0 foam to 3.53% for P3.6 foam (Figure 4). Figure 5. Dependence between absorbability, water absorption and bio-polyol content Figure 5. Dependence between absorbability, water absorption and bio-polyol content An important feature of porous polyurethane materials is their fl ammability. Rigid polyurethane-polyisocyanurate foams are characterized by lower fl ammability than polyu- rethane foams due to the presence of their isocyanurate ring. However, due to stringent fi re safety requirements, they must have a fl ame retardant (Antiblaze TMCP in foam series P3). Flammability tests of obtained polyurethane materials (vertical test, horizontal test, oxygen index) showed that the increase of the content of bio-polyol based on white mustard oil infl uenced on the increase of fi re resistance of these materials. Pol. J. Chem. Tech., Vol. 20, No. 2, 2018 Pol. J. Chem. Tech., Vol. 20, No. 2, 2018 Pol. J. Chem. Tech., Vol. 20, No. 2, 2018 29 Studies of accelerated aging i.e. change of linear di- mensions (Δl) and change of geometric volume (ΔV) showed that bio-polyol modifi ed foam up to 0.4 R had the same value like a reference foam, 1.3% and 6%, respectively. The increase in bio-polyol content resulted in an increase of Δl to 5.6% and ΔV to 10.8%. This was due to the migration of the blowing agent closed in the foam cells. The increased amount of long elastic segments encouraged easier escape from gas fumes such as 1,1,1,3,3-pentafl uorobutane and 1,1,1,2,3,3,3-heptafl uo- ropropane (Solkane HFC 365/227). Mass loss studies have shown that the addition of white mustard based bio-polyol was conducive to lowering this parameter from 3.09% for reference foam to 1.37% for foam with 0.6 R. This meant that the bio-polyol has a higher aging resistance than petrochemical polyol. measured during foaming, showed a tendency to elongate (Table 5). However, up to 0.4 R technology times were similar to the reference foam. With the use of more bio-polyol, a noticeable increase in start, growth and gelling time. This suggests that the new white mustard- -based bio-polyols have less reactivity than petrochemical polyols. As long as its contribution to the formulation is less than 0.5 R, it does not signifi cantly affect the reactivity of the polyol premix (component A). measured during foaming, showed a tendency to elongate (Table 5). However, up to 0.4 R technology times were similar to the reference foam. With the use of more bio-polyol, a noticeable increase in start, growth and gelling time. This suggests that the new white mustard- -based bio-polyols have less reactivity than petrochemical polyols. As long as its contribution to the formulation is less than 0.5 R, it does not signifi cantly affect the reactivity of the polyol premix (component A). Table 5. Processing times of PUR-PIR foams with bio-polyol Table 5. Processing times of PUR-PIR foams with bio-polyol Absorbability and water absorption are important pa- rameters in porous materials. The fi rst one refers to the amount of water that is in the material immediately after being removed from the dipping. The second one refers to the water that has got into its interior. In both cases, a signifi cant decrease in the value of this parameter was noted (Figure 5). Properties of rigid polyurethane-polyisocyanurate foams The second part of the experiment consisted of the synthesis of polyurethane-polyisocyanurate foams using a new white mustard oil based bio-polyol (PG3) in an amount of 0 to 0.6 molar equivalents. The formulation of rigid PUR-PIR foams are shown in Table 2. The effect of polyol on a number of properties of new polyurethane foams was investigated. Processing times, Figure 3. 13C NMR spectrum of bio-polyol based on mustard oil Figure 3 13C NMR spectrum of bio polyol based on mustard oil Figure 3. 13C NMR spectrum of bio-polyol based on mustard oil Figure 3. 13C NMR spectrum of bio-polyol based on mustard oil CONCLUSION New bio-polyol based on white mustard oil was synthe- sized in a two-step method: epoxidation of double bonds and opened it with 2,2’-mercaptodiethanol (Pat. Appl. PL422888). After testing, the obtained product showed suitability for the synthesis of rigid polyurethane-polyiso- cyanurate foams. It was characterized by i.e. a hydroxyl number of 371.70 mg KOH/g, an acid value of 4.71 mg KOH/g or a water content of 0.8%. The presented parameters were similar to those of commercial petro- chemical polyols used in rigid foams. The bio-polyol was used as a raw material for PUR-PIR foams, in amounts of 0 to 0.6 equivalents in admixture with a polyol based on product of oxypropylation of sorbitol. Properties of the obtained materials were tested. Studies were shown that bio-polyol based foams had lower apparent density, compressive strength and brittleness. The presence of polyols containing sulphur signifi cantly affected the fl ame retardancy of these materials. 11. Garrison, T., Murawski, A. & Quirino, R.L. (2016). Bio- -based polymers with potential for biodegradability. Polymers, 8 (7), 262. DOI: 10.3390/polym8070262. 12. Ibrahim, S., Ahmad, A. & Mohamed, N.S. (2015). Characterization of Novel Castor Oil-Based Polyurethane Polymer Electrolytes. Polymers, 7 (4), 747–759. DOI: 10.3390/ polym7040747. 13. Abdolhosscini, F. & Besharati Givi, M.K. (2016). Characterization of a Biodegradable Polyurethane Elastomer Derived from Castor Oil. Am. J. Polym. Sci, 6 (1), 18–27. DOI: 10.5923/j.ajps.20160601.03. 14. 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CONCLUSION Rapeseed oil as main component in synthesis of bio- -polyurethane-polyisocyanurate porous materials modifi ed with carbon fi bers. Polymer Testing, 59, 478–486. DOI: 10.1016/j. polymertesting.2017.03.006. Pol. J. Chem. Tech., Vol. 20, No. 2, 2018 & Wang, P. (2013). Syn- thesis of bio-based polyurethanes from epoxidized soybean oil and isopropanolamine. J. App. Polym. Sci, 10, 1929–1936. DOI: 10.1002/app.37564. 2. Lubczak, J., Chmiel-Szukiewicz, E., Duliban, J., Głowacz- -Czerwonka, D., Lubczak, R., Łukasiewicz, B., Zarzyka, I., Łodyga, A., Tyński, P., Kozioł, M., Majerczyk, Z. & Minda- -Data, D. (2014). Polyurethane foams with 1,3,5-triazine ring of improved thermal stability. Przem. Chem., 93 (10), 1690–1697. DOI: 10.12916/przemchem.2014.1690. [in Polish]. 1. Prociak, A., Rokicki, G. & Ryszkowska, J. (2014). Po- lyurethane materials. Warszawa, Poland: Wydaw. Naukowe PWN. [in Polish]. 2. Lubczak, J., Chmiel-Szukiewicz, E., Duliban, J., Głowacz- -Czerwonka, D., Lubczak, R., Łukasiewicz, B., Zarzyka, I., Łodyga, A., Tyński, P., Kozioł, M., Majerczyk, Z. & Minda- -Data, D. (2014). Polyurethane foams with 1,3,5-triazine ring of improved thermal stability. Przem. Chem., 93 (10), 1690–1697. DOI: 10.12916/przemchem.2014.1690. [in Polish]. LITERATURE CITED 18. Bueno-Ferrer, C., Hablot, E., del Carmen Garrigos, M., Bocchini, S., Averous, L. & Jimenez, A. (2012). Relationship between morphology, properties and degradation parameters of novative biobased thermoplastic polyurethanes obtained from dimer fatty acids. Polym. Deg. Stab., 97, 1964–1969. DOI: 10.1016/j.polymdegradstab.2012.03.002. 1. Prociak, A., Rokicki, G. & Ryszkowska, J. (2014). Po- lyurethane materials. Warszawa, Poland: Wydaw. Naukowe PWN. [in Polish]. 2. Lubczak, J., Chmiel-Szukiewicz, E., Duliban, J., Głowacz- -Czerwonka, D., Lubczak, R., Łukasiewicz, B., Zarzyka, I., Łodyga, A., Tyński, P., Kozioł, M., Majerczyk, Z. & Minda- -Data, D. (2014). Polyurethane foams with 1,3,5-triazine ring of improved thermal stability. Przem. Chem., 93 (10), 1690–1697. DOI: 10.12916/przemchem.2014.1690. [in Polish]. 19. Malewska, E., Bąk, S., Kurańska, M. & Prociak, A. (2016). The effect of various rapeseed oil-based polyols on selected properties of fl exible polyurethane foams. Polimery, 61, 799–806. DOI: 10.14314/polimery.2016.799. p y 20. Tu, Y., Kiatsimkul, P., Suppes, G. & Hsieh, F. (2007). Physical properties of water-blown rigid polyurethane foams Pol. J. Chem. Tech., Vol. 20, No. 2, 2018 31 from vegetable oil-based polyols. J. Appl. Polym. Sci., 105, 453–459. DOI: 10.1002/app.26060. from vegetable oil-based polyols. J. Appl. Polym. Sci., 105, 453–459. DOI: 10.1002/app.26060. 21. Veronese, V.B., Menger, R.K., de C. Forte, M.M. & Petzhold, C.L. 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Polimery, 63 (3), 185–190. DOI: 14314/polimery.2018.3.3. [in Polish]. 31. Smagowicz, A. (2011). Obtaining of epoxidized rapeseed oil. Pubished doctoral dissertation, Zachodniopomorski Uni- wersytet Technologiczny, Szczecin, Poland. [in Polish].
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Nutrient and Trace Element Contributions from Drained Islands in the Sacramento–San Joaquin Delta, California
San Francisco estuary and watershed science
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Authors Copyright Information Copyright 2022 by the author(s).This work is made available under the terms of a Creative Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ UC Davis San Francisco Estuary and Watershed Science Title Nutrient and Trace Element Contributions from Drained Islands in the Sacramento–San Joaquin Delta, California Permalink https://escholarship.org/uc/item/8bk6n2wq Journal San Francisco Estuary and Watershed Science, 20(2) Authors Richardson, Christina M. Fackrell, Joseph K. Kraus, Tamara E. C. et al. Publication Date 2022 DOI 10.15447/sfews.2022v20iss2art5 Supplemental Material https://escholarship.org/uc/item/8bk6n2wq#supplemental Copyright Information Copyright 2022 by the author(s).This work is made available under the terms of a Creativ Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Davis San Francisco Estuary and Watershed Science Title Nutrient and Trace Element Contributions from Drained Islands in the Sacramento–San Joaquin Delta, California Permalink https://escholarship.org/uc/item/8bk6n2wq Journal San Francisco Estuary and Watershed Science, 20(2) Authors Richardson, Christina M. Fackrell, Joseph K. Kraus, Tamara E. C. et al. Publication Date 2022 DOI 10.15447/sfews.2022v20iss2art5 Supplemental Material https://escholarship.org/uc/item/8bk6n2wq#supplemental Copyright Information Copyright 2022 by the author(s).This work is made available under the terms of a Creativ Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ Peer reviewed Powered by the California Digital Library University of California eScholarship.org eScholarship.org Sponsored by the Delta Science Program and the UC Davis Muir Institute RESEARCH Nutrient and Trace Element Contributions from Drained Islands in the Sacramento–San Joaquin Delta, California Christina M. Richardson* 1, Joseph K. Fackrell1,2, Tamara E. C. Kraus2, Megan Young3, Adina Paytan4 Christina M. Richardson* 1, Joseph K. Fackrell1,2, Tamara E. C. Kraus2, Megan Young3, Adina Paytan4 ABSTRACT concentrations in island drainage on three Delta islands and surrounding rivers from June 2017 to September 2018. These data were used to calculate island-level fluxes and then upscaled to estimate Delta-wide contributions from island drains. Based on these results, we present (1) new estimates of gross and net nutrient and trace element fluxes from Delta island drains, and (2) concomitant N stable isotope data to improve our understanding of island N cycling. Over 60% of nearly all island drainage gross nutrient and trace element loads occurred in winter and spring. Upscaled island drainage net annual total nitrogen (TN), total dissolved nitrogen (TDN), and NH4 + loads comprised an estimated 9%, 7%, and 4%, respectively, of annual inputs to this system in 2018, before the SRWTP upgrade. Under a post-upgrade scenario, we estimated net annual island drainage TDN contributions to increase to 11% and NH4 + contributions to 45% of total Delta inputs as the SRWTP NH4 + load diminished to near zero. Our results suggest that island drainage is a measurable N source that has likely become increasingly important now that the SRWTP upgrade is complete. With over 200 potential active outfalls, these inputs may affect aquatic biogeochemical cycling in many regions of the Delta, especially in areas with long residence times. Inventorying nutrient and trace element sources in the Sacramento-San Joaquin Delta (the Delta) is critical to understanding how changes—including alterations to point source inputs such as upgrades to the Sacramento Regional Wastewater Treatment Plant (SRWTP) and landscape-scale changes related to wetland restoration—may alter the Delta’s water quality. While island drains are a ubiquitous feature of the Delta, limited data exist to evaluate island drainage mass fluxes in this system. INTRODUCTION Over the last 4 decades, the San Francisco Bay and Sacramento-San Joaquin Delta (the Delta) have experienced drastic ecological changes. From the 1970s to the 1990s, primary production and phytoplankton biomass decreased by 40% and 60%, respectively (Jassby 2008). More recent work shows that cholorphyll a concentrations have declined by over 70% since 1975 (Cloern 2019). At the same time, less desirable phytoplankton species that produce cyanotoxins, like Microcystis, are increasing in abundance (Lehman et al. 2013). The introduction of a number of invasive species, including two clams, Potamocorbula amurensis and Corbicula fluminea, are commonly thought of as important catalysts of structural ecological change in this system, and many studies suggest that their introductions have led to at least some of the observed declines in phytoplankton biomass as a result of high grazing rates (Jassby 2008; Jassby et al. 2002). Winder and Jassby (2011) showed zooplankton community shifts over a 37-year period and associated this shift with the clam invasion. Other invasive species, including several aquatic macrophytes (e.g., Eichhornia crassipes, water hyacinth, and Ludwigia hexapetala, water primrose), persist in the Delta today and are affecting both habitat and water quality (Dahm et al. 2016; Ta et al. 2017). Dominant N sources to the Delta at present include upstream rivers and wastewater treatment plants. The Sacramento River and the San Joaquin River generally represent about 84% and 13% of water inflow to the Delta, respectively (Jassby and Cloern 2000). Together, they deliver over 17 million kg of total N (TN), as particulate and dissolved inorganic and organic N annually (Jassby and Cloern 2000; Saleh and Domagalski 2015). Diffuse (non-point source) agricultural sources upstream of the Delta account for the majority of TN in these rivers (Saleh and Domagalski 2015). A major anthropogenic point source of TN to the Delta is the Sacramento Regional Wastewater Treatment Facility (SRWTP), which underwent upgrades from advanced secondary treatment to tertiary treatment with biological nutrient removal that were close to Higher trophic-level species have experienced similar widespread declines. Decreases over the past 2 decades in pelagic fish abundance, often referred to as Pelagic Organism Decline, have prompted a number of food web studies (Sommer et al. 2007). However, master controls on biomass and production trends remain elusive, likely owing to the hydrologic and biogeochemical complexity of the estuary. KEY WORDS worldwide (Howarth et al. 2011; Paerl et al. 1998; Paerl et al. 2006; Seitzinger and Sanders 1997). Drainage water quality, agricultural drainage, return flow, diversions, Delta island groundwater, nitrogen, phosphorous, metals A number of studies have attempted to assess the effects of nutrient forms and ratios on primary productivity in the Delta environment (see reviews by Senn and Novick [2014]; Dahm et al. [2016]; Ward and Paerl [2016]). Most research on nutrients in the Delta has focused on nitrogen (N) biogeochemistry because of both its ubiquitous presence in human-affected watersheds and because of the widely debated importance of N speciation for primary production in the Delta (Cloern 2021; Kraus et al. 2017; Ward and Paerl 2016). Cloern (2019) showed that ammonium (NH4 +) and nitrate plus nitrite (NO3 – + NO2 –) concentrations in the Delta have changed significantly since the mid-1970s, with mean annual concentrations increasing over 50%. While previous work had suggested that nutrients were at saturation levels for phytoplankton in the Delta (Jassby et al. 2002), more recent work has found that N forms and concentrations as well as N ratios to other nutrients may play an important role in phytoplankton ecology and uptake kinetics (Dugdale et al. 2015; Glibert et al. 2016). These complex relationships between nutrients and Delta ecology highlight the importance of adequately characterizing and accounting for all internal and external nutrient sources in this system. ABSTRACT To better constrain inputs from island drains, we measured monthly discharge along with nutrient and trace element SFEWS  Volume 20 | Issue 2 | Article 5 https://doi.org/10.15447/sfews.2022v20iss2art5 * Corresponding author: cmrichar@ucsc.edu 1 University of California, Santa Cruz Earth and Planetary Sciences Santa Cruz, CA 95064 USA 2 US Geological Survey California Water Science Center Sacramento, CA 95819 USA 3 US Geological Survey Water Mission Area Laboratory and Analytical Services Division Menlo Park, CA 94025 USA 4 University of California, Santa Cruz Institute of Marine Sciences Santa Cruz, CA 95064 USA SFEWS  Volume 20 | Issue 2 | Article 5 https://doi.org/10.15447/sfews.2022v20iss2art5 * Corresponding author: cmrichar@ucsc.edu 1 University of California, Santa Cruz Earth and Planetary Sciences Santa Cruz, CA 95064 USA 2 US Geological Survey California Water Science Center Sacramento, CA 95819 USA 3 US Geological Survey Water Mission Area Laboratory and Analytical Services Division Menlo Park, CA 94025 USA 4 University of California, Santa Cruz Institute of Marine Sciences Santa Cruz, CA 95064 USA SFEWS  Volume 20 | Issue 2 | Article 5 https://doi.org/10.15447/sfews.2022v20iss2art5 * Corresponding author: cmrichar@ucsc.edu 1 University of California, Santa Cruz Earth and Planetary Sciences Santa Cruz, CA 95064 USA 2 US Geological Survey California Water Science Center Sacramento, CA 95819 USA 3 US Geological Survey Water Mission Area Laboratory and Analytical Services Division Menlo Park, CA 94025 USA 4 University of California, Santa Cruz Institute of Marine Sciences Santa Cruz, CA 95064 USA 2 US Geological Survey California Water Science Center Sacramento, CA 95819 USA 3 US Geological Survey Water Mission Area Laboratory and Analytical Services Division Menlo Park, CA 94025 USA 4 University of California, Santa Cruz Institute of Marine Sciences Santa Cruz, CA 95064 USA 1 SAN FRANCISCO ESTUARY & WATERSHED SCIENCE VOLUME 20, ISSUE 2, ARTICLE 5 INTRODUCTION This seasonality in carbon cycling and transport raises important questions regarding concomitant changes in N species and other nutrient and trace element concentrations in drainage waters. Fluctuations in water table elevation and flow can shift the oxic-anoxic boundary in the subsurface, with subsequent effects on biogeochemical processes that ultimately control island drainage water quality. Microbial processing of carbon in the saturated zone is largely controlled by the influx of oxygenated waters, water residence times, nutrient availability, and the availability of carbon for respiration (Limpens et al. 2008). When O2 demand is greater than O2 influx, anoxic conditions can develop given sufficient carbon substrate. Naturally reduced zones (NRZs) exist in these saturated areas where organic matter is abundant and oxidant-consuming reactions are continuous (Yabusaki et al. 2017). As a result, NRZs can affect concentrations of dissolved organic carbon (DOC), dissolved inorganic N (DIN), dissolved organic N (DON), and reduced metal species (Du Laing et al. 2009; Yabusaki et al. 2017). Past work has shown that both trace elements and organo-metal complexes, like methylmercury, can be mobilized in Delta soils (Alpers et al. 2014; Bachand et al. 2019; Stumpner et al. 2015). As such, island drainage in the Delta may contribute seasonally important fluxes of both oxidized and reduced N species as well as other macronutrients and trace elements mobilized in wet-dry cycled NRZs. completion in April 2021. Before the upgrade, SRWTP annually discharged around 4 million kg of TN into the Sacramento River in the northern portion of the Delta, and the SRWTP TN input comprised roughly 32% of the Sacramento River annual TN load, though the importance of the SRWTP TN load was amplified during low flow months when contributions from other sources decreased (Saleh and Domagalski 2015). The SRWTP TN input predominantly occurred as NH4 +, and SRWTP NH4 + inputs were thought to account for over 90% of NH4 + input into the Sacramento River (Jassby 2008). Post-upgrade, SRWTP NH4 + concentrations in the discharged effluent have decreased to near zero, and TDN concentrations have declined by roughly 60% to 70% (https://ciwqs.waterboards.ca.gov/). This decrease has substantially lowered N inputs from SRWTP. With this reduction, NH4 + and TDN contributions from other sources, such as drainage from subsided islands in the central Delta, may increase in relative importance as sources of N to Delta waterways. INTRODUCTION While no one variable has been able to fully account for the previously discussed changes, water quality, especially nutrient availability, remains an important control on ecosystem function in estuaries 2 JUNE 2022 Recent companion work by Richardson et al. (2020) found that island drainage is an important seasonal source of carbon to the Delta, and also showed that seasonal increases in dissolved carbon concentrations in island drainage are best explained by water table rises that help mobilize carbon in island soils. This seasonality in carbon cycling and transport raises important questions regarding concomitant changes in N species and other nutrient and trace element concentrations in drainage waters. Fluctuations in water table elevation and flow can shift the oxic-anoxic boundary in the subsurface, with subsequent effects on biogeochemical processes that ultimately control island drainage water quality. Microbial processing of carbon in the saturated zone is largely controlled by the influx of oxygenated waters, water residence times, nutrient availability, and the availability of carbon for respiration (Limpens et al. 2008). completion in April 2021. Before the upgrade, SRWTP annually discharged around 4 million kg of TN into the Sacramento River in the northern portion of the Delta, and the SRWTP TN input comprised roughly 32% of the Sacramento River annual TN load, though the importance of the SRWTP TN load was amplified during low flow months when contributions from other sources decreased (Saleh and Domagalski 2015). The SRWTP TN input predominantly occurred as NH4 +, and SRWTP NH4 + inputs were thought to account for over 90% of NH4 + input into the Sacramento River (Jassby 2008). Post-upgrade, SRWTP NH4 + concentrations in the discharged effluent have decreased to near zero, and TDN concentrations have declined by roughly 60% to 70% (https://ciwqs.waterboards.ca.gov/). This decrease has substantially lowered N inputs from SRWTP. With this reduction, NH4 + and TDN contributions from other sources, such as drainage from subsided islands in the central Delta, may increase in relative importance as sources of N to Delta waterways. Recent companion work by Richardson et al. (2020) found that island drainage is an important seasonal source of carbon to the Delta, and also showed that seasonal increases in dissolved carbon concentrations in island drainage are best explained by water table rises that help mobilize carbon in island soils. INTRODUCTION The Delta contains over fifty peat islands, many of which are artificially drained and commercially farmed. Long-term drainage of Delta islands for farming has resulted in extensive land subsidence from soil oxidation, with many islands now residing more than 3 m below sea level (Deverel and Leighton 2010). As a result, most Delta islands must artificially maintain water tables below the land surface via managed pumping. Water pumped off Delta islands is commonly referred to as agricultural drainage, return flow, and/or island drainage, and here we use “island drainage” to refer to this flow. Previous estimates of water discharge from island drainage (~ 1.5 × 106 m3d–1) are comparable in magnitude to those from major wastewater treatment plants, such as SRWTP (~ 5.5 × 105 m3d–1) (Templin and Cherry 1997). However, nutrient budgets for the Delta generally only account for upstream river and wastewater inputs, and Delta island drainage inputs remain elusive since they are widely under-studied. There may be over 200 active island drainage outfalls in the Delta; however, even our knowledge of drain locations is outdated (Siegfried et al. 2014). To address this gap in knowledge, we measured monthly island drainage nutrient (NH4 +, NO3–, NO2 –, PO4 3–, DON, PON, SiO4 4–) and trace element (total dissolved Fe, Mn, As) concentrations along with discharge on Sherman, Staten, and Twitchell islands in the Delta from June 2017 to September 2018 (Figure 1). Island drainage discharge volumes were used together with constituent concentrations to calculate island- https://doi.org/10.15447/sfews.2022v20iss2art5 3 SAN FRANCISCO ESTUARY & WATERSHED SCIENCE VOLUME 20, ISSUE 2, ARTICLE 5 (DOC, DIC), stable isotopes of nitrate (δ15N-NO3 – and δ18O-NO3 –), and select trace elements (total dissolved As, Mn, Fe) (Figure 1). Samples for total suspended solids (TSS), δ15N‑NH4 +, δ15N-PON, and δ15N-DON were collected quarterly during the same sampling events. A multi-parameter water- quality meter (YSI Pro Plus) was used to measure ancillary water parameters (pH, dissolved oxygen, conductivity, and temperature) at the time of sample collection. We do not report data for sites where monthly discharge was zero during the study period (e.g., May 2018 to September 2018 at SH-P4 and all of SH-P1). Similarly, all carbon data are presented separately in the Richardson et al. (2020) companion paper. level gross fluxes. Site Description p Drainage from three Delta islands was sampled in this study: Sherman (SH), Staten (ST), and Twitchell (TW) islands (Figure 1A through 1D). Island drainage from each of these Delta islands is discharged via outfalls that connect island pump stations to Delta channels. Sherman Island has five pump stations and is dominated by pastureland (67%) and cropland (30%) (Table A1). Staten Island has two pump stations and is predominantly cropland ( 93%). Twitchell Island has one pump station and is mixed land use made up approximately of cropland (48%), pastureland ( 22%), and several managed wetlands ( 30%). These islands are surrounded by river channels and sloughs from which water is siphoned or pumped for irrigation, a practice commonly referred to as diversion. These channels are also a source of seepage waters onto the islands. To characterize nutrient and element inputs onto the islands, surrounding river channels were also sampled. All nutrient and trace element concentrations were determined at the Marine Analytical Laboratory at the University of California at Santa Cruz. Nutrients were measured on a Lachat QuikChem 8000 Flow Injection Analyzer. DON was determined indirectly by conversion to inorganic N using Kjeldahl digestions and run on a Lachat QuikChem 8000 Flow Injection Analyzer. Dissolved trace element concentrations were determined on a Thermo ElementXR High-Resolution Inductively Coupled Plasma Mass Spectrometer and run together with certified reference materials (NIST Standard Reference Material 1643f). DOC concentrations were measured as non-purgeable organic carbon (NPOC) on a Shimadzu TOC-VCPH TOC/TN Analyzer. Island drainage particulate organic matter (POM) concentrations were estimated from TSS concentrations as described in Richardson et al. (2020), and molar ratios of C to N (C:N)m of POM values were used to INTRODUCTION Inflow water volumes, taken as the collective sum of seepage and diversions, to Delta islands were calculated using a water budget approach, and river nutrient and trace element concentrations were used with the calculated water inflow volumes to estimate inflow gross fluxes. Island drainage net fluxes were subsequently calculated by subtracting the drainage fluxes (off-island) from the river inflow fluxes (on-island). We then upscaled island-level fluxes to the entire Delta to establish baseline estimates of (1) the timing and magnitude of this poorly characterized input to the larger Delta environment and (2) the regional importance of island drainage in the context of other major freshwater inputs under pre- and post-SRWTP upgrade conditions. Water samples for all analyses, except trace elements, were vacuum filtered in the lab to 0.7 µm (pre-combusted Whatman GF/F) first and then 0.2 µm (Millipore Nylon Membrane). Water samples for trace elements were filtered on-site to 0.45 µm using trace-clean certified capsule filters (Geotech) and immediately acidified to pH < 2 with triple-distilled trace clean HCl. Samples were kept on ice until filtered and subsequently frozen or refrigerated, as dictated by their storage requirements. Water Analyses Water samples were collected monthly from all drains on the three islands as well as from seven surrounding river channels from June 2017 through September 2018 for nutrients (NH4 +, NO3 –, NO2 –, DON, PO4 3–, SiO4 4–), carbon species 4 JUNE 2022 JUNE 2022 Figure 1  (A) Overview of the Sacramento–San Joaquin Delta showing the location of, the Sacramento River at Freeport, Sacramento Regional Wastewater Treatment Plant, San Joaquin River at Vernalis, and the three islands sampled in this study. Island drain locations for the entire Delta are shown as blue circles based on a digitized map from CDWR (1995). (B-D) Maps of Sherman, Staten and Twitchell Island indicating locations of island drain (blue circles) and river (orange triangles) sites sampled on each island. All study area maps are oriented such that North is up. Site abbreviations are as follows: Sherman Island (SH), Staten Island (ST), and Twitchell Island (TW). Figure 1  (A) Overview of the Sacramento–San Joaquin Delta showing the location of, the Sacramento River at Freeport, Sacramento Regional Wastewater Treatment Plant, San Joaquin River at Vernalis, and the three islands sampled in this study. Island drain locations for the entire Delta are shown as blue circles based on a digitized map from CDWR (1995). (B-D) Maps of Sherman, Staten and Twitchell Island indicating locations of island drain (blue circles) and river (orange triangles) sites sampled on each island. All study area maps are oriented such that North is up. Site abbreviations are as follows: Sherman Island (SH), Staten Island (ST), and Twitchell Island (TW). https://doi.org/10.15447/sfews.2022v20iss2art5 5 SAN FRANCISCO ESTUARY & WATERSHED SCIENCE VOLUME 20, ISSUE 2, ARTICLE 5 TW-P1 on Twitchell Island were cross-checked with 1.5 years of daily flow meter data (AgriFlo XCi ultrasonic sensor) collected across the 2017 and 2018 water years. This cross-comparison indicated that the unit-power consumption method is a relatively robust approximation of discharge (slope  =  0.87, R2   =  0.75) that slightly underestimates actual discharge. As such, mass fluxes generated using these discharge estimates herein are considered conservative estimates (e.g., actual fluxes are likely higher). estimate PON concentrations. For river samples, we used the previously published relationship between TSS and POC for the Delta’s rivers from Murrell and Hollibaugh (2000) to calculate POC concentrations, and then used (C:N)m ratios to generate PON concentrations. Water Analyses This method assumes around 5% of riverine TSS is POC, which is in line with more recent work by Hernes et al. (2020). Precision and accuracy were below 5% for all nutrients and below 8% for trace elements. Annual means and standard deviation of geochemical data are presented for the 2018 water year (October 2017 to September 2018), a below-normal water year which we refer to herein as “dry.” Island drainage fluxes (mass per unit of time) or loads (mass) off-island into Delta waterways are referred to herein as gross fluxes or gross loads. Island drainage gross fluxes were calculated from monthly measured concentrations and discharge data for each drainage site. For islands with more than one drain (Sherman and Staten islands), monthly loads were summed from all drainage sites. All fluxes in this study are reported as elemental mass per unit of time. PON fluxes were calculated quarterly, at the same frequency as sample collection, and these fluxes were assumed to represent their respective seasonal contributions. Seasons were defined as follows: fall (September through November), winter (December through February), spring (March through May), and summer (June through August). Stable isotope samples (δ15N-NH4 +, δ15N-NO3 –, δ18O-NO3 –, δ15N-PON, and δ15N-DON) were run at the USGS Menlo Park Stable Isotope Facility using the methods described in Kendall et al. (2015). All values are presented in permil notation (‰) relative to Vienna Air (VAIR) for δ15N and Vienna Standard Mean Oceanic Water (VSMOW) for δ18O. Analytical precision for δ15N-NH4 +, δ15N- NO3 –, and δ18O-NO3 – was 1.1‰, 0.3‰, and 0.7‰, respectively. Analytical precision for δ15N-DON and PON was 0.4‰. Statistical analysis was performed in two steps: (1) a Kruskal-Wallis One Way Analysis of Variance on Ranks was completed to determine if seasonal values were statistically significantly different and (2) a coupled pairwise multiple comparisons procedure was then conducted using Dunn’s Method to determine which seasonal values were different from one another. P values less than 0.05 were considered as significant for all statistical tests. Island drainage gross mass fluxes were upscaled to Delta-wide contributions using seasonal mean geochemistry data from this study and an annual volumetric estimate of drainage discharge, ~5.3 × 108 m3, from Templin and Cherry (1997). Water Analyses First, to calculate monthly regional drainage volumes, the annual discharge volume was scaled to monthly time-steps using monthly flow percentages calculated from discharge data in this study, where discharge for each month was calculated as a percentage of total water year discharge (Table A2). Flow percentages were generated for Sherman Island, a pastureland- dominated land use, and Staten Island, a cropland-dominated land use. This assumes that the hydrologic regime of each island is, generally, regionally representative of these two land- use groups. Monthly discharge values for these Island drainage gross mass fluxes were upscaled to Delta-wide contributions using seasonal mean geochemistry data from this study and an annual volumetric estimate of drainage discharge, Discharge and Mass Flux Estimates This method gives greater weight to stations that have higher discharge rather than averaging across stations that may not discharge as much as nearby sites. The gross flux calculations resulted in two unique estimates: (1) upscaled gross mass fluxes based on Staten Island (cropland) flow percentages and geochemistry, and (2) upscaled gross mass fluxes based on Sherman Island (pastureland) flow percentages and geochemistry. Twitchell Island data were not used for upscaling because of its mixed land use. Around 82% of the region within the legal boundary of the Delta is cropland and 18% is pastureland, idle, or grassland (based on spatial data available online from the California Crop Mapping database https://data.cnra.ca.gov/ dataset/statewide-crop-mapping). These spatial coverage percentages were used to weight the previously discussed upscaled fluxes based on dominant land use in the Delta, and the data presented herein are the spatially weighted averages of these two estimates. Upscaled net flux estimates are discussed below. To estimate mass fluxes at greater temporal resolution than yearly, we also assumed that change in water storage was negligible seasonally across the 2018 water year. We recognize that this assumption probably does not reflect actual seasonal changes in water storage on Delta islands. The consequence of this assumption is that our seasonal water budgets may be overestimating water inflow when on-island water storage decreases (likely in the spring/ summer) and underestimating water inflow when on-island water storage increases (likely in the fall/winter). However, the use of these seasonal water inflow volumes is roughly supported by inflow values estimated by the DCD model, which were generally the same order of magnitude and positively correlated to one another (R2 = 0.88, slope = 0.96). As such, we believe our seasonal net fluxes, made possible by the seasonal water budgets, provide useful new insights that Discharge and Mass Flux Estimates Change in storage was assumed to be negligible on an annual scale, based on previous studies that show island water tables are generally stable at this time-scale (Deverel et al. 2015; Deverel et al. 2016). Since our outflow values were measured directly, the close alignment of ET and P estimates with DCD suggests that our island-level inflow estimates are relatively robust at the annual scale. Our island- level outflow values did differ substantially and non-linearly from DCD estimates though, with seasonal comparisons of measured outflow from this study vs. DCD estimates poorly correlated (R2 =  0.02). two land-use types in the Delta were summed to seasonal scales and then used together with seasonal mean concentration data for each island in subsequent gross and net flux calculations. The seasonal mean concentration data were not averaged equally across these islands, which have multiple drainage outlets; instead, we divided the island-scaled seasonal gross loads by their seasonal outflow volumes to obtain a spatially integrated mean concentration. This method gives greater weight to stations that have higher discharge rather than averaging across stations that may not discharge as much as nearby sites. The gross flux calculations resulted in two unique estimates: (1) upscaled gross mass fluxes based on Staten Island (cropland) flow percentages and geochemistry, and (2) upscaled gross mass fluxes based on Sherman Island (pastureland) flow percentages and geochemistry. Twitchell Island data were not used for upscaling because of its mixed land use. Around 82% of the region within the legal boundary of the Delta is cropland and 18% is pastureland, idle, or grassland (based on spatial data available online from the California Crop Mapping database https://data.cnra.ca.gov/ dataset/statewide-crop-mapping). These spatial coverage percentages were used to weight the previously discussed upscaled fluxes based on dominant land use in the Delta, and the data presented herein are the spatially weighted averages of these two estimates. Upscaled net flux estimates are discussed below. two land-use types in the Delta were summed to seasonal scales and then used together with seasonal mean concentration data for each island in subsequent gross and net flux calculations. The seasonal mean concentration data were not averaged equally across these islands, which have multiple drainage outlets; instead, we divided the island-scaled seasonal gross loads by their seasonal outflow volumes to obtain a spatially integrated mean concentration. Discharge and Mass Flux Estimates Water discharge from islands was measured as discussed by Richardson et al. (2020). Briefly, records documenting electrical usage, P (kW‑hr), from each pump station were used together with pump efficiency, U (kW‑hr m-3), to calculate discharge, D (m -3), using the unit-power consumption method where D  =  P/U (Ogilbee 1966; Ogilbee and Mitten 1970; Diamond and Williamson 1983). Discharge estimates from 6 JUNE 2022 from Station 247 for Sherman Island, Station 242 for Staten Island, and Station 140 for Twitchell Island via California Irrigation Management Information System (CIMIS; https://cimis.water. ca.gov/). ET was calculated at a monthly scale and summed to seasonal and annual scales by correcting monthly reference ET rates using crop coefficients for land use cover on each island for the dry 2018 water year (http://www.itrc.org/etdata/ index.html). Our seasonal ET and P estimates at the island level were in close agreement with values estimated by the Delta Channel Depletion (DCD) model (R2 = 0.98 for ET and R2  =  0.92 for P; 2020 email between L Liang and CM Richardson, unreferenced, see “Notes”). Change in storage was assumed to be negligible on an annual scale, based on previous studies that show island water tables are generally stable at this time-scale (Deverel et al. 2015; Deverel et al. 2016). Since our outflow values were measured directly, the close alignment of ET and P estimates with DCD suggests that our island-level inflow estimates are relatively robust at the annual scale. Our island- level outflow values did differ substantially and non-linearly from DCD estimates though, with seasonal comparisons of measured outflow from this study vs. DCD estimates poorly correlated (R2 =  0.02). from Station 247 for Sherman Island, Station 242 for Staten Island, and Station 140 for Twitchell Island via California Irrigation Management Information System (CIMIS; https://cimis.water. ca.gov/). ET was calculated at a monthly scale and summed to seasonal and annual scales by correcting monthly reference ET rates using crop coefficients for land use cover on each island for the dry 2018 water year (http://www.itrc.org/etdata/ index.html). Our seasonal ET and P estimates at the island level were in close agreement with values estimated by the Delta Channel Depletion (DCD) model (R2 = 0.98 for ET and R2  =  0.92 for P; 2020 email between L Liang and CM Richardson, unreferenced, see “Notes”). Inflow Water Flux Estimates To calculate net fluxes for Delta islands, we used a water budget approach to first estimate annual and seasonal water inflow to each island as follows: I = O + ET – P where I is total inflow (or import), including groundwater infiltration and diversions that bring river water onto the island (m3), O is outflow (or export) from island drainage pumps (m3), ET is evapotranspiration (m3), and P is precipitation (m3). Water budget data are provided in Appendix A, Tables A3 (annual) and A4 (seasonal). P was based on measured data where I is total inflow (or import), including groundwater infiltration and diversions that bring river water onto the island (m3), O is outflow (or export) from island drainage pumps (m3), ET is evapotranspiration (m3), and P is precipitation (m3). Water budget data are provided in Appendix A, Tables A3 (annual) and A4 (seasonal). P was based on measured data https://doi.org/10.15447/sfews.2022v20iss2art5 7 SAN FRANCISCO ESTUARY & WATERSHED SCIENCE VOLUME 20, ISSUE 2, ARTICLE 5 for water year (WY) 2018 (USGS 2019). The Sacramento River at Freeport site is located just upstream of the SRWTP discharge point. Concentration and flow data for SRWTP fluxes for WY 2018 were downloaded via the California Integrated Water Quality System and used in pre-upgrade SRWTP flux calculations (https:// www.waterboards.ca.gov/ciwqs/). Additionally, SRWTP only had data released for TP for the first 3 months of WY 2018, while all other N species had data availability for the full water year; our TP estimates for WY 2018 are thus based on these 3 months. Predicted post-upgrade dissolved N (TDN = 528 µM, NH4 + = 11.4 µM, NO3 – = 480 µM) and P concentrations (TP = 73 µM, unchanged) in SRWTP effluent were used together with WY 2018 discharge volumes to estimate post-upgrade SRWTP mass fluxes (LWA 2014). We assume reported SRWTP TP concentrations are roughly equivalent to PO4 3– as PO4 3– is the dominant component of TP in SRWTP effluent. warrant discussion herein. At the same time, we also recognize that there is uncertainty in the estimated seasonal water inflow volumes. This uncertainty could be better assessed with island groundwater level data, which are generally not currently available. To estimate Delta-wide inflow, we used the above island-level inflow data and calculated seasonal inflow-to-outflow ratios for pastureland- dominated islands (Sherman Island) vs. cropland- dominated islands (Staten Island). Net Mass Flux Estimates River nutrient and trace element concentrations were averaged seasonally from monthly samples for (1) only the river sites that surround each island, to calculate island-level inflow mass flux estimates, and (2) all river sites, to calculate river inflow mass fluxes at the Delta-wide scale (Table A5). The island-level and Delta-wide inflow water volumes, used to calculate inflow mass fluxes, were taken directly from water budget calculations as discussed above. The inflow mass fluxes were then subtracted from gross outflow fluxes at the island and Delta-wide level to calculate net mass fluxes. Monthly averaged island drainage nutrient and trace element concentrations showed seasonal trends for many, though not all, constituents (Figure 3). TDN, DON, NH4 +, SiO4 4–, and total dissolved Mn were significantly higher in the winter and spring across all sites compared to the summer (p < 0.05), while NO3 – and NO2 – concentrations were more variable, but generally higher in the winter and spring. Island drainage TDN concentrations averaged 201 ± 104 µM (defined as the mean ± one standard deviation) in fall, 240 ± 109 µM in winter, 199 ± 60 µM in spring, and 93 ± 22 µM in summer. A majority of TDN was comprised of DON, and DON concentrations were RESULTS Island Drainage Discharge and Water Quality Island drainage discharge was highly variable across sites and water years, though seasonal trends were apparent (Figure 2). Discharge was generally greatest in the winter across all three islands, with 49 ± 6 and 32 ± 10% of annual discharge occurring in winter of WY 2017 and 2018, respectively (Table A2). Cumulative discharge was 1.2 to 2.2 times greater in wet WY 2017 than dry WY 2018 across all islands. Inflow Water Flux Estimates These were used to scale previously reported Delta-wide island drainage water outflow (~ 5.3 × 108 m3) from Templin and Cherry (1997) to inflow (Table A4). Our annualized Delta-wide inflow-to-outflow ratio generally agreed with the value estimated by the DCD model, though volumetric magnitudes differed, with the DCD model estimating about two times the outflow volume we use herein for regional upscaling from Templin and Cherry (1997). The volumetric difference between the two methods suggests that our results can be considered conservative estimates that likely under-value the contribution of these waters. Pre- and Post-Upgrade Comparison of Wastewater, Island Drainage, and River Mass Fluxes NH4 + concentrations averaged 14 ± 11 µM across all sites in summer months and were notably higher at 26 ± 17 µM in fall, 61 ± 35 µM in winter, and 60 ± 44 µM in spring, respectively (p < 0.05). NO3 – concentrations in island drainage averaged 10 ± 10 µM across all sites in summer months, and were significantly higher in winter (34 ± 58 µM) (p < 0.05), while fall and spring had mean concentrations of 15 ± 34 µM in fall and 23 ± 35 µM in spring, respectively. Island drainage PON concentrations did not show a consistent seasonal pattern and averaged 210 ± 70 µM in fall, 120 ± 61 µM in winter, 156 ± 74 µM in spring, and 180 ± 58 µM in summer. PO4 3– concentrations in drainage were variable across sites and through statistically significantly different across seasons at 161 ± 99, 132 ± 56, 115 ± 42, and 79 ± 39 µM, in fall, winter, spring, and summer, respectively (p < 0.05). Relative proportions of DIN and DON shifted seasonally as well, with DIN generally increasing in relative proportion during winter and spring compared to summer and fall. NH4 + concentrations were typically higher than NO3 – and thus comprised a larger proportion of DIN in island drainage, except for some dates on Staten Island where NO3 – concentrations were elevated. NH4 + concentrations averaged 14 ± 11 µM across all sites in summer months and were notably higher at 26 ± 17 µM in fall, 61 ± 35 µM in winter, and 60 ± 44 µM in spring, respectively (p < 0.05). NO3 – concentrations in island drainage averaged 10 ± 10 µM across all sites in summer months, and were significantly higher in winter (34 ± 58 µM) (p < 0.05), while fall and spring had mean concentrations of 15 ± 34 µM in fall and 23 ± 35 µM in spring, respectively. Island drainage PON concentrations did not show a consistent seasonal pattern and averaged 210 ± 70 µM in fall, 120 ± 61 µM in winter, 156 ± 74 µM in spring, and 180 ± 58 µM in summer. Pre- and Post-Upgrade Comparison of Wastewater, Island Drainage, and River Mass Fluxes Monthly concentration (dissolved N and P) and discharge data were aggregated from the Sacramento River at Freeport (USGS 11447650), San Joaquin River at Vernalis (USGS 11303500), and SRWTP for seasonal mass flux comparisons to our Delta-wide island drainage net fluxes 8 JUNE 2022 JUNE 2022 Figure 2  (A) Monthly precipitation and (B) discharge from Sherman, Staten, and Twitchell Islands. Precipitation data were acquired from Station 242 via the California Irrigation Management Information System (CIMIS). Discharge data were determined using the unit-power consumption method and cross- checked with measured flow meter estimates discussed in Methods. Figure 2  (A) Monthly precipitation and (B) discharge from Sherman, Staten, and Twitchell Islands. Precipitation data were acquired from Station 242 via the California Irrigation Management Information System (CIMIS). Discharge data were determined using the unit-power consumption method and cross- checked with measured flow meter estimates discussed in Methods. time as well, with concentrations only slightly higher in summer months (2.7 ± 1.2 µM) relative to winter (1.6 ± 0.8 µM). Concentrations of SiO4 4– in island drainage were significantly lower in the summer (380 ± 130 µM) relative to fall, winter, and spring, when means ranged between 540 ± 160 µM to 600 ± 140 µM (p < 0.05). Total dissolved Mn concentrations were significantly higher during fall (670 ± 210 µg L–1), winter (760 ± 370 µg L–1), and spring (1100 ± 740 µg L–1) compared to summer (310 ± 300 µg L–1) as well (p < 0.05). Total dissolved Fe and As concentrations showed no significant seasonal trends, with means ranging between 820 ± 970 to 1550 ± 1450 µg L–1 for Fe and 5.5 ± 5.0 to 6.9 ± 5.0 µg L–1 for As across all seasons. statistically significantly different across seasons at 161 ± 99, 132 ± 56, 115 ± 42, and 79 ± 39 µM, in fall, winter, spring, and summer, respectively (p < 0.05). Relative proportions of DIN and DON shifted seasonally as well, with DIN generally increasing in relative proportion during winter and spring compared to summer and fall. NH4 + concentrations were typically higher than NO3 – and thus comprised a larger proportion of DIN in island drainage, except for some dates on Staten Island where NO3 – concentrations were elevated. Pre- and Post-Upgrade Comparison of Wastewater, Island Drainage, and River Mass Fluxes 10 JUNE 2022 Table 1  Mean and standard deviation of river and island drainage geochemistry collected monthly during water year (WY) 2018 between October 2017 and September 2018. WY 2017 data are not included so as not to bias the annual mean. Rivers SH-P2 SH-P3 SH-P4a SH-P5 ST-P1 ST-P2 TW-P1 TN (µM) mean 65 386 468 230 328 352 370 227 stdev 46 74 98 115 62 114 134 12 TDN (µM) mean 56 183 272 168 113 188 262 126 stdev 25 87 136 52 44 105 155 45 NO3 – + NO2 – (µM) mean 22 10 24 12 12 49 35 10 stdev 11 12 48 9 5 55 65 8 NH4 + (µM) mean 6 53 56 39 24 29 48 36 stdev 5 56 58 19 13 20 23 20 DON (µM) mean 29 119 192 113 75 107 174 80 stdev 27 35 105 48 32 67 109 39 PON (µM) mean 8 218 210 53 204 175 129 105 stdev 7 28 28 28 48 86 53 51 SiO4 4– (µM) mean 250 310 440 790 670 510 540 500 stdev 60 120 100 60 130 140 180 110 PO4 3– (µM) mean 1.7 1.0 1.8 2.5 2.4 2.2 1.6 3.1 stdev 0.6 0.8 1.3 1.6 0.8 2.0 1.2 1.3 As (µg L–1) mean 1.6 3.1 6.1 3.8 2.4 8.5 13.5 4.8 stdev 0.5 1.2 3.0 3.3 0.8 3.3 9.2 1.4 Mn (µg L–1) mean 30 1120 860 780 620 640 600 430 stdev 30 820 550 710 250 350 380 180 Fe (µg L–1) mean 60 820 310 1820 1030 1020 1650 2020 stdev 60 830 200 2070 740 770 1230 1000 δ15N-PON (‰) mean 4.9 3.0 3.3 0.1 -0.1 3.2 2.4 1.6 stdev 2.1 3.4 3.2 3.0 0.5 0.9 1.5 1.4 δ15N-DON (‰) mean 1.8 0.1 1.8 0.9 0.3 1.7 1.6 1.8 stdev 2.1 2.1 2.3 0.4 1.0 0.7 0.5 1.7 δ15N-NH4 + (‰) mean 9.9 9.0 9.8 9.3 9.0 11.9 11.2 10.3 stdev 5.6 4.3 2.3 1.4 1.8 1.7 2.3 2.4 δ15N-NO3 – (‰) mean 7.0 4.7 3.5 5.2 4.0 12.8 15.2 3.8 stdev 1.4 3.4 2.3 7.5 1.7 6.6 6.7 3.9 a. SH-P4 water year data are incomplete as data collected during net zero discharge months were not included. SH-P4 water year data are incomplete as data collected during net zero discharge months were not included. Pre- and Post-Upgrade Comparison of Wastewater, Island Drainage, and River Mass Fluxes δ15N values of PON, DON, NH4 +, and NO3 – indicated clear differences in stable isotope composition amongst N pools that were common to all island drainage sites (Figure 4A). DON and PON pools overlapped in concentration range and N stable isotope composition, ranging from 120 to 150 µM and 1.2 ± 0.5‰ to 2.2 ± 1.6‰, on average, respectively. Concentrations of NH4 + were similar or lower than organic N pools and generally had higher δ15N values, around 10.3 ± 1.1‰ on average, compared to PON and DON. Concentrations of NO3 – and δ15N-NO3 – values were generally lower and more variable than the NH4 + pool, with mean δ15N-NO3 – values of 6.7 ± 3.2‰. Pre- and Post-Upgrade Comparison of Wastewater, Island Drainage, and River Mass Fluxes PO4 3– concentrations in drainage were variable across sites and through At an annual scale, mean island drainage TDN, NH4 +, NO2 –, DON, PON, and SiO4 4– concentrations for WY 2018 were always greater than surrounding rivers at all sites, while NO3 – and PO4 3– concentrations were more variable, with concentrations both higher and lower than nearby rivers (Table 1). Mean annual dissolved Mn, Fe, and As concentrations were generally higher in island drainage, by up to two orders of magnitude, relative to river water (Table 1). https://doi.org/10.15447/sfews.2022v20iss2art5 9 SAN FRANCISCO ESTUARY & WATERSHED SCIENCE VOLUME 20, ISSUE 2, ARTICLE 5 Box plots of monthly island drainage concentrations from all sites, starting in June 2017 through September 2018, for (A) NO3 – d organic N (DON), (E) total dissolved N (TDN), (F) SiO4 4–, (G) PO4 3–, (H) total dissolved Fe, (I) total dissolved Mn, and (J) total d ent the bounds of the middle quartiles, and lines represent median values. Whiskers show the bounds of the outer quartiles s, starting in June 2017 through September 2018, for (A) NO3 – 4 3–, (H) total dissolved Fe, (I) total dissolved Mn, and (J) total dian values. Whiskers show the bounds of the outer quartiles Figure 3  Box plots of monthly island drainage concentrations from all sites, starting in June 2017 through September 2018, for (A) NO3 – (B) NO2 –, (C) NH4 +, (D) dissolved organic N (DON), (E) total dissolved N (TDN), (F) SiO4 4–, (G) PO4 3–, (H) total dissolved Fe, (I) total dissolved Mn, and (J) total dissolved As. Boxes represent the bounds of the middle quartiles, and lines represent median values. Whiskers show the bounds of the outer quartiles (5th and 95th) of the data. Figure 3  Box plots of monthly island drainage concentrations from all sites, starting in June 2017 through September 2018, for (A) NO3 – (B) NO2 –, (C) NH4 +, (D) dissolved organic N (DON), (E) total dissolved N (TDN), (F) SiO4 4–, (G) PO4 3–, (H) total dissolved Fe, (I) total dissolved Mn, and (J) total dissolved As. Boxes represent the bounds of the middle quartiles, and lines represent median values. Whiskers show the bounds of the outer quartiles (5th and 95th) of the data. Island Drainage Nutrient and Trace Element Fluxes Island-level gross TN and TDN fluxes, calculated from monthly concentration and discharge data, https://doi.org/10.15447/sfews.2022v20iss2art5 11 SAN FRANCISCO ESTUARY & WATERSHED SCIENCE VOLUME 20, ISSUE 2, ARTICLE 5 Figure 4 (A) δ15N values versus N species concentration for NH4 + (purple), NO3 – (blue), particulate organic N (PON) (dark green), and dissolved organic N (DON) (light green). Circular markers represent inorganic N pools and triangular markers represent organic N pools. (B) Conceptual model of the relative relationship between δ15N values and concentration for major dissolved and particulate N pools on Delta islands. Model shows interconnecting processes, but arrows are ranged from 70 to 230 kg d–1 and 20 to 100 kg d–1 in the summer and 170 to 320 kg d–1 and 120 to 200 kg d–1 in the winter, respectively (Table 2, Figure 5). Organic N accounted for ~79 to 81% of gross annual TN fluxes across all sites (with 40% to 48% and 29% to 39% of the gross TN flux as PON and DON, respectively), while DIN accounted for the remaining ~19% to 21%. Island drainage gross SiO4 4– fluxes peaked in the winter (610 to 820 kg d–1), while gross PO4 3– fluxes were generally greatest during the summer (1 to 7 kg d–1). Island drainage gross total dissolved Mn and Fe fluxes ranged between 10 and 70 kg d–1 and 10 to 110 kg d–1, respectively, and were seasonally greatest in the winter on all islands (Table 2). Island drainage gross total dissolved As fluxes ranged between 0.1 to 0.5 kg d–1 across all sites and seasons. While nutrient and trace element gross fluxes generally peaked in the winter, Staten and Twitchell islands also experienced secondary peaks in mass fluxes during summer. After accounting for inflow fluxes, island-level net drainage fluxes were highest in the winter and spring for all measured species (Table 2). Islands were generally net sinks in the summer for TDN (0 to –130 kg d–1), NO3 – + NO2 – (–20 to –50 kg d–1), SiO4 4– (–440 to –1310 kg d–1), and PO4 3– (-3 to -12 kg d–1), and Sherman Island was a temporary sink for all DIN species and DON in the summer. Annual net island level fluxes were positive for both TDN and TN across all islands, though NO3 – + NO2 – fluxes were net negative on both Sherman and Twitchell islands. Island Drainage Nutrient and Trace Element Fluxes Figure 4 (A) δ15N values versus N species concentration for NH4 + (purple), NO3 – (blue), particulate organic N (PON) (dark green), and dissolved organic N (DON) (light green). Circular markers represent inorganic N pools and triangular markers represent organic N pools. (B) Conceptual model of the relative relationship between δ15N values and concentration for major dissolved and particulate N pools on Delta islands. Model shows interconnecting processes, but arrows are qualitative and do not refer to the direction of concentration or δ15N change. DNRA represents dissimilatory nitrate reduction to ammonium. Double curved arrows represent conversions to or from various gaseous (g) N forms. Upscaled to annual Delta-wide contributions, island drainage contributed an estimated total annual gross TN load of 2.7 × 106 kg to Delta waterways in WY 2018 (Table 3). Similar to island- level estimates, the annual gross TN load was compositionally dominated by PON (43%) and DON (34%), with DIN comprising the remaining 24% (see Figure 6 for seasonal percentages). The annual total island drainage gross SiO4 4– load was estimated to be about 7.6 × 106 kg, while the PO4 3- load was about 2.8 × 104 kg (Table 3). Annual gross total dissolved Mn and Fe loads from all islands were similar in magnitude, 3.7 × 105 to 5.9 × 105 kg, while total dissolved As contributions were 12 JUNE 2022 Table 2  Seasonal gross and net island drainage nutrient and trace element fluxes for each island for water year 2018 calculated by season (Fall: September to November; Winter: December to February; Spring: March to May; Summer: June to August), and for the water year (annual). the smallest of all loads and averaged around 4.4 × 103 kg annually. Annual Delta-wide net island drainage fluxes were positive for all constituents measured except PO4 3– (Table 3). Delta-wide net island drainage fluxes for TN and TDN averaged 5,030 kg d–1 and N fluxes totaled 570 kg d–1 for NH4 +, 170 kg d–1 for NO3 – + NO2 –, 1,520 kg d–1 for DON and 2,740 kg d–1 for PON. Seasonally, Delta-wide island drainage fluxes were net negative in the summer for all constituents except for total dissolved Mn, suggesting that islands may act as temporary sinks for many constituents (Table 4). These September to November; Winter: December to February; Spring: March to May; Summer: June to August), and for the water year (annual). Island Drainage Nutrient and Trace Element Fluxes Season TN (kg d–1) TDN (kg d–1) NO3 – + NO2 – (kg d–1) NH4 + (kg d–1) DON (kg d–1) PON (kg d–1) SiO4 4– (kg d–1) PO4 3– (kg d–1) As (kg d–1) Mn (kg d–1) Fe (kg d–1) Gross Sherman Fall 140 90 0 10 80 50 370 2 0.1 20 20 Winter 320 200 30 60 110 110 820 2 0.2 60 40 Spring 340 150 10 50 80 190 660 2 0.2 70 50 Summer 70 20 0 0 20 50 150 1 0.1 10 10 Annual 220 110 10 30 70 100 500 2 0.1 40 30 Staten Fall 150 60 10 10 50 80 260 2 0.2 10 20 Winter 170 140 30 30 80 30 610 1 0.4 30 80 Spring 250 110 40 30 40 140 550 1 0.3 30 20 Summer 230 100 30 20 60 130 860 7 0.5 10 40 Annual 200 100 30 20 60 100 570 3 0.4 20 40 Twitchell Fall 110 70 0 10 50 40 460 4 0.2 20 70 Winter 200 120 10 40 70 80 820 5 0.2 20 110 Spring 90 60 10 20 40 20 570 3 0.1 20 50 Summer 130 60 0 10 40 70 440 5 0.3 10 100 Annual 130 80 10 20 50 50 570 4 0.2 20 80 Net Sherman Fall 50 10 –30 0 0 40 –300 –4 0.0 20 20 Winter 260 150 10 60 90 110 430 0 0.1 60 40 Spring 190 10 –40 50 10 170 –230 –6 0.0 60 30 Summer –140 –130 –50 –10 –70 –20 –1170 –12 –0.4 –10 –10 Annual 90 10 –30 20 10 80 –320 –6 –0.1 30 20 Staten Fall 140 50 10 10 40 80 170 1 0.2 10 20 Winter 160 140 30 30 80 30 580 1 0.4 30 80 Spring 190 60 30 20 10 130 –20 –2 0.1 30 20 Summer 50 –60 –40 –40 20 110 –1310 –8 –0.1 10 30 Annual 140 50 10 0 40 90 –150 –2 0.2 20 40 Twitchell Fall 70 20 –20 10 30 40 20 0 0.1 20 70 Winter 130 60 –20 30 50 70 220 1 0.1 20 100 Spring 20 0 –20 10 10 20 100 –2 0.0 20 50 Summer 60 0 –20 10 10 60 –440 –3 0.0 10 100 Annual 70 20 –20 20 30 50 –20 –1 0.0 20 80 the smallest of all loads and averaged around 4.4 × 103 kg annually. Island Drainage Nutrient and Trace Element Fluxes N fluxes totaled 570 kg d–1 for NH4 +, 170 kg d–1 for NO3 – + NO2 –, 1,520 kg d–1 for DON and 2,740 kg d–1 for PON. Seasonally, Delta-wide island drainage fluxes were net negative in the summer for all constituents except for total dissolved Mn, suggesting that islands may act as temporary sinks for many constituents (Table 4). These upscaled net negative fluxes were overcome by the smallest of all loads and averaged around 4.4 × 103 kg annually. N fluxes totaled 570 kg d–1 for NH4 +, 170 kg d–1 for NO3 – + NO2 –, 1,520 kg d–1 for DON and 2,740 kg d–1 for PON. Seasonally, Delta-wide island drainage fluxes were net negative in the summer for all constituents except for total dissolved Mn, suggesting that islands may act as temporary sinks for many constituents (Table 4). These upscaled net negative fluxes were overcome by Annual Delta-wide net island drainage fluxes were positive for all constituents measured except PO4 3– (Table 3). Delta-wide net island drainage fluxes for TN and TDN averaged 5,030 kg d–1 and 2,290 kg d–1 annually, respectively. Delta-wide net https://doi.org/10.15447/sfews.2022v20iss2art5 13 SAN FRANCISCO ESTUARY & WATERSHED SCIENCE VOLUME 20, ISSUE 2, ARTICLE 5 SAN FRANCISCO ESTUARY & WATERSHED SCIENCE VOLUME 20, ISSUE 2, ARTICLE Figure 5  Seasonal (A-C) gross and (D-F) net island drainage nitrogen (N) species loads for (A, C) Sherman, (B, E) Staten, and (C, F) Twitchell Islands. Bar color refers to N species (NO3 – + NO2 –, NH4 +, dissolved organic nitrogen (DON), particulate organic N (PON). See Table 2 for values. Figure 5  Seasonal (A-C) gross and (D-F) net island drainage nitrogen (N) species loads for (A, C) Sherman, (B, E) Staten, and (C, F) Twitchell Islands. Bar color refers to N species (NO3 – + NO2 –, NH4 +, dissolved organic nitrogen (DON), particulate organic N (PON). See Table 2 for values. Controls on Island Drainage Nutrient and Trace Element Composition Under oxic conditions, a portion of NH4 + can also be converted to NO3 – via nitrification, which will also leave behind a 15N-enriched NH4 + pool. The spatial and temporal variability in NO3 – and NO2 – concentrations along with δ15N-NO3 – values across all sites suggests that these processes change irregularly and are not spatially or temporally consistent. Island Drainage Nutrient and Trace Element Fluxes stable isotope signature of mineralized organic N—which would have NH4 + with lower δ15N-NH4 + values relative to its organic source—is overprinted by other biogeochemical processes common to all sites (Nadelhoffer and Fry 1994). This unexpected elevation in δ15N-NH4 + values relative to organic N pools is best explained by a combination of nitrification, uptake, and volatilization of NH4 +, all of which would lead to preferential loss of 14NH4 + and/or 14NH3 that leaves remaining NH4 + enriched in 15N (Ostrom et al. 1998; Clark 2015). The NH4 + pool is likely subject to uptake and volatilization in the unsaturated zone during summer and fall when water tables are low and plant biomass is high. The seasonality in NH4 + concentrations across all sites suggests that increases in NH4 + concentrations coincide with known winter and spring periods of water table rises from seasonal shifts in island hydrology (evapotranspiration, precipitation, etc.). As such, drainage outlets receive mineralized NH4 + that is mobilized and transported from shallow soil stores during winter and spring that was previously subjected to uptake and volatilization during the summer and fall. Some of the material transported during this time could be pulsed off-island as “first flush” events, but the sustained elevation in N content in drainage suggests that seasonal shifts in primary water sources are the major driver of change. Figure 6  Relative proportion of individual N species as a percentage of the seasonal upscaled Delta-wide island drainage total nitrogen (TN) flux for WY 2018 (see text for details). Figure 6  Relative proportion of individual N species as a percentage of the seasonal upscaled Delta-wide island drainage total nitrogen (TN) flux for WY 2018 (see text for details). greater net positive fluxes during other seasons though, as indicated by the positive annual island drainage net fluxes for all constituents except PO4 3– (Table 4). Island Drainage Nutrient and Trace Element Fluxes 14 JUNE 2022 Table 3  Upscaled Delta-wide island drainage gross fluxes, river inflow fluxes, and net fluxes for WY 2018 before any upgrades to the Sacramento Regional Wastewater Treatment Plant Wastewater Treatment Plant Annual island drainage load (kg) Island drainage gross flux (kg d–1) River inflow flux onto islands (kg d–1) Mean annual net flux (kg d–1) Annual net load (kg) TN 2.7 x 106 7,390 2,360 5,030 1.8 x 106 TDN 1.5 x 106 4,240 1,950 2,290 8.4 x 105 NO3 – + NO2 – 3.3 x 105 910 750 170 6.2 x 104 NH4 + 3.0 x 105 830 260 570 2.1 x 105 DON 9.1 x 105 2,500 970 1,520 5.6 x 105 PON 1.1 x 106 3,150 410 2,740 1.0 x 106 SiO4 4– 7.6 x 106 20,770 19,270 1,490 5.4 x 105 PO4 3– 2.8 x 104 80 170 – 90 –3.3 x 104 As 4.4 x 103 10 10 10 2.3 x 103 Mn 3.7 x 105 1,010 80 930 3.4 x 105 Fe 5.9 x 105 1,620 180 1,440 5.3 x 105 Table 4  Upscaled Delta-wide seasonal mean island drainage gross and net fluxes based on water year 2018 geochemistry data Fall (kg d–1) Winter (kg d–1) Spring (kg d–1) Summer (kg d–1) Gross TN 6,240 9,620 9,590 4,100 TDN 3,800 7,030 4,500 1,630 NO3 – + NO2 – 520 1,500 1,270 380 NH4 + 360 1,500 1,190 270 DON 2,930 4,040 2,040 980 PON 2,440 2,590 5,100 2,470 SiO4 4– 14,930 31,890 22,750 13,500 PO4 3– 70 70 50 110 As 10 20 10 10 Mn 770 1,620 1,430 200 Fe 1,200 3,390 1,200 670 Net TN 5,470 9,150 6,910 – 1,410 TDN 3,120 6,600 2140 – 2,680 NO3 – + NO2 – 250 1,330 320 – 1,220 NH4 + 280 1,460 1,000 – 470 DON 2,450 3,870 770 – 990 PON 2,350 2,560 4,780 1,270 SiO4 4– 8,470 28,540 4,560 – 35,600 PO4 3– 20 50 – 100 – 330 As 10 20 10 – 10 Mn 750 1,620 1,330 10 Fe 1,180 3,360 960 260 Upscaled Delta-wide seasonal mean island drainage gross and net fluxes based on water year 2018 geochemistry data https://doi.org/10.15447/sfews.2022v20iss2art5 15 SAN FRANCISCO ESTUARY & WATERSHED SCIENCE VOLUME 20, ISSUE 2, ARTICLE 5 Figure 6  Relative proportion of individual N species as a percentage of the seasonal upscaled Delta-wide island drainage total nitrogen (TN) flux for WY 2018 (see text for details). Trace Elements We found high concentrations of total dissolved Mn and Fe in island drainage during winter and spring months when compared to summer (Figure 3H and 3I). Mobilization of Fe and Mn in water is commonly associated with redox state, and the observed seasonal increases in total dissolved Fe and Mn concentrations suggest that drainage waters receive contributions from a reduced water source seasonally. This seasonality has been observed for other trace elements in temporarily flooded fields of the Delta, where re-wetting periods are thought to mobilize mercury species previously formed in unsaturated soils during dry phases (Marvin-DiPasquale et al. 2014). Regionally, reduction of Fe- and Mn-oxides commonly leads to increases in soluble Fe and Mn species in groundwater (Bennett et al. 2006). However, we found no relationship between total dissolved Fe and Mn concentrations across all sites, though site-specific trends were evident at some locations (but weakly correlated) (Figure 7). Interestingly, total dissolved Mn and DOC concentrations (R2 = 0.29) were weakly positively correlated, while total dissolved Fe and dissolved oxygen concentrations (R2 = 0.38) along with pH (R2 = 0.37) were weakly negatively correlated when considering all sites (Figure 7). This dynamic relationship suggests that controls on total dissolved Mn and organic matter inputs may be At the individual site level, N species stable isotope values were highly variable, both spatially and temporally (Table 1). Such variability in individual N species stable isotope values, without context relative to other N pools, shows that biogeochemical controls and sources are complex at small spatial and temporal scales. However, the clear distinctions among the N pools and stable isotope composition in island drainage as a whole show that there are indeed broad, common links in N cycling across Delta islands. Nitrogen We generated estimates of N fertilizer application amounts to each island using areal crop cover estimates and the N application rate associated with each crop, as available, from Rosenstock et al. (2013); these rates are not based on data from Delta islands and not all land cover types have N application rate data, so estimates are considered preliminary. Estimates of fertilizer inputs on Staten (~800,000 kg N) and Sherman (~230,000 kg N) islands eclipsed TDN gross fluxes by an order of magnitude, while Twitchell Island fertilizer application estimates were much smaller (~38,000 kg N). These estimates suggest that fertilizer N may be an important source of “new” island N annually, especially on cropland- dominated islands such as Staten Island. Importantly, the N stable isotope data generally suggests that any fertilizer N that leaves via drainage is generally highly recycled. the system were also evidenced by high δ15N- NO3 – values in several samples on Staten Island that overlapped or were higher than δ15N-NH4 + values. The high NO3 – concentrations and δ15N- NO3 – values of these samples show the influence of a N input that is most consistent with a high- concentration, partially-denitrified fertilizer source (Kendall and McDonnell 2012; Clark 2015). We generated estimates of N fertilizer application amounts to each island using areal crop cover estimates and the N application rate associated with each crop, as available, from Rosenstock et al. (2013); these rates are not based on data from Delta islands and not all land cover types have N application rate data, so estimates are considered preliminary. Estimates of fertilizer inputs on Staten (~800,000 kg N) and Sherman (~230,000 kg N) islands eclipsed TDN gross fluxes by an order of magnitude, while Twitchell Island fertilizer application estimates were much smaller (~38,000 kg N). These estimates suggest that fertilizer N may be an important source of “new” island N annually, especially on cropland- dominated islands such as Staten Island. Importantly, the N stable isotope data generally suggests that any fertilizer N that leaves via drainage is generally highly recycled. Nitrogen The multi-species stable isotope data we collected provides new insight into the dominant biogeochemical processes that control N species concentrations and stable isotope composition in island drainage. The clear distinctions in δ15N values of inorganic and organic N pools in drainage from all islands suggests that N is cycled in a relatively consistent biogeochemical manner across Delta islands (Figure 4). Similarity between δ15N values of PON and DON indicates that DON is mainly derived from the breakdown of larger OM, such as PON. Drainage POM likely originates from soil as discussed by Richardson et al. (2020), which showed that annual mean (C:N)m ratios of POM were generally above 10 at these sites. The higher δ15N-NH4 + values in nearly all samples relative to δ15N-PON and δ15N-DON values suggests that the Values of δ15N-NO3 – were generally low with lower NO3 – concentration relative to the NH4 + pool, which is consistent with partial nitrification of NH4 +. Some of the NH4 + appears to be nitrified locally in the subsurface and/or in the drainage waters under sub-oxic to oxic conditions, possibly from hot spots and hot moments of NO3 – and NO2 – production (McClain et al. 2003). Additional inorganic N sources external to 16 JUNE 2022 JUNE 2022 as a result of water-mineral interactions in the subsurface. In the case of island hydrology, mineral accumulation from evaporation may also contribute to higher SiO4 4– in the subsurface. In contrast, controls on drainage PO4 3– concentrations (Figure 3G) were not clear, although some sites showed higher concentrations in the summer, which may indicate that most PO4 3– is sourced from fertilizer application during the growing season. Phosphorous is subject to complex sorption reactions in the subsurface that can significantly limit mobility, which may account for the generally low drainage PO4 3– concentrations year-round (Schoumans 2013). Studies on fertilizer applications of soluble phosphorous show that over 50% of the added PO4 3– is immobilized in under 3 days (do Nascimento et al. 2018). the system were also evidenced by high δ15N- NO3 – values in several samples on Staten Island that overlapped or were higher than δ15N-NH4 + values. The high NO3 – concentrations and δ15N- NO3 – values of these samples show the influence of a N input that is most consistent with a high- concentration, partially-denitrified fertilizer source (Kendall and McDonnell 2012; Clark 2015). Silicon and Phosphorous Concentrations of SiO4 4– were seasonally elevated in the fall and winter in island drainage (Figure 3F) and suggestive of increased groundwater contributions in line with expected water table fluctuations (Richardson et al. 2020). This finding is not surprising because SiO4 4– concentrations in groundwater are commonly high relative to those in river water https://doi.org/10.15447/sfews.2022v20iss2art5 17 SAN FRANCISCO ESTUARY & WATERSHED SCIENCE VOLUME 20, ISSUE 2, ARTICLE 5 Figure 7  Island drainage total dissolved Fe, Mn, and As concentrations versus (A, D, G) dissolved oxygen, (B, E, H) pH, and (C, F, I) DOC concentration for all 16 months of sampling in WY 2017 and WY 2018. Inset figures show cumulative R2 value for exponential regressions when considering all sites. Light blue circles, dark blue triangles, and purple squares represent drainage sites on Sherman, Staten, and Twitchell islands, respectively. broadly related across all islands, which is not surprising given that organic matter has a high be a more important driver of Fe solubility and speciation than OM in drainage waters Since Figure 7  Island drainage total dissolved Fe, Mn, and As concentrations versus (A, D, G) dissolved oxygen, (B, E, H) pH, and (C, F, I) DOC concentration for all 16 months of sampling in WY 2017 and WY 2018. Inset figures show cumulative R2 value for exponential regressions when considering all sites. Light blue circles, dark blue triangles, and purple squares represent drainage sites on Sherman, Staten, and Twitchell islands, respectively. Figure 7  Island drainage total dissolved Fe, Mn, and As concentrations versus (A, D, G) dissolved oxygen, (B, E, H) pH, and (C, F, I) DOC concentration for all 16 months of sampling in WY 2017 and WY 2018. Inset figures show cumulative R2 value for exponential regressions when considering all sites. Light blue circles, dark blue triangles, and purple squares represent drainage sites on Sherman, Staten, and Twitchell islands, respectively. broadly related across all islands, which is not surprising given that organic matter has a high retention capacity for trace elements (Aiken et al. 2011). The negative relationship between total dissolved Fe and dissolved oxygen as well as pH, but lack of a relationship between total dissolved Fe and DOC, suggests that redox processes may be a more important driver of Fe solubility and speciation than OM in drainage waters. Island Drainage Nutrient and Trace Element Contributions to Delta Waterways Sherman, Staten, and Twitchell islands were each a net annual source of TN, TDN, NH4 +, DON, PON, total dissolved Mn and Fe, and a net annual sink for SiO4 4– and PO4 3– (Table 2). Sherman and Twitchell islands were also sinks for NO3 – + NO2 –, while Staten Island was a net source of NO3 – + NO2 – (Table 2). Upscaled to Delta-wide contributions, calculated mean annual island drainage gross TN fluxes were 7390 kg d–1 (or 2.7 × 106 kg annually), and net TN fluxes were 5,030 kg d–1 (or 1.8 × 106 kg annually) (Table 3). This finding complicates many existing N box models in the Delta, which commonly assume island drainage N inputs are negligible or net zero (Novick et al. 2015). The net and gross annual island drainage TN load for WY 2018 was about 9% and 13%, respectively, of previously reported annual TN loads from the Sacramento River (including SRWTP) and San Joaquin River combined (~1.8 × 107 kg) (Saleh and Domagalski 2015) (Table A6). To further examine the relative importance of island drainage TN and NH4 + inputs, we revised three existing box models (“SFEI,” “DSM2,” and “EPA”) described in Novick et al. (2015) and TetraTech (2006) to include our new estimates of (1) island drainage TN and NH4 + fluxes off- island, and (2) river TN and NH4 + fluxes on-island (Table A6). We found that gross island drainage contributions could account for ~13% to 17% of annual TN loads into the Delta, while TN loads from river inflow onto islands could account for ~8% to 10% of TN flow out of Delta waters. These existing models also suggest that the Delta is a sink for NH4 + and TN that enters the region from riverine inputs. Taking island drainage inputs into account, we estimated whole-Delta TN losses of 33% to 35%, which is slightly higher in range than original estimates of 25% from Novick et al. (2015) and in close agreement with original estimates of 35% from TetraTech (2006) (Table A6). Silicon and Phosphorous Since island drainage integrates the effects of both solute source contributions and biogeochemical processes that change solubility and speciation, it is hard to assess the importance of each of these processes without detailed porewater studies. be a more important driver of Fe solubility and speciation than OM in drainage waters. Since island drainage integrates the effects of both solute source contributions and biogeochemical processes that change solubility and speciation, it is hard to assess the importance of each of these processes without detailed porewater studies. 18 JUNE 2022 JUNE 2022 in source geochemistry and biogeochemical transformations during transport to drainage ditches (and ultimately surrounding river channels). Total dissolved As concentrations in island drainage were more variable than Fe and Mn, with no clear seasonal trends across sites (Figure 3J). Reduction of As-bearing Fe- and Mn-oxides is the primary mechanism for As contamination of groundwater in the Delta and areas nearby (e.g., northern San Joaquin Basin) (Izbicki et al. 2008; Bennett and Belitz 2010). In fact, some of the highest concentrations of acid- extractable As—a measure of As available for desorption from mineral surfaces—in this region are from Delta sediments (Izbicki et al. 2008). Similar to PO4 3–, complex sorption reactions affect As mobilization (Herath et al. 2016), and the variability in As concentrations observed across sites in our study is likely a reflection of the complex As biogeochemistry in both the subsurface and surface waters of Delta islands. Broadly, total dissolved As concentrations in drainage were higher under low oxygen conditions, aside from a subset of samples collected on Sherman Island (Figure 7G). While reductive dissolution reasonably explains As mobilization under low oxygen conditions, this subset of Sherman Island samples may actually represent As mobilization from a different biogeochemical process. In high pH oxic waters, As can be mobilized via alkali desorption and sulfide oxidation (Herath et al. 2016). SAN FRANCISCO ESTUARY & WATERSHED SCIENCE SAN FRANCISCO ESTUARY & WATERSHED SCIENCE VOLUME 20, ISSUE 2, ARTICLE 5 likely shift inter-annually from differences in island-level and Delta-wide hydrology. Inflow to outflow ratios at the island level were highly variable across islands and water years (Table A3, A4). Inflow and drainage constituent concentrations may also change year to year, and even slight changes could affect the magnitude of net fluxes. Richardson et al. (2020) estimated that wet water years see greater mass fluxes of carbon from island drainage, and these results may scale to N. Future work should consider the importance of water year variability, which can affect both concentrations and discharge, when estimating the relative contributions of different nutrient sources internal and external to the Delta. Seasonality also matters because the fate and effects of these inputs will vary with environmental conditions (e.g., flow, temperature, and solar radiation). For example, flows in the Delta are higher in the winter and spring, which may facilitate rapid transport and dilution through the system relative to other seasons. In the summer and fall, changes in nutrient concentrations and forms may be of greater consequence as they overlap with warmer temperatures and higher solar radiation that can promote phytoplankton growth. to 87% and were similar to past estimates of 65% to 85% from Novick et al. (2015). These revisions to existing box models to include island drainage loads from this study, which is a net source of both TN and NH4 +, suggests that the Delta is a slightly larger sink for TN (+1.9 × 105 kg to 1.8 × 106 kg) and NH4 + (+ 2.1 × 105 kg) than previous estimates suggest (Table A6). In contrast to N dynamics, Delta islands were a net sink for PO4 3– during WY 2018 (Table 3). The annual gross PO4 3– load in drainage waters pumped off islands was 2.8 × 104 kg, which is similar in magnitude to past estimates of ~5.1 × 104 kg (TetraTech 2006). However, island drainage exports of PO4 3– were negated by the larger inflow of PO4 3– onto islands, resulting in islands being a net sink for PO4 3– to the order of –3.2 × 104 kg annually (Table 3). Relative to previously reported annual estimates of PO4 3- inputs to the Delta, the gross island drainage PO4 3– contribution comprised just 1% of existing PO4 3– contributions to the Delta (TetraTech 2006). SAN FRANCISCO ESTUARY & WATERSHED SCIENCE Delta islands were, annually, a net source of total dissolved Mn (3.4 × 105 kg), Fe (5.3 × 105 kg), and As (2.3 × 103 kg) to the larger Delta environment (Table 3). Most of these loads likely precipitate in the Delta’s oxic river waters and are deposited in the sediments. Similar to concerns about methylmercury in the Delta, re-suspension of sediments via dredging or other physicochemical processes could remobilize elements deposited from island drainage for downstream transport, with ultimate fate depending on concentrations and speciation (Shipley et al. 2011). Island Drainage Nutrient and Trace Element Contributions to Delta Waterways Our annual NH4 + losses in the Delta ranged from 64% Interestingly, drainage waters on Staten Island had mean annual dissolved As concentrations that were almost double the other drainage sites and, at times, exceeded recommended thresholds of total dissolved As set by the US Environmental Protection Agency (USEPA) (> 10 µg L–1) and World Health Organization (> 30 µg L–1). We suspect that these high levels of total dissolved As, which were specific to Staten Island, are related to seasonal flooding of fields that contribute to localized anoxic conditions. This is also evidenced by past studies showing large CH4 fluxes during these times, that likely allow for release of As via reductive dissolution (Pellerin et al. 2013). Taken together, trace element geochemistry broadly suggests that island drainage receives water from a seasonally reduced water source or NRZ; future work could explicitly sample along possible flow paths to better account for differences https://doi.org/10.15447/sfews.2022v20iss2art5 19 Importance of Island Drainage Nutrient Contributions Using Pre- and Post-Upgrade SRWTP Scenarios Importance of Island Drainage Nutrient Contributions Using Pre- and Post-Upgrade SRWTP Scenarios To better understand the relative magnitude of island drainage nutrient contributions in the context of the larger Delta environment under pre- and post-upgrade scenarios, we first compared island drainage nutrient contributions to other major Delta inflows (Sacramento and San Joaquin rivers) along with contributions from SRWTP, pre-upgrade, for WY 2018. We focus on dissolved N and P based on data availability from the above river sites and SRWTP. Annual net island drainage TDN, NH4 +, NO3 – + NO2 –, and PO4 3– contributions were 7%, 4%, 2%, and –4% of total inputs to the Delta, respectively (Figure 8, Table A7). During WY 2018, SRWTP NH4 + contributions were 92% of all NH4 + inputs, and this mass flux percentage is nearly identical to past estimates by Jassby (2008). Seasonally, we estimated island drainage contributed around 16% of TDN, 9% of NH4 +, 10% of NO3 – + NO2 –, and A large fraction of the previously discussed nutrient and trace element exports from Delta islands occurred in the winter and spring, from increases in both concentrations and discharge (Figure 3, Figure A1). Seasonality in the delivery of island drainage nutrients and trace elements to Delta waters has important implications for mass flux and net flux comparisons. For example, nearly 65% and 68% of the annual upscaled gross TN and TDN load, respectively, was delivered in winter and spring of WY 2018 (Table 4). Island drainage nutrient and trace element fluxes also 20 JUNE 2022 JUNE 2022 Calculated mean seasonal fluxes to the Delta from the Sacramento River (SR) at Freeport, the San Joaquin River (SJR) at Verna nage (taken as net contributions), and Sacramento Regional Wastewater Treatment Plant (SRWTP) under (A-D) pre-upgrade an onditions. Fluxes were calculated using flow and generally monthly concentration data from water year (WY) 2018. River sites h data, depending on species, and, as such, we present these fluxes as baseline seasonal estimates for WY 2018, a dry year. Study Limitations and Applications This study is an important first step toward better constraining and evaluating the importance of island drainage nutrient contributions to the Delta. Future work should work toward resolving water budget uncertainties and issues relating to scale, both in space and time. Island water budgets, which are commonly modeled in this system, would benefit from ground truthing where possible. In the context of this study, explicit accounting of water inflow volumes could improve a source of uncertainty in both net flux estimates and island water budgets. Specifically, our study was limited by two key assumptions that warrant further work and improvement: (1) that island water budgets are at steady-state, and (2) that the regional estimates of island drainage from Templin and Cherry (1997) are accurate. This study is an important first step toward better constraining and evaluating the importance of island drainage nutrient contributions to the Delta. Future work should work toward resolving water budget uncertainties and issues relating to scale, both in space and time. Island water budgets, which are commonly modeled in this system, would benefit from ground truthing where possible. In the context of this study, explicit accounting of water inflow volumes could improve a source of uncertainty in both net flux estimates and island water budgets. Specifically, our study was limited by two key assumptions that warrant further work and improvement: (1) that island water budgets are at steady-state, and (2) that the regional estimates of island drainage from Templin and Cherry (1997) are accurate. The steady state water budget assumptions used herein affect the resulting mass flux estimates. A simple sensitivity analysis shows that changing inflow based on expected directional shifts in on-island water storage seasonally (decreases in spring/summer, increases in fall/winter) increases annual and seasonal net mass fluxes for nearly all constituents; many analytes become smaller sinks and/or larger sources, especially in the summer (Table A8). Additionally, if Delta-wide drainage outflow is closer to DCD estimates (2 times greater, 1.2 109 m3 for WY 2018), the mass flux estimates herein are clear underestimates. Predicted effluent NO3 – + NO2 –, NH4 +, PO4 3–, and TDN concentrations for SRWTP—once fully upgraded to tertiary treatment with biological nutrient removal (i.e., nitrification and denitrification)—were used to forecast possible changes in dominant N and P sources to and within the Delta under a post-upgrade SRWTP scenario. Importance of Island Drainage Nutrient Contributions Using Pre- and Post-Upgrade SRWTP Scenarios Pre- RWTP were calculated from three months (Oct-17 to Dec-17) of data in WY 2018 due to limited concentration data, and the decre oss upgrade scenarios for SRWTP is a residual effect of bias in the WY 2018 mean as SRWTP PO4 3– fluxes are not expected to ch Figure 8  Calculated mean seasonal fluxes to the Delta from the Sacramento River (SR) at Freeport, the San Joaquin River (SJR) at Vernalis, Delta-wide island drainage (taken as net contributions), and Sacramento Regional Wastewater Treatment Plant (SRWTP) under (A-D) pre-upgrade and (E-H) post- upgrade conditions. Fluxes were calculated using flow and generally monthly concentration data from water year (WY) 2018. River sites had some months of missing data, depending on species, and, as such, we present these fluxes as baseline seasonal estimates for WY 2018, a dry year. Pre-upgrade PO4 3– fluxes at SRWTP were calculated from three months (Oct-17 to Dec-17) of data in WY 2018 due to limited concentration data, and the decrease in PO4 3– fluxes across upgrade scenarios for SRWTP is a residual effect of bias in the WY 2018 mean as SRWTP PO4 3– fluxes are not expected to change significantly. Figure 8  Calculated mean seasonal fluxes to the Delta from the Sacramento River (SR) at Freeport, the San Joaquin River (SJR) at Vernalis, Delta-wide island drainage (taken as net contributions), and Sacramento Regional Wastewater Treatment Plant (SRWTP) under (A-D) pre-upgrade and (E-H) post- upgrade conditions. Fluxes were calculated using flow and generally monthly concentration data from water year (WY) 2018. River sites had some months of missing data, depending on species, and, as such, we present these fluxes as baseline seasonal estimates for WY 2018, a dry year. Pre-upgrade PO4 3– fluxes at SRWTP were calculated from three months (Oct-17 to Dec-17) of data in WY 2018 due to limited concentration data, and the decrease in PO4 3– fluxes across upgrade scenarios for SRWTP is a residual effect of bias in the WY 2018 mean as SRWTP PO4 3– fluxes are not expected to change significantly. https://doi.org/10.15447/sfews.2022v20iss2art5 21 SAN FRANCISCO ESTUARY & WATERSHED SCIENCE VOLUME 20, ISSUE 2, ARTICLE 5 biogeochemical processing and incorporation into the food web. 2% of PO4 3– in winter months under pre-upgrade conditions (Table A7). Importance of Island Drainage Nutrient Contributions Using Pre- and Post-Upgrade SRWTP Scenarios In the summer and fall, net contributions from island drainage represented –12% and 10% of TDN, –3% and 2% of NH4 +, –47% and 2% of NO3 – + NO2 –, and –21% and –1% of PO4 3– total inputs under pre-upgrade conditions (Table A7). CONCLUSIONS For more explicit source tracking, coupled physicochemical instrumentation (e.g., water level, chemical sensors) of island groundwater and drainage waters across multiple islands could provide a better understanding of biogeochemical transformations as they occur from initial diversion or infiltration to final discharge. Our use of stable isotope tracers provided new insight into N cycling on Delta islands and a similar application of isotopic tracers could be used in conjunction with the suggested monitoring regime to better understand N sources and transformations. We estimated island-level and upscaled Sacramento-San Joaquin Delta (Delta)-wide drainage fluxes using monthly nutrient (PON, DON, NO3 – + NO2–, NH4 +, PO4 3–, SiO4 4–) and trace element (total dissolved Fe, Mn, and As) concentrations with discharge data. Upscaled island drainage estimates for the entire Delta suggested that islands are annual net sources of total nitrogen (TN), total dissolved nitrogen (TDN), NH4 +, NO3 – + NO2 –, DON, PON, SiO4 4–, total dissolved Mn, Fe, and As, and sinks for PO4 3–. Island drainage net annual TN and TDN exports were 1.8 × 106 and 8.4 × 105 kg, respectively. Delta-wide island drainage gross and net TN contributions were roughly 13% and 9% of previously reported TN loads to the system. Our results complicate existing nutrient budgets in the Delta—which commonly assume that N inputs from island drainage are negligible or net zero— and provide new information on under-studied trace element inputs to the Delta. Beyond nutrients, work on contaminants in the Delta suggests farmed Delta islands may also be a source of a number of ecologically consequential pesticides, transported in dissolved forms and/ or sorbed on soil particulates (Kuivila and Hladik 2008; De Parsia et al. 2019; Weston et al. 2019). With over 200 possible active drainage sites in this system, new studies could examine the possibility of delivery of these contaminants via island drains in the Delta. Drainage waters may also be a potential source for harmful algae and cyanotoxins that has not been studied; Richardson et al. (2020) found several island drainage sites had seasonal algal blooms. Using forecasted changes in post-upgrade Sacramento Regional Wastewater Treatment Plant (SRWTP) dissolved N and P loads, we also estimated how relative contributions of N and P from island drainage and other major freshwater inflow sources could shift in importance in the Delta environment. Study Limitations and Applications Post-upgrade, annual net island drainage contributions were predicted to comprise 11% of the TDN, 45% of the NH4 +, and 1% of the NO3 – + NO2 – delivered to and within the Delta relative to inputs from major inflows and SRWTP (Figure 8, Table A7). Since SRWTP does not anticipate changes to its PO4 3– loads, net island drainage PO4 3– percent contributions remained relatively similar, around –4% to –5%, under pre- and post-upgrade scenarios. Pre- versus post-upgrade seasonal percentages for winter net island drainage N contributions shifted from 16% to 23% for TDN, and from 9% to 65% for NH4 +, while NO3 – + NO2 – percent contributions remained around 8% to 10% (Table A7). Because overall NH4 + inputs to the Delta will be reduced as SRWTP transitions to be a more advanced treatment plant, following the upgrade, a large fraction of NH4 + that enters the Delta may originate internally from island drainage. Importantly, a majority of island drainage N delivery to Delta waterways will occur seasonally, in the winter and spring, when carbon and trace element contributions from drainage are similarly elevated (Richardson et al. 2020) (Figure 8, Table 4). Though NO3 – will likely dominate external inorganic N loads to the Delta post-upgrade, the seasonal delivery of N from island drainage will be measurable and may be locally relevant. The spatially diffuse locations of drainage outfalls may mean that these seasonal loads are delivered to regions of the Delta with long residence times that allow for extended High-frequency monitoring of drainage outlets— for nutrient concentrations and related ancillary water-quality parameters such as temperature, dissolved oxygen, and pH—could be used for better resolution of mass flux estimates and shifting nutrient biogeochemistry in island drainage. Similar recent work, enabled by deployment of high-frequency sensor networks, showed nutrient dynamics can change at time- scales of hours, days, and weeks in the Delta (Downing et al. 2017; Kraus et al. 2017), and we suspect a similar high-frequency data set for multiple island drainage sites could help resolve some of the variability seen in this study and 22 JUNE 2022 JUNE 2022 companion work by Richardson et al. (2020). Such monitoring would also likely generate more refined and accurate load estimates both within and across water years. how different land uses affect constituent source and sink dynamics could also help inform best management practices on Delta islands. CONCLUSIONS Under a post-upgrade scenario, annual island drainage net TDN and NH4 + contributions to the Delta—relative to inputs from the San Joaquin River, the Sacramento River, and SRWTP—increased in relative importance from 7% to 11% and 4% to 45% based on data from 2018 (a dry water year), respectively. Both pre- and post-upgrade NO3 – + NO2 – and PO4 3– percent contributions from island drainage—relative to other considered sources—were similar, suggesting that the recent SRWTP upgrade, which reduces total N loads while maintaining similar P loads to the Delta, will also shift dominant sources of N species in different ways. Of these sources, island drainage may become Finally, this study and past work show that Delta islands are spatially heterogenous, both within islands and across islands. This has been observed in studies of (1) gas fluxes, which can be remarkably variable across identical land- use types on multiple Delta islands (Hemes et al. 2019), and (2) aqueous fluxes of carbon, nutrients, and trace elements, with clear site- to-site variability in concentrations as shown in this study and Richardson et al. (2020). Finally, this study and past work show that Delta islands are spatially heterogenous, both within islands and across islands. This has been observed in studies of (1) gas fluxes, which can be remarkably variable across identical land- use types on multiple Delta islands (Hemes et al. 2019), and (2) aqueous fluxes of carbon, nutrients, and trace elements, with clear site- to-site variability in concentrations as shown in this study and Richardson et al. (2020). Better resolution of controls on this spatial heterogeneity—which affects system-wide assessments of gaseous, aqueous, and particulate fluxes—could provide more accurate upscaling of lateral and vertical fluxes. Similarly, identifying https://doi.org/10.15447/sfews.2022v20iss2art5 23 SAN FRANCISCO ESTUARY & WATERSHED SCIENCE VOLUME 20, ISSUE 2, ARTICLE 5 Science, Inc. (CUAHSI) at http://www.hydroshare. org/resource/90464d9f9bb34c43b9c6b13eafb5e843. Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the US Government. the dominant source of NH4 + to Delta waterways, at least during dry water years, though more work is needed to assess how NH4 + fluxes from other sources, like wetlands and sediments, may factor into the shifting N budget. REFERENCES Aiken GR, H Hsu-Kim H, Ryan JN. 2011. Influence of dissolved organic matter on the environmental fate of metals, nanoparticles, and colloids. Environ Sci Tech. [accessed 2020 Jan 1]. https://doi.org/10.1021/es103992s Alpers CN, Fleck JA, Marvin-DiPasquale M, Stricker CA, Stephenson M, Taylor HE. 2014. Mercury cycling in agricultural and managed wetlands, Yolo Bypass, California: Spatial and seasonal variations in water quality. Sci Total Environ. 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A report prepared for the California Energy Commission. [accessed 2020 Jan 1]. Pellerin B, Anderson F, Bergamaschi B. 2013. Assessing the role of winter flooding on baseline greenhouse gas fluxes from corn fields in the Sacramento-San Joaquin Bay Delta. Energy Sommer T, Armor C, Baxter R, Breuer R, Brown L, Chotkowski M, Culberson S, Feyrer F, Gingras M, Herbold B. 2007. The collapse of pelagic fishes in the upper San Francisco Estuary. Fisheries. [accessed 2020 Jan 1];32(6):270-277. https://doi. org/10.1577/1548-8446(2007)32[270:TCOPFI]2.0.CO;2 https://doi.org/10.15447/sfews.2022v20iss2art5 27 SAN FRANCISCO ESTUARY & WATERSHED SCIENCE VOLUME 20, ISSUE 2, ARTICLE 5 Stumpner EB, Kraus TEC, Fleck JA, Hansen AM, Bachand SM, Horwath WR, DeWild JF, Krabbenhoft DP, Bachand PA. 2015. Mercury, monomethyl mercury, and dissolved organic carbon concentrations in surface water entering and exiting constructed wetlands treated with metal-based coagulants, Twitchell Island, California. Data Series 950. [Reston (VA)]: US Geological Survey. 26 p. [accessed 2020 Jan 1];26. NOTES Liang L. 2020. Email communication between C. Richardson and Lan Liang, California Department of Water Resources, regarding island discharge comparisons in the Delta in July 2020. Tetra Tech, Inc. 2006. Conceptual model for nutrients in the Central Valley and Sacramento- San Joaquin Delta. A technical report prepared for the US Environmental Protection Agency and the Central Valley Drinking Water Public Policy Workgroup. [accessed 2020 Jan 1]. Available from: https://www.waterboards.ca.gov/centralvalley/water_ issues/drinking_water_policy/final_nutrient_report_ lowres.pdf [USGS] United States Geological Survey. 2019. National Water Information System: US Geological Survey Web Interface. [accessed 2020 Jan 1]. Available from: https://doi.org/10.5066/F7P55KJN Ward AK, Paerl HW. 2016. Delta nutrients forms and ratios public workshop: “Role of nutrients in shifts in phytoplankton abundance and species composition in the Sacramento-San Joaquin Delta.” Sacramento (CA): November 29-30, 2016. [accessed 2020 Jan 1]. Available from: https:// www.waterboards.ca.gov/centralvalley/water_issues/ delta_water_quality/delta_nutrient_research_plan/ science_work_groups/2017_0530_phyto_wp.pdf 28
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https://eprints.leedsbeckett.ac.uk/id/eprint/9728/1/LocalAgencyForThePublicPurposeDissectingAndEvaluatingTheEmergingDiscoursesOfMunicipalEntrepreneurshipInTheUkAM-BARNETT.pdf
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Local agency for the public purpose? Dissecting and evaluating the emerging discourses of municipal entrepreneurship in the UK
Local government studies
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public-domain
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Citation: Barnett, N and Griggs, S and Hall, S and Howarth, D (2021) Local agency for the public purpose? Dissecting and evaluating the emerging discourses of municipal entrepreneurship in the UK. Local Government Studies, 48 (5). pp. 907-928. ISSN 0300-3930 DOI: https://doi.org/10.1080/03003930.2021.1988935 Link to Leeds Beckett Repository record: https://eprints.leedsbeckett.ac.uk/id/eprint/9728/ Document Version: Article (Accepted Version) Creative Commons: Attribution-Noncommercial 4.0 This is an Accepted Manuscript of an article published by Taylor & Francis in Local Government Studies on 31st Oct 2021, available at: https://doi.org/10.1080/03003930.2021.1988935 The aim of the Leeds Beckett Repository is to provide open access to our research, as required by funder policies and permitted by publishers and copyright law. The Leeds Beckett repository holds a wide range of publications, each of which has been checked for copyright and the relevant embargo period has been applied by the Research Services team. We operate on a standard take-down policy. If you are the author or publisher of an output and you would like it removed from the repository, please contact us and we will investigate on a case-by-case basis. Each thesis in the repository has been cleared where necessary by the author for third party copyright. If you would like a thesis to be removed from the repository or believe there is an issue with copyright, please contact us on openaccess@leedsbeckett.ac.uk and we will investigate on a case-by-case basis. 1 Local Agency for the Public Purpose? Dissecting and evaluating the emerging discourses of municipal entrepreneurship in the UK Neil Barnett1 Steven Griggs2 Stephen Hall3 David Howarth4 1 Leeds Beckett University, UK De Montfort University, UK 3 University of the West of England, UK 4 University of Essex, UK 2 Abstract This article explores the contested politics and interpretations of the new practices of municipal entrepreneurship across local government in the UK. Drawing on empirical evidence from six cases studies of entrepreneurship in local councils, selfcharacterisations of income-generating projects in thirty authorities, and a series of semi-structured interviews, we identify, name and characterise an emergent discourse of municipal entrepreneurship for the public purpose. We argue that this novel strand of discourse within the wider field of urban entrepreneurialism confers a degree of political agency to local authorities under austerity, while redescribing and attaching commercialism and entrepreneurship to the public good. In so doing, we challenge overly reductionist accounts of local state agency under austerity, and articulate and evaluate the potentials and obstacles for a progressive interventionism in this discursive space. Key words Municipalism Entrepreneurship Commercialisation Local government Agency Austerity Discourse Word count 7998 2 Austerity governance has transformed embedded practices of budgetary stewardship and service delivery across local authorities in the UK (Ferry and Eckersley, 2020). 1 Reductions in grant support from central government, only partially offset by business rates and increases in council tax, have seen council spending on local services drop by 24 per cent in England (from 2009 to 2017), 11.5 per cent in Scotland, and 12 per cent in Wales (Gray and Barford, 2018, p. 554). Working within highly centralised financial and legal regimes, with few powers of local taxation, and often limited local resource bases, especially in disadvantaged communities in ‘old’ industrial towns and cities, local authorities have increasingly turned towards new strategies of incomegeneration and commercialisation, as they endeavour to fill the ‘funding gaps’ left by reductions in traditional sources of revenue (Gray and Barford, 2018). The generation of alternative revenue streams - the ‘marketisation of income’ (Taylor, Haynes and Darking, 2021) - has embraced a multiplicity of commercial and entrepreneurial logics and practices of municipal action (Thompson, 2020). Councils have entered into partnerships with real estate developers, while investing some £6.6 billion in commercial property such as hotels, offices and shopping centres from 2016/17 to 2018/19 (National Audit Office, 2020, p.4). They have also made novel trading and charging interventions in local markets, launching direct ‘for profit’ trading companies in municipal goods and services; creating public service cooperatives and mutuals in collaboration with communities; exploiting procurement policies as a tool to support local businesses and social enterprises; and driving authority-wide culture change towards entrepreneurship and financial self-sufficiency (Ferry et al., 2018). This article evaluates the under-researched motivations and meanings attached by local actors to such commercial and entrepreneurial forms of income-generation (Shearmur and Poirier, 2017). Answering calls for a ‘greater sensitivity’ to local agency under conditions of austerity (Fuller, 2018), it reveals and assesses how 1 We thank the Editor and two anonymous reviewers for their constructive engagement with our work and for their helpful suggestions on how we might refine and develop our arguments. Of course, responsibility for the claims remains with the authors. 3 officers make sense of entrepreneurship in their everyday practices; the role of local agency in relation to the demands of austerity governance; and the reconfiguration of the local state. In so doing, we recognise and build upon existing accounts that have identified global varieties of urban entrepreneurship (Phelps and Miao, 2020). While we acknowledge variations in cross-national patterns and uneven geographical ‘reach’, we identify and name four dominant problematisations of entrepreneurial practices which are alleged to best fit the case of UK local government: municipal financialization, progressive interventionism, social innovation, and progressive selforganisation. Secondly, we discern and characterize a particular mode or variant of the emergent discourse of ‘municipal entrepreneurialism’, which we name ‘municipal entrepreneurialism for the public purpose’, where entrepreneurship is assocated with the provision of a diverse array of commercial services, municipal stewardship and public goods. Thirdly, we detect a number of potential tensions in this discourse of municipal entrepreneurship, including its amplificiation of political risks; clashes in organisational culture over demands for flexibility; grievances over the naming of ‘surpluses’ and traditional budgetary logics; and the transformation of the roles and responsibilities of officers. Fourthly, while recognising the risks of drawing narrow definitions of such phenomena, we conclude that this emerging discourse creates new possibilities of agency for local authorities that resonate with and contribute to problematisations of what we have named ‘progressive interventionism’. RESEARCH STRATEGY In developing a research strategy to address our objects of inquiry, our initial task was to describe the core elements and features that constituted the discourse of municipal entrepreneurship. This involved an analysis of the interpretations and statements of representative subjects and institutional actors - chief officers, project leads, local councillors, and frontline staff - who spoke or wrote about the practices and activities in which they were engaged. Here the main aim of the exercise was to characterize the beliefs and interpretations of these selected subjects and institutional actors in different contexts, using our judgement to discern and test the underlying rules and logics of the discourses. More fully, drawing on the resources of poststructuralist 4 policy analysis, we focussed on the signifier of ‘entrepreneurship’, tracking and tracing its various iterations across multiple contexts, while using these descriptions to construct the discourse. Our approach also contained an implicit logic of comparison, as our descriptions sought to bring out similarities and differences in the multiple articulations of ‘entrepreneurship’ across local authorities (cf. Robinson, 2016). Here the objective was to sketch out a grammar of the different usages of ‘municipal entrepreneurship’, so as to provide a perspicuous representation of the discursive field, and to determine the different variants at work in this field. Of course, this strategy presupposes a particular conception of discourse and statements. In our approach, discourses are best defined as linguistic and nonlinguistic practices that connect ideas, things and activities together to produce specific systems of meaning. This perspective contrasts with other methods of discourse analysis, which focus either on the more restricted analysis of speeches or texts, or more broadly on the role of arguments and the semiotic dimension of social practice (e.g. Fairclough, 2013; Hajer, 2005). Discourses are thus sets of articulatory practices that connect and modify the meaning of contingent elements to form systems of signification, where such systems are demarcated and unified by the creation of boundaries with other discourses. Moreover, in seeking to determine the regularities and linkages between different statements, beliefs and actions in a particular field of discourse, our conception focusses on the way differences are established between contending positions and the creation of political divisions between discourses (XXXX, TBI). Finally, using the work of Foucault, we define statements as ‘serious speech acts’ that are enunciated by officers and policymakers when describing their practices and programmes in particular local contexts (Foucault, 1972). Here the notion of a ‘speech act’ highlights the performative dimension of their utterances and written expressions – ‘saying as doing’ – while the idea of ‘serious’ captures the way these linguistic performances seek to accurately and licitly describe their beliefs, thoughts and practices (Dreyfus and Rabinow, 1982, pp. 45-56). Our aim was to establish the core statements in the discourse, and to explore their repetition, resonance, reiteration and 5 transformation in particular spaces and places, as well as their dissemination and takeup in other comparable cases. More practically, empirical fieldwork and data generation was undertaken in three steps. We began by discerning and naming four dominant problematisations of entrepreneurial practices in local government in different contexts. Secondly, we analysed the council initiatives nominated for the Association for Public Service Excellence’s annual award for commercialisation and entrepreneurship. The Association is a member-owned local authority body, which works with over 300 councils across the UK. As an advocate of new forms of income-generation, it introduced the award for commercialisation and entrepreneurship in 2015, and by 2019 over 37 initiatives had been shortlisted as finalists for the award, with nominations spread across 30 authorities. Thirdly, we undertook case studies of entrepreneurship in six local councils, which have been widely identified as being at the forefront of income-generation activities (see Table 1). The empirical cases were not selected as ‘critical cases’, which would enable us to generate or test universal explanations or predictions, but rather because they were considered to be exemplary sites through which to explore the discursive work of ‘entrepreneurship’. INSERT TABLE 1 HERE For each case study, the research team assembled and analysed an archive of policy documents, including briefings, strategic plans, project reports and evaluations. This was followed by a series of semi-structured interviews with a total of 21 participants drawn from project and corporate teams. Interviews lasted an hour, and the sample included chief officers, project leads and frontline staff. Questions explored the competing rationales underpinning initiatives, everyday practices of implementation, and the barriers and opportunities for change. Interviews were then coded thematically, as we focused on exemplary statements pertaining to entrepreneurship, the articulation of demands, and the equivalences and differences between elements of the discourse. 6 In analysing this archive, we focussed on the way actors interpreted their situations, and the repetition of statements that constituted the specific goals, subjects, strategies, and objects of the discourse of entrepreneurship. At each stage, we undertook repeated readings of the texts, using ‘manual processing’ to isolate and describe the core statements that emerged or disappeared in different contexts (Keller, 2013, p.97). Through this to-and-fro movement between our empirical data and our research puzzles, we engaged in a process of articulation. We did not ‘find’ the discourse of municipal entrepreneurship ‘hiding’ in the text. Rather, we assembled and named its logic, character, and value through our judgements and our situated knowledge of local government, as we mediated and negotiatied our research between our theoretical assumptions and the four problematisations of entrepreneurship that we identified at the outset of the study. It is to these problematisations that we now turn. PROBLEMATISING COMMERCIALISM AND ENTREPRENEURSHIP UNDER AUSTERITY GOVERNANCE Having outlined our research strategy, we now turn to the problematisation of the existing interpretations of commercialism and entrepreneurship under austerity governance in the UK. Here we identify and analyse the four main problematisations of entrepreneurial practices within existing accounts of these ideas and practices. Municipal Financialisation The first problematisation emphasizes the way that practices of local commercialisation have transformed urban infrastuctures into financial assets and revenue streams for local government. In the process, the local state has evolved from that of a ‘facilitator and enabler’ of the private sector to that of an ‘active executor’ of neoliberal financialisation (Beswick and Penny, 2018). This transformation of the local state is a consequence of the logics of austerity governance and top-down cuts to public funding, which do not ‘enabl[e]’ local actors ‘to behave differently’, if they are to strengthen the fiscal and political capacities of the local state (Christophers, 2019, p. 583; see also Penny, 2018). Subordinated to central government, they are left with 7 little freedom of manoeuvre as to ‘where and how they can raise or generate funds’, except through the financialisation of local assets (Beswick and Penny, 2018, p. 624). This financialisation, however, arguably increases the contradictions facing the local state, as it is caught between the demands of value-extraction, speculation, risk-taking and those of public intervention, policy coordination, planning and regulatuion (Beswick and Penny, 2018, p. 612; Pike et al., 2019, p. 4; Raco and De Souza, 2018). In pessimistic renditions of this approach, local actors adopt entrepreneurial practices as ‘much by dull compulsion as ideological zeal’, so that the actions of local actors ultimately mask the implementation of an ‘aggressively commercial and speculative mode of governance new to local government’ (Beswick and Penny, 2018, p. 625; Penny, 2017, p. 1370). Such interpretations resonate with ‘austerian realist’ accounts of local agency, where actors in local government deny their own agency, leading to the consolidation of state power (Davies et al., 2020, p.63). More optimistic interpretations privilege the uneven nature of processes of financialisation, drawing attention to the agency of local actors, as well as the political and economic contexts, which ultimately shape outcomes (O’Brien, O’ Neil and Pike, 2019). They thus characterise practices of local entrepreneurship as part of a ‘financialisation-in-motion’ (Pike et al., 2020, p.792), that is, a set of messy, incomplete and recursive processes, which are marked by the continued interactions of managerial, financialised and entrepreneurial logics (p. 793), where actors are ‘actively financialising and being financialised’ (p. 792). In this view, the motivations of local actors and the potential outcomes of commercialisation and entrepreneurship are highly contingent and contextual, so that until the outcomes of programmes prove otherwise, ‘there is no compelling reason not to give councils the benefit of the doubt’ (Christophers, 2019, p. 582). Progressive Interventionism In arguing that the pro-active entrepreneurial strategies of the local state can drive forward civic and collective provision of goods and services as an integral component of a ‘more inclusive, holistic and integrated place-based economic strategy’, the second problematisation goes further than merely giving councils the ‘benefit of the 8 doubt’ (Thompson et al., 2020, p. 1191; Gardner and Lowndes, 2016). Typically, Thompson et al. (2020) mobilise a Polanyian understanding of the socially embedded economy to foreground the agency of the local state in developing ‘accelerators’ of economic growth in combination with ‘stabilisers’ of social welfare and protection. Characterising such practices as ‘entrepreneurial municipalism’, they argue that the local state goes beyond generating funds to fill the ‘holes’ in central grants, to invest ‘directly in self-sustaining projects, which harness the value of (de-commodified) land, (cooperative) labour and (patient) capital to ground economic development in people and place’ (p. 1188). This approach thus suggests that interventions in the foundational economy represent an emergent and nuanced, yet distinct, strategy available to local state actors, who are duly assigned agency on route to the production of locally-embedded social value. Similar conclusions are drawn by Shearmur and Poirier (2017) and Aldag et al. (2019), who assert that the origins of municipal entrepreneurship are not necessarily intertwined with competition between cities and local authorities to attract inward investment. On the contrary, they argue that local government can exercise its agency to protect public services in a ‘progressive push back’ against central cuts. Indeed, Shearmur and Poirier (2017) suggest that municipal entrepreneurship can be driven and sanctioned by the demands of the local population, civil society and businesses. Social Innovation The claims of Shearmur and Poirier (2017) act as a bridge to our third problematisation. In this approach, entrepreneurship is tied to public value creation, thus privileging local officers and political leaders as the key agents of change in the response to austerity, as well as longer-term shifts in the policy environment, which have been caused by deregulation, privatisation, asset sales, and collaboration (Bello et al., 2018). Local state actors are perceived to exercise innovative forms of agency within a ‘relational’ form of governance, which stands in marked contrast to a bureaucratic, ‘traditional’ top-down government (Liddle and McElwee, 2019). Local actors, it is claimed, are increasingly ‘freed’ within such relational modes of governance to facilitate and leverage public value, as they are encouraged to pursue forms of ‘collective entrepreneurship [that have] the capacity to provide greater 9 options, and allow bottom up solutions to emerge’ (Liddle and McElwee, 2019; Klein et al., 2010). In such spaces, the local state assumes the role of an ‘animateur’, which in part acts as a catalyst for economic development by ensuring network leadership (Quinn and Courtney, 2016, p. 144). In this perspetive, practices of entrepreneurship and innovation are thus framed as ‘co-operative in nature, emphasising mutuality rather than voracious profit-making’ (Johnson and Fenwick, 2018, p. 212). Progressive Self-organisation The fourth problematisation tends to eschew any explicit reference to the signifier ‘entrepreneurship’. Practices of ‘smart procurement’ and interventions into the foundational cooperative economy, which might otherwise be characterised as the activity of the municipal ‘entrepreneur’, are understood in terms of ‘community wealth building’ and ‘progressive new municipalism’ (O’Neil and Howard, 2018; Russell, 2019). Rejecting urban entrepreneurialism or the regime of inter-urban competition over inward investment (Harvey, 1989), this problematisation promotes the democractisation of the economy, alternative forms of ownership and service delivery (Russell, 2019). Importantly for our analysis the innovative forces of social change are thus situated ‘outside’ the local state in the prefigurative micro-settings of self-management and the spaces of informality created by the accelerated disaggregation of the local state under austerity (Beveridge and Koch, 2019, pp.11-12). The local state remains a flawed and somewhat damaged vehicle, hampered by weak powers, financial scarcity, and a lack of capabilities to address the issues and constraints generated by the global political economy (Blanco, Salazar and Bianchi, 2020). More pessimistically, it is replete with bureaucratic hierarchies, asymmetrical power relations and illegitimate forms of authority (see Russell, 2019). Proponents of progressive self-organisation thus advocate the further hybridisation of the local state so as to exploit its organisational resources to the advantage of community activists and to embed the local state in ‘everyday urban politics’ (Beveridge and Koch, 2019, pp.11-12). THE EMERGING DISCOURSES OF MUNICIPAL ENTREPRENEURSHIP 10 In order to evaluate these problematisations, we now set out the results of our empirical research of municipal entrepreneurship. Our initial empirical mapping and analysis of the emerging discourse of municipal entrepreneurship yields six interconnected themes, which function as the quilting points that knit together the overlapping strands and threads. Together they form a loose system of storylines, often in contradiction with one another, which weave together the different strands of the emerging discourse in a dispersed regularity. The internal unities and limits of this ‘dispersed regularity’ are in turn shaped by a series of suble divisions and exclusions, which mark the boundaries between this formation and others. Income-Generation and Local Agency One core strand of the discourse of municipal entrepreneurship constructed commercialisation and income-generation as ‘necessary’ strategic responses to the ‘perfect storm’ of rising demands on council services (Statement 1) and reduced government funding ( Statements 8). In this context, entrepreneurial narratives portrayed local government as the ‘triumphant underdog’ winning out against the odds (like the eponymous hero in the Rocky boxing films) in a challenge to the ‘mentality of cuts’ (Statement 7). Aspirations for greater local agency were clearly emphasized in the naming of the initiatives, with repeated references to ‘taking control’ (Statement 8), ‘shaping the future’ (Statement 6) and challenging the ‘mentality of cuts’ (Statement 12). INSERT FIGURE 1 HERE For the Public Purpose The narrow instrumental goal of income-generation was framed as a means for generating public value or social benefit, be it tackling social care demands (Statement 3), addressing fuel poverty (Statement 2) or mitigating climate change (Statement 4). Entrepreneurship was thus firmly anchored in the public domain, while a series of equivalences were drawn in the discourse between practices of commercialisation, income-generation, and innovation and resilience in service delivery. Manifesting itself in multiple forms in our case studies, the leitmotif of 11 ‘entrepreneurship for the public purpose’ shaped diverse initiatives across different local authority contexts from house building through to property investment, promoting healthy lifestyles, and smart procurement. The East Riding Leisure Services programme, for example, working in partnership with local GPs, referred patients identified at risk of inactive lifestyles or poor diet to leisure services for physical exercise and support via a bespoke on-line system, rather than recommending costly clinical care. But, importantly, the programme was couched in an entrepreneurial narrative, which connected support for patients to become longterm participants in physical activity to the economic case for reducing demand on over-stretched public services through the prioritisation of prevention and the collaborative ‘buy-in’ of multiple partners. The initiative foregrounded savings of some £800,000 per annum to the NHS, evident in reduced numbers of bariatric surgery procedures and a £200,000 accrual to the local authority from increased takeup of leisure services. Stewards of the Local Economy A third theme of the discourse evoked images of the authority as the stewards of the local economy, ‘nurturing the growth of local people and businesses’ (Statement 5). When asked to explain their role in service delivery in Enterprising DG, the direct service arm of Dumfries and Galloway council, one officer initially described it narrowly as ‘basically making money for the council’, yet immediately countered that their work also aimed to make the most of local authority assets within communities, and building local supply chains. Such legitimising narratives ultimately resonated with place-based appeals of the council as an ‘energiser’ authority, driving forward local growth by supporting local companies to retain economic benefits within what was deemed to be a peripheral locality poorly served by the market. Local procurements of goods and services, which rose from £44 million in 2015/16 to £62.4 million in 2018/19, while developing a network of preferred supplier relationships with over 300 local small and medium-sized companies (Dumfries and Galloway Council, 2020), was regularly portrayed as a means of creating social value and promoting social cohesion within local communities. 12 Allusions to place-based stewardship were constructed in opposition to markets through the rhetoric of negative exteralities and market failures, prioritising ‘stakeholder not shareholder’ value (Statement 6). The mission of Birmingham Municipal Housing Trust (BMHT) was thus legitimised by senior officers as a means of countering the failure of private developers to provide an adequate quantity and quality of new homes across the city. One outome of this initiative, for example, was the provision of housing in disadvantaged inner-city neighbourhoods with large BAME communities, where it was wrongly assumed that there was low levels of demand. For BMHT officers, this market failure stemmed from: the flawed business model adopted by the national, private house building sector, which privileged development on large sites in areas of ‘demonstrable’ demand (typically suburbs); the primacy of shareholder value, requiring a high return on investment (typically 25 per cent); and the provision of standardised house types, notwithstanding local context. In this context, BMHT positioned itself as a distinctive niche as a bespoke, local alternative to the ‘budget and scarper’ blueprint of volume house builders (interview with senior officer). It opposed its development model against those of its private competitors, thus offsetting market failure by absorbing risks traditionally borne by the private sector. The strategy involved the development of homes designed by council architects, exclusively on council owned land (with planning consent presecured), with construction, sales and marketing contracted to the private sector, with whom surpluses were shared. In this process, BMHT has built to date more than 3,000 homes for sale or rent, becoming the largest house builder in the West Midlands. Importantly, this discursive strand also established divisions between local and national/global companies and markets. In Dumfries and Galloway, such antagonisms typically rested on demands to tackle the ‘Klondike economy’, whereby national contractors and global companies ‘come into our authority […] take the money and the benefits [..] and then disappear’ (interview with senior officer). ‘Othering’ large external providers in an emergent discourse of patriotic localism, the local was privileged over the national and global, while autonomous strategies of local development were prioritised over external interdependency. It followed that networking with local suppliers and contractors was framed as a means of promoting place-based inclusive economies, ensuring that the benefits of growth were retained 13 locally, notably by using local authority procurement rules to generate demand for goods and services from local suppliers. In short, working alongside local firms, councils used their political powers to open up access to new markets, from which previously, local businesses might have been excluded, because of their obligations to bid for large contracts and compete against national contractors. Redefining Risk The affirmations of local agency and market intervention were made possible by the reframing of risk and risk-taking by local authorities, as they drew equivalences between, on the one hand, the often conflicting institutional logics of budgetary stewardship and, on the other hand, service improvement and innovation (Ferry and Eckersley, 2020). In the first instance, municipal entrepreneurship was reframed as ‘astute’ risk-taking with ‘a clear sense of purpose’ (Statement 11), implicity drawing boundaries with forms of allegedly ‘uncalculated’ risk-taking, while foregrounding the capacity of councils to manage risk and contingencies. In our case studies, commercial risks attached to the use of public funds were compared to the ‘horrific’ risks of inaction and the failure to defend services and local communities under austerity. In this way, the property investment strategy of Sevenoaks District Council weighed the risks of commercial investments against the generation of a revenue stream (a minimum yield of 5 per cent per annum on investments), the value of interventions in local markets to maintain employment property, and the aspiration of the authority to be become financially self-sufficient within ten years (from 2013). In short, the meaning of stewardship was itself redescribed to incorporate budgetary demands, the defence of public services, and the risks of inaction. But it is also noteworthy that in this discourse the notion of ‘risk’ is primarily constructed as political risk, and is conceptualised in terms of negative electoral outcomes for local councillors, including, for example, fears that the council would be seen to be competing with local business. In Birmingham, one officer noted, ‘commercialism has always been classified as a “dirty word”, for [it assumes] council departments competing against anybody who is local […]’. Such political risks were negated by appeals to collaboration with local small and medium firms, and niche market provision (Statement 9), which it was claimed met specifically local needs, 14 while avoiding direct competition with established local businesses. The ventures thus opened up opportunities to compete against national contractors in local and external markets. For example, Oxford City Council rebranded itself as a ‘social enterprise council’, as it explored different ways to trade and charge for its services within and, crucially, beyond Oxford itself. It also recycled dividends produced for local benefit via a municipal trading company - Oxford Direct Services - which generated in 2018/19, a £1.3m dividend for the Council, while employing over 600 employees and supporting 1,251 jobs (Oxford Direct Services, 2020). In discursive terms, then, collaborative and niche provision serves as a logic that can incorporate the defence of local business into the basket of other aims and demands advocated by the appeals to municipal entrepreneurship. It thus seeks to negate political risk surrounding market interventions, while allaying the fears of elected members that competition with local businesses might undermine local political support. Driving Forward Cultural Change Appeals to commercialisation and entrepreneurship were frequently couched in the rhetoric of ‘behavioral change’, ‘embedding a commercial culture’ and ‘working for our citizens’ (Statement 10). In our case studies, municipal entrepreneurship was defined in opposition to standard ‘checks and balances’, which were interpreted as a barrier to exploiting new opportunities or as a competitive disadvantage. In response to such demands, authorities introduced new managerial models and practices of delegation that were deemed to fit with the different business demands and modes of intervention of municipal entrepreneurship. Councils established practices for signing off initiatives by key individuals - chief officers and portfolio holders - without recourse to cabinet, full council, or relevant committees. Most importantly, however, practices of municipal entrepreneurship introduced new financial and budgetary logics into the running of the council, triggering new conflicts and antagonisms. In particular, these tensions concerned the reinvestment of surpluses, when only part of the council was engaged in such entrepreneurial practices. Indeed, the discourse of municipal entrepreneurship embeds a logic of needs-led funding that establishes local needs and levels of service provision and then seeks to generate funding to meet such outcomes. It redirects thinking away from 15 standard budget-setting processes that assume ‘fixed’ levels of resources and expenditure patterns, while privileging the generation of efficiencies to meet service outcomes. In so doing, it challenges protectionist practices of silo funding. As such, the City & County of Swansea inverted traditional budgetary mechanisms, adopting a method of ‘co-produced’ budgeting that brought senior officers, policy officers and frontline service staff into dialogue in outcome-focussed service reviews. These reviews assumed zero-based budgets and explored service delivery as part of an ‘endto-end, needs-based’ process. Yet practices of income-generation also triggered conflicts between departments about the use of ‘additional incomes’, and the absence of incentives to generate income, if any such resources were not re-invested in the department that generated them. Across authorities, such conflicts about the ‘ownership’ of commercial funding triggered the construction of rival discursive projects which sought to name additional income as ‘surpluses’, ‘savings’ or ‘profits’ - each term legitimising different uses of additional incomes. The Innovative Puzzler The emergent discourse also constructed novel subject-positions and voices for officers. Practices of municipal entrerpreneurship were not typically associated with innovative ruptures or breaks with established ways of working. Practitioners spoke of a ‘long journey’, which involved the morphing of services by building upon past activities or by bringing existing assets together in new combinations to respond to local needs. In Birmingham, such narratives resonated with appeals to ‘start small and have a plan B’ (interview with senior officer). In fact, such practices privileged the subject-position of the ‘innovative puzzler’, where the ‘puzzle’ to be solved was represented not as some intellectual conundrum, but the contradictions between - and the exclusions of - customary practices, organisational styles, and policy commitments (see Spinosa, Flores, Dreyfus, 1997, pp. 22-9). Across all case studies, many local officers thus criticized existing practices and organisational styles of councils, using phrases and tropes that highlighted the inconsistencies between traditional ways of working, the council stated aims, and the service-delivery outcomes within communities. New fora and dialogues emerged across councils to accommodate such ‘voices’. Typically, one senior officer spoke of how it is vital that all staff are ‘not afraid to come up with a duff idea’. Another referred to the value of 16 working in environments in which there is ‘permission to fail’ and a ‘no handcuffs policy’, which gives frontline staff the confidence to work outside risk adverse cultures which stifle innovation. To this end, Dumfries and Galloway council established its own centre of excellence, which was a two-way learning space to engage other council services, as well as to transfer ‘good practice’ lessons across the authority and facilitate culture change. Its operational values typically sought to enshrine ‘promoting a responsibility culture not a blame culture’ (DG First, undated, p.15). Importantly for our analysis, such practices are intrinsically tied to perceptions of local agency, for ‘puzzling’ opens up the perspective of making political choices, re-politicising taken-for-granted ways of working, and imagining new visions. RE-INTERPRETING MUNICIPAL ENTREPRENEURSHIP AND LOCAL AGENCY UNDER AUSTERITY In the light of our empirical evidence, and our mapping of the emergent discourse, we now turn back to the critical evaluation of our findings. These are developed in relation to the four rival problematisations that we identified at the start of this article. The Dangers of Municipal Financialisation We began by noting that pessimistic interpretations of municipal financialisation criticize the way that practices of commercialisation and entrepreneurship accelerate the neoliberalization of the local state, transforming the local state from a facilitator of private entrepreneurship into an active entrepreneur in its own right. However, our interpretation of the empirical evidence suggests an alternative possibility, in which the local state is a site and driver of progressive interventions in local markets. Indeed, this strand of the emergent discourse of municipal entrepreneurship challenges the long-held assumptions of the neoliberal model of the enabling council, which advocated councils contracting out and divesting themselves of local services (Smith, 2000). In contrast, municipal entrepreneurship – whether in the form of house building, smart procurement, promotion of active and healthy lifestyles, property investment or social enterprise - leads authorities to adopt interventionist stewardship 17 strategies that reconnect with the ‘big ticket’ policy issues facing local communities (Stoker, 2011). At the same time, the discourse of municipal entrepreneurship also potentially moves beyond the confines of optimistic accounts of municipal financialisation. Such accounts argue that commercialisation generates additional funding to fill the gaps in local authority budgets due to austerity. But we argue that in advancing the progressive capabilities of the local state, municipal entrepreneurship for the public purpose goes further, articulating demands for income-generation with market intervention, stewardship, and the advancement of the public good. It thus severs the enterprise narrative from connotations of neo-liberal, market-led growth and favours thinking about commercialisation and entrepreneurship in ways in which ‘take back’ the local state from capital (Cumbers, 2015, p. 74). That is to say, the emerging discourse recognises the contingency of the institutional configurations of the local state and local economies, so that under certain conditions the state can reframe commercialisation to advance the common interest and social well-being, notably through the tackling of public problems (Sheamur and Poirier, 2017, pp. 721-4). The Agency of the Local State Seen in this way, the discourse of municipal entrepreneurship resonates with the demands of the new municipalism of ‘self-organising progressives’, as well as with the public value orientation of ‘social innovators.’ Indeed, in all our case studies, the rhetoric of municipal entrepreneurship often endorses appeals to practices of: community wealth building; local inclusive growth; the use of procurement policy to support local businesses and social enterprises; and the facilitation and leverage of public and private capabilities in the pursuit of public value. We argue that such demands are advanced by both self-organising progressives and social innovators (see Johnson and Fenwick, 2018; O’Neil and Howard, 2018). Yet, in contrast to these two problematisations, our research suggests that the discourse of municipal entrepreneurship discloses an alternative reading of the agency of the local state, opening up new avenues of inquiry for the transformative role of local agency, and the formal arenas and practices of the local state. In fact, the 18 discourse of municipal entrepreneurship for the public purpose, which we discern in our findings, calls into question the risk of ‘state phobia’ and ‘the fear of the formal’ often associated with accounts of progressive self-organisation and social innovation (Lopdrup-Hjorth and du Gay, 2019). Indeed, although the latter two problematisations acknowledge the progressive potentials that are afforded by the control of the local state, they still ultimately ground the origins of innovation and entrepreneurship in civil society movements and the informal politics that operate beyond the local state, and the local state remains dominated by the hierarchy of the centre and the ‘push’ for formality. Progressive Pragmatism By contrast, our empirical analysis shows that the drive for innovation and entrepreneurship cannot be disentangled from the agency of local state actors within the formal arena of local authorities. The practices of local enterprise that characterised our our case studies were driven by actors inside the formal apparatus of the local state, working in and against established practices of local policymaking. The logics of municipal entrepreneurship challenged established cultures and budgetary practices, while redefining perceived risks and subject positions for officers. Such logics went beyond the ‘tinkering’ and the ‘active politics of the present’. Instead, they privileged the subject-position of the officer as the ‘innovative puzzler’, who is generally intent on challenging the rupture between the declared strategic aims of councils and everyday practices and outcomes. Our case study participants thereby rearticulated the demands of commercialisation and incomegeneration, as they rhetorically redescribed such demands as entrepreneurship for the public purpose. Our analysis thus exposes the political work of local agents and the rhetorical reframing of ‘commercialisation’ and ‘entrepreneurship’ in and through their articulation with municipalism. We foreground how local actors within the local state were able to move beyond the practices of ‘tinkering’ to generate an alternative mobilising vision for local government in response to the demands of austerity governance. Importantly, such evidence supports calls for a more actor-focussed 19 orientation that trawls beneath the ‘surface appearances’ of financialisation (Pike et al, 2020, p. 794). Stewards of Place The upshot of our interpretation is that the discourse of municipal entrepreneurship resonates with and supports the problematisation of progressive interventionism. This problematisation recognises the capacity of the local state to act as a ‘countermovement’ to the predominant logics of competitive, market-led growth, implementing entrepreneurial strategies, which disrupt local markets by investing in foundational services and infrastructures, so as to address economic inequalities across communities (Thompson et al., 2020, pp. 1178-80). The interpretation of our evidence suggests that markets can operate according to different logics, so that municipal interventions in local economies can challenge logics of economic necessity or technocracy to harness markets for the delivery of the public good (Hay and Payne, 2015). Indeed, the analysis of our case studies adds weight to claims that local state actors act as ‘activist-entrepreneurs’, who are able to intervene to address market failure, sometimes through the logic of market disruption, as they seek to ‘reshape’ or redesign the operations of local markets. At the same time, they can mobilise and generate resources in the form of pragmatic public actions that challenge economic injustices, rather than extending the harmfully competitive and exclusionary logics of neoliberalism (Thompson et al., 2020; Aldag et al., 2019). Having said this, the evidence generated in our study also questions the ideological grounding and temporal fixing of progressive interventionism, opening up a dialogue about why such interventions ‘grip’ local officers and politicians. It will be recalled that Thompson et al characterise progressive intervention as practices of ‘entrepreneurial municipalism’, associating such practices with the project of democratic socialism (2020, p. 1180). This definition rests on their aim of intregrating social justice into economic structures, identifying entrepreneurial municipalism as a more or less progressive and left-leaning set of practices, which can be aligned with the so-called ‘new municipalism’ and the fearless cities movement. However, it is clear that in the local authorities that we have studied the patterns of political leadership cannot be narrowly confined to the left of the political spectrum. The party 20 affiliations of council leaders included the Labour Party and Scottish Labour, as well as the Conservative Party and the Scottish National Party (not to mention their different internal party positionings). Equally, officers within the local state also played a primary role in advancing the discourse of municipal entrepreneurship, so that the practical interventions cannot easily be defined as politically-led. We thus posit an alternative reading of entrepreneurship across local authorities, which foregrounds the potential resonance of practices of municipal entrepreneurship for the public purpose with the embedded discourse of stewardship of place. Our evidence suggests that innovative practices and visions of public entrepreneurship traverse party political allegiances, and that the ‘grip’ of such practices rests less on the political alignment with progressive politics, and more on the sedimented discourse of place stewardship embedded within political and administrative leaderships in the different cultures and systems of local government. CONCLUSION: MUNICIPAL ENTREPRENEURSHIP, LOCAL DEMOCRACY AND THE PUBLIC PURPOSE In characterising and naming an emergent discourse of municipal entrepreneurship for the public purpose, this article has critically assessed the income-generation practices of local government authorities. Adding to our knowledge of how local actors interpret their practices and roles when they engage in entrepreneurship, the analysis also challenges the continued salience of ‘hollow’ accounts of local agency under austerity. Although it is tempting to show a clear complicity between these new discursive practices and other tendencies, including logics of neoliberalization, the rise of the ‘new municipalism’ or the constraints of ‘austerian realism’, our analysis discloses a more nuanced and richer set of processes, which have complex lines of descent. One upshot of our findings is to caution against a too rapid desire to overgeneralise, leading to the positing of universal forms that embody a clear essence and a fixed set 21 of features. Instead, we argue that the discourses and practices of commercialisation and entrepreneurship can take multiple forms. This is not to say that this discourse cannot slip into logics of financialisation or forms of social innovation, which have been identified in ther problematisations we have evaluated. On the contrary, under certain conditions, commercialisation and entrepreneurship may manifest itself as a form of self-organising progression or social innovation. In equal fashion, the discourse of municipal entrepreneurship for the public purpose is not without its tensions and contradictions, as it is always open to contestation and re-articulation, as well as to economic risk and market competition. Indeed, it remains open to counteraccusations of providing ideological cover for growing inequalities and government retreat, harbouring the real risks that the narrow income-generation concerns of commercialisation come to dominate over the public purpose. Under such circumstances, municipal entrepreneurship carries the danger of being rearticulated as little more than a justificatory narrative for ‘go-it-alone’ competitive localism and reductionist strategies of community betterment and divestment. Of course, the sustainability of such entrepreneurial practices has also been called into question by COVID-19 and its impacts on commercialisation revenues. At the time of writing, the Local Government Association (2020) has predicted that local authorities in England will lose £2.8 billion in falling commercial income due to the pandemic. Equally, the National Audit Office (2020) has raised concerns about the exposure of local authorities to economic and market risk, particularly where they rely on rental incomes to fund services. Yet, our study suggests that the stewardship garned by the discourse of municipal entrepreneurship for the public purpose could offer local government a privileged means of supporting local economies, particularly the pillars of the foundational economy, in a post-COVID recovery. REFERENCES Aldag, A., Kim, Y. and Warner, M. (2019) ‘Austerity urbanism or pragmatic municipalism?’, Environment and Planning A, 51(6): 1287-1305. 22 Bello, B., Downe, J., Andrews, R. and Martin, S. (2018) ‘Does austerity drive public service innovation?’, Public Money & Management, 38(2): 131-138. Beswick, J, and Penny, J. 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Dumfries and Galloway Council (2020) Procurement annual report, 2018/19, https://www.dumgal.gov.uk/media/20817/Procurement-Annual-Report-for-20181/pdf/Procurement_Annual_Report_for_Financial_Year_18-19__appendix.pdf?m=637038048383900000 accessed 30 June 2020. Fairclough, N. (2013) ‘Critical discourse analysis and critical policy studies’, Critical Policy Studies, 7(2): 177-97. 23 Ferry, L., Andrews, R., Skelcher, C. and Wegorowski, P. (2018) ‘New development: Corporatization of local authorities in England in the wake of austerity, 2010-2016’, Public Money & Management, 38(6): 477-480. Ferry, L. and Eckersley, P. (2020) ‘Hybridizing the institutional logics of performance management and budgetary stewardship in English and Welsh local government’ Public Policy & Administration, 35(1), 45-64. Foucault, M. (1972) The Archaeology of Knowledge, London: Tavistock. Fuller, C. 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(2016), ‘The Public Sector as an Entrepreneur?’, New Perspectives on Research, Policy & Practice in Public Entrepreneurship Vol. 6, Bradford: Emerald, pp. 127-146. Raco, M and de Souza, T.M. (2018) ‘Urban development, small business communities and the entrepreneurialisation of English local government’ Town Planning Review 89(2), 145-165. Robinson,W. (2016) ‘Comparative Urbanism’, International Journal of Urban and Regional Research 40(1): 187-199. Russell, B., (2019) ‘Beyond the Local Trap’, Antipode 51(3): 989–1010 Shearmur, R. and Poirier, V. (2017) ‘Conceptualising non-market municipal entrepreneurship’, Urban Affairs Review, 53(4): 718-51. Smith, B. (2000) ‘The concept of the “enabling” local authority’, Environment and Planning C, 18(1): 79-94. Spinosa, C. Flores, F. and Dreyfus, H. (1999) Disclosing New Worlds, Cambridge, Mass.: MIT. 26 Stoker, G. (2011) ‘Was local governance such a good idea?’, Public Administration, 89(1): 15-31. Taylor, L. Haynes, P. and Darking, M. (2021) ‘English local government finance on transition’, Public Management Review, 32(7): 1081-1106. Thompson, M. (2020) ‘What so new about New Municipalism?’ Progress in Human Geography, 45(2): 317-342. Thompson, M., Nowak, V., Southern, A., Davies, J. and Furmedge, P. (2020) ‘Regrounding the city with Polyani’, Environment and Planning A, 52(6): 1171-1194. 27 Table 1: Case Study Councils Birmingham City Municipal Housing Trust, local authority housing company Dumfries and Galloway Enterprising DG, in-house service delivery arm East Riding of Yorkshire Leisure services partnership with GPs Oxford City Oxford Direct Services, local authority social enterprise Sevenoaks District Property investment strategy Swansea City and House building programme County 28 Figure 1: Statements: Commercialisation and Entrepreneurship, APSE Nominees 2015-2019 1. ‘Commercialising our services to meet the challenge of reduced Government funding.’ (Nottingham City Council, New DLO for Commercial Gas and Electrics, 2015) 2. ‘To deliver free residential solar PV to residents in the city and deliver a community benefit fund and income to the Council whilst offering some of the City’s residents an opportunity to address fuel poverty issues.’ (Peterborough City Council, Empower Peterborough, 2015) 3. ‘Business reengineering project that has (…) already helped safeguard and enhance the lives of over 5,000 residents and has delivered cashable savings of over 70% to the public purse.’ (North Hertfordshire District Council, Hertfordshire Careline - Social Entrepreneurship in Practice, 2016) 4. ‘Growth of 110% in external income, financial savings of £168k a year, reduction annually in 17,000 tons of emissions and an on target, projected additional income stream of over £2.9 million over the next five years.’ (GS Plus Ltd – Royal Borough of Greenwich, Creating a Prosperous Future!, 2016) 5. ‘The company was created to nurture the growth of people, businesses and the region.’ (Cheshire East Council, The Skills and Growth Company, 2017) 6. ‘Our USP as the only social caterer in town: ‘Stakeholders NOT Shareholders’ whilst maximising quality in delivery, income growth and penetration.’ (Birmingham City Council, Building on the Past - Shaping the Future, 2017) 7. ‘The Rocky Balboa-style story that has taken KWL from being the underdog to a wellrespected champion in its field.’ (Kingstown Works Ltd, Ten Years On and Trading Strong, 2017) 8. ‘Commercialisation is a key enabler in tackling the perfect storm of austerity and rising demand on council services.’ (Warrington Borough Council, Taking Control of Our Future, 2018) 9. ‘We were able to assist 200 new clients and deliver nearly £200K in surpluses back to our host authority in just one year.’ One West, Bath and North East Somerset Council, Delivering Good Governance Through Commercialisation, 2019) 10. ‘To influence substantial behavioural change, positively impact upon trading imperatives and embed a commercial culture – changing the perception of every council employee from ‘I work for the Council’ to ‘I work for our Citizens’.’ (Birmingham City Council, The Commercial Business Hub - Providing the Oxygen for Growth, 2019) 11. ‘Astute commercial investments with a clear sense of purpose.’ (Cheltenham Borough Council, Calculated Risk Taking, 2019) 12. ‘Being an effective commercial business managing its budget, generating income, reducing costs and expenditure, effectively commissioning and procuring and ensuring a focus on outcomes and value for money rather than ‘the mentality of cuts’.’ (Thurrock Council, The Commercial Council, 2019)
https://openalex.org/W2960196667
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Expression of components of the urothelial cholinergic system in bladder and cultivated primary urothelial cells of the pig
BMC urology
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Leonhäuser et al. BMC Urology (2019) 19:62 https://doi.org/10.1186/s12894-019-0495-z Leonhäuser et al. BMC Urology (2019) 19:62 https://doi.org/10.1186/s12894-019-0495-z Open Access Expression of components of the urothelial cholinergic system in bladder and cultivated primary urothelial cells of the pig Dorothea Leonhäuser1, Jasmin Kranz2, Regina Leidolf2, Patrick Arndt1, Ulrich Schwantes3, Joachim Geyer2 and Joachim O. Grosse1* Abstract Background: Porcine urinary bladders are widely used for uro-pharmacological examinations due to their resemblance to the human organ. However, characterisations of the porcine urothelium at the molecular level are scarce up to now. As it has become clear over the last years that this tissue plays an important role in the signaling-pathways of the bladder, we examined whether the transporter and receptor pattern (with focus on the transmitter acetylcholine) is comparable to the human urothelium. With regard to in vitro studies, we also investigated if there is a difference between the native tissue and cultivated primary urothelial cells in culture. Methods: Urothelium from German Landrace and Göttingen Minipig bladders was collected. One part of the German Landrace tissue was used for cultivation, and different passages of the urothelial cells were collected. The actual mRNA expression of different transporters and receptors was examined via quantitative real-time PCR. These included the vesicular acetylcholine transporter (VAChT), the choline acetyl transferase (ChAT), organic cation transporters 1–3 (OCT1–3), organic anion transporting polypeptide 1A2 (OATP1A2), P-glycoprotein (ABCB1), the carnitine acetyl- transferase (CarAT), as well as the muscarinic receptors 1–5 (M1–5). Results: There is a strong qualitative resemblance between the human and the porcine urothelium with regard to the investigated cholinergic receptors, enzymes and transporters. CarAT, OCT1–3, OATP1A2 and ABCB1 could be detected in the urothelium of both pig races. Moreover, all 5 M-receptors were prominent with an emphasis on M2 and M3. VAChT and ChAT could not be detected at all. Cultures of the derived urothelial cells showed decreased expression of all targets apart from ABCB1 and CarAT. Conclusions: Based on the expression pattern of receptors, transporters and enzymes of the cholinergic system, the porcine urinary bladder can be regarded as a good model for pharmacological studies. However, cultivation of primary urothelial cells resulted in a significant drop in mRNA expression of the targets. Therefore, it can be concluded that the intact porcine urothelium, or the whole pig bladder, may be appropriate models for studies with anticholinergic drugs, whereas cultivated urothelial cells have some limitation due to significant changes in the expression levels of relevant targets. Abstract Keywords: Urothelium, Urinary bladder, Acetylcholine, Transporter, Muscarinic receptor, German landrace pig, Göttingen Minipig Urothelium, Urinary bladder, Acetylcholine, Transporter, Muscarinic receptor, German landrace pig, Minipig * Correspondence: joachim.grosse1963@gmail.com 1Department of Urology, RWTH Aachen University Hospital, Pauwelsstraße 30, 52074 Aachen, Germany Full list of author information is available at the end of the article Harvest and storage of urothelial tissue and cell culture of urothelial cells (UC) Harvest and storage of urothelial tissue and cell culture of urothelial cells (UC) All experiments on animals were performed in com- pliance with German legislation governing animal stud- ies and the Guide for the Care and Use of Laboratory Animals (National Institutes of Health (NIH), Publica- tion No 85–23, revised 2011). The pig urinary bladders were obtained from other working groups in our animal facility in order to reduce the number of animal experi- ments (3-R principle). These animal experiments were approved by the Governmental Animal Care and Use Committee (LANUV Recklinghausen). Göttingen Mini- pigs (GM) were about 2 years old whereas the German Landrace (GL) pigs were 6 months old. Besides the anaesthetics, no other drugs or treatments were applied to the pigs which could affect the bladder tissue. The pigs were euthanized by the original working group using 0.16 g/kg barbiturate i.v. (Narcoren®, Merial, Hall- bergmoos, Germany), and the intact bladders were transported to the cell culture facility in a 37 °C pre- warmed Modified Eagle’s Medium (MEM, Life Tech- nologies, Braunschweig, Germany). Some molecular examinations of detrusor and urothe- lial tissue have been performed on human material concerning transporters and receptors of anticholinergic drugs, such as trospium chloride, which are used as the clinical standard in the treatment of overactive bladder (OAB) [6–9]. Although it is clear that anticholinergic drugs block muscarinic (M-) acetylcholine (ACh) recep- tors, it is not completely understood how these drugs interact with the cellular machinery of the urothelium and the detrusor. Goepel et al. [10], as well as Sellers et al. [11], showed that muscarinic receptors M2 and M3 are prominent in porcine as well as human detrusor tissue, and in the human urothelium all five known M- receptor subtypes are present [12–14]. g g y) Via a Y-shaped incision, bladders from GM and GL were opened, and the urothelial tissue was carefully dissected and minced with scissors. Whole urothelial tissue of GM and one segment of GL tissue was frozen in liquid nitro- gen and stored at −80 °C. The other segment of GL tissue was used for cell culture as described previously [20]. Briefly, the urothelium was incubated in MEM containing 400 μg/ml collagenase (Liberase®, Roche Applied Sciences, Penzberg, Germany) for 1 h at 37 °C. © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Leonhäuser et al. BMC Urology (2019) 19:62 Page 2 of 8 Leonhäuser et al. BMC Urology (2019) 19:62 Page 2 of 8 Page 2 of 8 Methods Over the last years, the use of urinary pig bladders, and the pig itself as a large animal model, have be- come extremely popular for urological examinations. Especially pharmacological studies depend on the test- ing of drugs using porcine material [1–3]. This is justified on the basis that the porcine bladder closely resembles the human organ both anatomically and physiologically [4, 5]. Nevertheless, not much research on the molecular basics has as yet been conducted which justifies unlimited use and comparison of the porcine material [5]. Harvest and storage of urothelial tissue and cell culture of urothelial cells (UC) The cell suspension was then filtered, washed with MEM containing 10% fetal calf serum (FCS, Thermo Scientific) and transferred into collagen-coated (Biochrom AG, Berlin, Germany) cell culture flasks (Nunclon™, Thermo Scientific). Incubation of the urothelial cells (UC) for the first 24 h was per- formed in MEM containing 20% FCS, 1% gentamicin (PAA, GE Healthcare, Frankfurt am Main, Germany) and 1% amphotericin B (PAA). After 24 h, the medium was changed to Keratinocyte-SFM (Life Technologies) and subsequently changed twice a week. Examination of the cells in culture was performed with a Leica DMI 4000B (Leica Microsystems GmbH, Wetzlar, Germany) with integrated software Diskus (4.80.5909, Hilgers, Tech- nisches Büro, Königswinter, Germany). The cells were split at confluence and 1 × 106 cells were transferred into a new 75 cm2 flask. The remaining cells were frozen in liquid nitrogen and stored at −80 °C. Besides the M-receptors, other possible drug targets exist in the human bladder. Lips et al. [15] found proof for different transporters and enzymes responsible for synthesis and storage of ACh in the murine and human urothelium, including the carnitine acetyl-transferase (CarAT), whereas the classical ACh synthesising enzyme of neuronal cells, choline acetyltransferase (ChAT), seems to be absent in the urothelium. Furthermore, they demonstrated expression of the organic cation trans- porters OCT1–3 in the human urothelium. Interestingly, all of them are active in transporting TrCL [16]. More- over, the solute carrier organic anion transporting poly- peptide 1A2 (OATP1A2), involved in the cellular uptake of TrCL, as well as P-glycoprotein (syn. ATP-binding cassette transporter ABCB1, encoded by the multi-drug resistance gene MDR1), involved in the efflux of TrCL, are expressed in the normal urothelium [17–19]. The vesicular ACh transporter VAChT, which transports ACh in synaptic vesicles of neuronal cells, appears to be absent in the urothelium [13]. With regard to the pig as a model for pharmaco- logical studies on anticholinergic drugs in the bladder, our aim was to determine whether these relevant transporters, enzymes and receptors are present in the porcine urothelium with the same expression pattern and to the same extent as in the human. Fur- thermore, their molecular stability during cell culture was to be examined. Results Cell culture of urothelial cells and proof of cell type During the cultivation period, UCs from GL could be passaged up to three times. This resulted in five samples per GL pig; the untreated bladder biopsy and four immediately consecutive passages of UCs. From these GL pig bladders and three additional GM blad- ders, untreated urothelial tissue samples were frozen to compare the native tissues of the different races. Immu- nohistochemical staining of the cells against panCK confirmed an urothelial phenotype (Fig. 1). The pres- ence of muscarinic receptors M2 and M3, most relevant for storing and voiding of urine in the bladder, could also be visualized in the native urothelium of the German Landrace pig (Fig. 2a+b). Antigen retrieval of deparaffinised sections was per- formed using citrate-buffer (Zytomed Systems GmbH, Berlin, Germany) in a steamer for 30 min. Primary monoclonal and polyclonal antibodies (Table 1) were incubated for 1 h, and secondary antibody and chromo- gen development (DAKO Real EnVision HRP rabbit/ mouse with DAB) were applied according to the manu- facturer’s protocol. Counterstaining was performed using haemalaun (Merck). Staining was observed using a Leica DM6000B and integrated software Diskus (4.80.5909, Hilgers, Technisches Büro, Königswinter, Germany). Immunohistochemical staining of tissue and cells Validation of the cell type was performed via immu- nohistochemistry. Therefore, confluent UC were de- tached with trypsin-EDTA (Life Technologies), washed Page 3 of 8 Leonhäuser et al. BMC Urology (2019) 19:62 was performed using OriginPro (2017G, Origin Lab Corporation, Northampton, USA). The Shapiro-Wilks test was used to test for normal distribution, and One- Way ANOVA with the Tukey-Post Hoc test was used to determine statistical differences. Values of p ≤0.05 were considered to be significant. with phosphate buffered saline (PBS) (Life Technologies) and fixed in 4% (w/v) phosphate buffered formaldehyde (Merck). After further centrifugation, supernatant for- maldehyde was removed and the cells were mixed with 3% (w/v) agarose (Biozym Scientific GmbH, Hessisch Old- endorf, Germany). The agarose cell-hybrids were cooled for 3 min. in a fridge at 4 °C. Native bladder tissue served as the control and was also used for staining of muscar- inic receptors M2 and M3 and therefore fixed in phos- phate buffered formaldehyde. The cell-hybrids and the native tissue were dehydrated, embedded in paraffin and cut into 3 μm sections. Quantitative real-time polymerase chain reaction In general, quantitative real-time PCR expression analysis of native porcine urothelium was very similar concerning the presence of the examined transporters and receptors as in humans [13, 15]. The enzyme CarAT and the trans- porter ABCB1 were most commonly expressed. The or- ganic cation transporters OCT1–3, as well as OATP1A2, could be detected, but to a lesser extent (Fig. 3a). The mRNA expression for all muscarinic receptors was found in the porcine urothelium, especially M2 and M3 (Fig. 3b). VaChT and ChAT, as in human urothelium, were not detectable. There were no significant differences in the expression pattern between the two pig breeds. Quantitative real time polymerase chain reaction TriReagent (Sigma Aldrich) was used to extract RNA from cultured cells and tissues according to the manufacturer’s protocol. The isolated RNA was reverse-transcribed using the SuperScript III system (Life technologies GmbH, Darmstadt, Germany). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using the Taq- Man GEX Master Mix (Life Technologies). Four of the ob- tained assays (VAChT, OCT3, CHRM4, and CHRM5) had to be custom-made as there were no such probes available for the pig. All other TaqMan assays were ordered according to the reference numbers shown in Table 2. Glyercinaldehyde-3-phosphate-dehydrogenase (GAPDH) was used as the housekeeping gene. All assays were tested using a pig tissue cDNA panel (BioCat GmbH, Heidelberg, Germany) prior to the initial examinations. Subsequently, it was analysed whether the enzyme/ transporter/receptor expression changed during culti- vation of the porcine urothelium. In general, most of the analysed targets were down-regulated following repeated passaging of the cells. This down-regulation was most noticeable for OCT1 and OCT2 as well as M1-M3, whereas the expression level of the M4 and M5 recep- tors was almost maintained up to passage 3 (P3) (Fig. 4). Statistical analysis Cell cultures from three different animals were exa- mined using technical triplicates. Statistical evaluation Table 1 Primary antibodies for immunohistochemistry Antibody Clone number Reactivity Dilution Incubation time Company PanCK AE1/AE3 Monoclonal mouse, anti- human 1:300 1 h Dako GmbH, Hamburg, Germany Anti-Muscarinic Acetylcholine Receptor 2 31-1D1 Monoclonal mouse, anti- human 1:100 1 h Abcam, Cambridge, United Kingdom Anti-Muscarinic Acetylcholine Receptor M3 antibody – Polyclonal rabbit, anti-pig 1:100 1 h Abcam, Cambridge, United Kingdom Table 1 Primary antibodies for immunohistochemistry Leonhäuser et al. BMC Urology (2019) 19:62 Page 4 of 8 Leonhäuser et al. BMC Urology Table 2 TaqMan gene expression assays used for real-time PCR expression analysis Assay Order No. VAChT-Pig Custom Gene ex assay, AIWR3FN, referred to the cDNA sequence with GenBank Accession No. XM_013983446 ChAT-Pig Taqman Gene ex assay MTO, sm, Ss03391504_m1 CarAT-Pig Taqman Gene ex assay MTO, sm, Ss03389781_m1 ABCB1-Pig Taqman Gene ex assay MTO, sm, Ss03373435_m1 OCT1-Pig Taqman Gene ex assay MTO, sm, Ss03391173_m1 OCT2-Pig Taqman Gene ex assay MTO, sm, Ss03390935 OCT3-Pig Custom Gene ex assay, JD89KCA, referred to the cDNA sequence with GenBank Accession No. XM_003121106 OATP1A2-Pig Taqman Gene ex assay MTO, sm, Ss03375623_u1 CHRM1-Pig Taqman Gene ex assay MTO, sm, Ss03393581_u1 CHRM2-Pig Taqman Gene ex assay MTO, sm, Ss03383697_u1 CHRM3-Pig Taqman Gene ex assay MTO, sm, Ss03387661_u1 CHRM4-Pig Custom plus Tqmn RNA Assays, AJPADK0, referred to the cDNA sequence with GenBank Accession No. XM_003122828 CHRM5-Pig Custom plus Tqmn RNA Assays, AJN1FES, referred to the cDNA sequence with GenBank Accession No. XM_021099908 GAPDH-Pig FG, Off the shelf GX Set, Ss03374854_g1 Table 2 TaqMan gene expression assays used for real-time PCR expression analysis Table 2 TaqMan gene expression assays used for real-time PCR expression analysis The downregulation of muscarinic receptors M2 and M3 could also be visualized by immunohistochemical staining (Fig. 2c+d). The downregulation of muscarinic receptors M2 and M3 could also be visualized by immunohistochemical staining (Fig. 2c+d). process have been overlooked. This might be due to the fact, that this special tissue was only assumed to be a barrier with no physiological impact on the bladder func- tion [21, 22]. As it has become increasingly clear that the urothelium plays an essential role, it is even more import- ant to re-examine the animal models already used for trans- lational examinations applicable to the human bladder. Discussion Previously, systematic examinations on the urothelium as an active component of the bladder storage and micturition Fig. 1 Isolation of urothelial cells from German Landrace pig urothelium. a Urothelial layer which was dissected from the underlying lamina propria (HE staining). b Urothelial cells in culture at confluence. c Immunostaining of UCs with panCK embedded in agarose. d Positive control of porcine urothelium, immunostained with panCK Fig. 1 Isolation of urothelial cells from German Landrace pig urothelium. a Urothelial layer which was dissected from the underlying lamina propria (HE staining). b Urothelial cells in culture at confluence. c Immunostaining of UCs with panCK embedded in agarose. d Positive control of porcine urothelium, immunostained with panCK Leonhäuser et al. BMC Urology (2019) 19:62 Page 5 of 8 Leonhäuser et al. BMC Urology Fig. 2 Immunohistochemical staining of muscarinic receptors in native bladder tissue and derived urothelial cells. a+c Muscarinic receptor M2 and b+d M3 could be visualized in a+b native bladder tissue of the German Landrace pig but c+d not in the derived urothelial cells at passage 1. Scale bar = 50 μm Fig. 2 Immunohistochemical staining of muscarinic receptors in native bladder tissue and derived urothelial cells. a+c Muscarinic receptor M2 and b+d M3 could be visualized in a+b native bladder tissue of the German Landrace pig but c+d not in the derived urothelial cells at passage 1. Scale bar = 50 μm Fig. 2 Immunohistochemical staining of muscarinic receptors in native bladder tissue and derived urothelial cells. a+c Muscarinic receptor M2 and b+d M3 could be visualized in a+b native bladder tissue of the German Landrace pig but c+d not in the derived urothelial cells at passage 1. Scale bar = 50 μm So far, we have only established the M2 and M3 immunohistostaining for the targets also analysed via qRT-PCR. As it is not easy to find antibodies that are applicable for pig tissue, it is often necessary to use anti- bodies for human or other species and hope for cross- reactions. Therefore, we also tried a GAPDH antibody which turned out to react very unspecific. As the use of immune staining is more for visual effects, we post- poned the establishment of other antibodies. cholinergic system, a comparable expression pattern was detected in the pig in the present study, as has pre- viously been described for humans [13, 15, 18, 19]. Discussion * Significant down-regulation compared to tissue with p < 0.05 limits the usability of these cells, for example, for trans- port or receptor binding experiments at higher passaging frequencies. Bexten et al. were able to show that, on the one hand, TrCL is a substrate of the aforementioned solute carriers OCT1 and OATP1A2 (uptake), but on the other hand also for the efflux carrier ABCB1 [19]. The in vitro downregulation of the uptake transporters in combination with the still relatively high amount of the ABCB1 efflux carrier would lead to a non-physiological shift compared to the actual in vivo situation. This cultivation-related down- regulation could also be observed for the muscarinic re- ceptors, especially M2 and M3, which play a crucial role in the storing and voiding mechanisms of the bladder. M3 is known to mediate the contractile response and thus is addressed by the main muscarinic receptor antagonist TrCL [12]. This has to be considered for uptake studies with anticholinergic drugs like TrCL. However, down- regulation of M1-M5 has also been demonstrated for hu- man urothelial cells in culture by Tyagi et al. [14]. tissue so far. Additionally, in the present study, expres- sion of the classical ACh synthesising enzyme ChAT could not be detected in the porcine urothelium as has previously been reported for the human [13]. However, the porcine urothelium showed high mRNA expression levels for CarAT, which is an alternative source of ACh synthesis in the urothelium [13]. In the pig, all three OCTs (OCT1–3) were detected in the urothelium in the order OCT3 > OCT2 > OCT1, and all of them were also present in the human urothelium [13]. The solute carrier OATP1A2, uptake carrier for endogenous substances and pharmaceuticals in the human urothelium, has been identified as a transporter for TrCL by Bexten et al. [19], and could also be detected in the pig urothelium by our group. ABCB1 plays an important role in the efflux of many drugs, also including the anticholinergic drug TrCL [23]. ABCB1 is highly expressed in normal human urothelium [18, 19] and the present study also con- firmed its expression in the porcine urothelium. Finally, all 5 M-receptors have been reported in the human urothelium [13, 14], and could be detected in the present study with a similar expression pattern in the pig, with the highest expression levels for M2 and M3. Discussion Although VAChT is an important vesicular transporter for ACh, it could not be detected in the porcine uro- thelium. This too, is in accordance with the findings in the human urothelium, where the presence of VAChT could not be proved [15]. However, as a limitation it has to be mentioned that the gene expression assay for VAChT was derived from a predicted cDNA sequence (see Table 1) and could not be confirmed in any porcine Using qRT-PCR expression analysis of selected en- zymes, transporters and receptors of the urothelial Fig. 3 Relative gene expression in the urothelium of German Landrace pigs and Göttingen minipigs (n = 3). VAChT and ChAT expression could not be detected. All enzymes/transporters (a) as well as receptors (b) relevant for anticholinergic therapy showed comparable expression pattern for GL and GM. Highest expression was detected for CarAT, ABCB1, OCT3, OATP1A2, M2 and M3. GAPDH expression was used for normalization pigs and Göttingen minipigs (n 3) VAChT and ChAT expression could Fig. 3 Relative gene expression in the urothelium of German Landrace pigs and Göttingen minipigs (n = 3). VAChT and ChAT expression could not be detected. All enzymes/transporters (a) as well as receptors (b) relevant for anticholinergic therapy showed comparable expression pattern for GL and GM. Highest expression was detected for CarAT, ABCB1, OCT3, OATP1A2, M2 and M3. GAPDH expression was used for normalization Leonhäuser et al. BMC Urology (2019) 19:62 Page 6 of 8 Leonhäuser et al. BMC Urology Fig. 4 Relative gene expression in the urothelium from German Landrace pigs (“Tissue”) as well as derived UCs (“P0-P3”) (n = 3). All enzymes/ transporters (a) as well as receptors (b) relevant for anticholinergic therapy were down-regulated during repeated passaging, with the exception of M4. In particular, expression levels of OCT2 and M1 dramatically dropped to nearly undetectable levels. GAPDH expression was used for normalization. * Significant down-regulation compared to tissue with p < 0.05 i (“Ti ”) ll d i d UC (“P0 P3”) ( 3) All / Fig. 4 Relative gene expression in the urothelium from German Landrace pigs (“Tissue”) as well as derived UCs (“P0-P3”) (n = 3). All enzymes/ transporters (a) as well as receptors (b) relevant for anticholinergic therapy were down-regulated during repeated passaging, with the exception of M4. In particular, expression levels of OCT2 and M1 dramatically dropped to nearly undetectable levels. GAPDH expression was used for normalization. Conclusions This study was able to show that the porcine urothelium of the GL and GM pig is very similar to the human urothelium concerning the investigated ACh-dependent targets. We therefore conclude that both pig races are appropriate as a pharmacological animal model for in vivo and ex vivo investigations. Nevertheless, the use of UCs from healthy animals is limited due to the down- regulation of the afore-mentioned targets. Therefore, the development and use of an animal model with OAB or IC could provide more insight into the working mechanism of these diseases. Funding h d This study was funded by the Dr. R. Pfleger GmbH, Germany. The Dr. R. Pfleger GmbH had no control over the study conduct, or the collection and analysis of the data. Abbreviations b d ABCB1: ATP-binding cassette B1; ACh: Acetylcholine; CarAT: Carnitine acetyl- transferase; ChAT: Choline acetyl transferase; GAPDH: Glyercinaldehyde-3- phosphate-dehydrogenase; GL: German Landrace pig; GM: Göttingen Minipig; IC: Interstitial cystitis; M1–5: Muscarinic receptors 1–5; MEM: Modified Eagle’s Medium; OAB: Overactive bladder; OATP1A2: Organic anion transporting polypeptide 1A2; OCT1–3: Organic cation transporters 1–3; PBS: Phosphate buffered saline; TrCL: Trospium chloride; UC: Urothelial cell; VAChT: Vesicular acetylcholine transporter 2. Yamanishi T, Chapple CR, Yasuda K, Chess-Williams R. The role of M-2- muscarinic receptors in mediating contraction of the pig urinary bladder in vitro. Br J Pharmacol. 2000;131(7):1482–8. 3. Parsons BA, Drake MJ, Gammie A, Fry CH, Vahabi B. The validation of a functional, isolated pig bladder model for physiological experimentation. Front Pharmacol. 2012;3:52. 4. Huppertz ND, Tolba RH, Grosse JO. Micturition in Gottingen minipigs: first reference invivo data for urological research and review of literature. Lab Anim. 2015;49(4):336–44. Discussion Interestingly, ABCB1 showed slight upregulation dur- ing the first round of cultivation in comparison to the tissue samples. This upregulation of ABCB1 might be triggered by cell culture supplements such as antibiotics, as the main role of this transporter is the efflux of po- tential hazardous substances out of the cell [23, 24]. Two different pig races were analysed i.e. GL and GM, and both showed comparable expression patterns to each other and the human. Taking this into account, it could be expected that pharmacological studies in pigs, for example with anticholinergic drugs, would sub- stantially reflect the situation in man. UCs in culture are a promising model for phar- macological in vitro studies with anticholinergic drugs. However, such studies would be disadvantaged by down- regulation of relevant transporters and receptors for ACh and / or anticholinergic drugs. Nevertheless, Mukerji et al. [25], as well as Gupta et al. [26], could show that urothelial cells of patients with interstitial cystitis (IC) retained their phenotype under cell culture conditions. Furthermore, the animals in this study were mature but not old [27, 28] and thus, do not represent the aged population which suffer from bladder dysfunc- tions. Therefore, a corresponding animal model with IC Cultivation of urothelial cells has previously been estab- lished in order to avoid repeated in vivo experiments in pigs, and to enable in vitro studies with urothelial derived cells. For this reason, the expression of the relevant en- zymes, transporters, and receptors was also analysed under cell culture conditions during repeated passaging. Unfortunately, most of the analysed targets revealed continuous downregulation over time, which in general Page 7 of 8 Leonhäuser et al. BMC Urology (2019) 19:62 Page 7 of 8 Page 7 of 8 Leonhäuser et al. BMC Urology (2019) 19:62 or OAB could provide more insight in the mechanisms of these diseases at a molecular level. References 1. Templeman L, Chapple CR, Chess-Williams R. Urothelium derived inhibitory factor and cross-talk among receptors in the trigone of the bladder of the pig. J Urol. 2002;167(2):742–5. 2. Yamanishi T, Chapple CR, Yasuda K, Chess-Williams R. The role of M-2- muscarinic receptors in mediating contraction of the pig urinary bladder in vitro. Br J Pharmacol. 2000;131(7):1482–8. 3. Parsons BA, Drake MJ, Gammie A, Fry CH, Vahabi B. The validation of a functional, isolated pig bladder model for physiological experimentation. Front Pharmacol. 2012;3:52. 4. Huppertz ND, Tolba RH, Grosse JO. Micturition in Gottingen minipigs: first reference invivo data for urological research and review of literature. Lab Anim. 2015;49(4):336–44. 5. Bode G, Clausing P, Gervais F, Loegsted J, Luft J, Nogues V, et al. The utility of the minipig as an animal model in regulatory toxicology. J Pharmacol Toxicol Methods. 2010;62(3):196–220. 6. Abrams P, Andersson KE. Muscarinic receptor antagonists for overactive bladder. BJU Int. 2007;100(5):987–1006. 7. Andersson KE, Yoshida M. Antimuscarinics and the overactive detrusor - which is the main mechanism of action? Eur Urol. 2003;43(1):1–5. 8. Abrams P, Andersson KE, Buccafusco JJ, Chapple C, de Groat WC, Fryer AD, et al. Muscarinic receptors: their distribution and function in body 1. Templeman L, Chapple CR, Chess-Williams R. Urothelium derived inhibitory factor and cross-talk among receptors in the trigone of the bladder of the pig. J Urol. 2002;167(2):742–5. 1. Templeman L, Chapple CR, Chess-Williams R. Urothelium derived inhibitory factor and cross-talk among receptors in the trigone of the bladder of the pig. J Urol. 2002;167(2):742–5. Authors’ contributions All authors have made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data: DL designed the study, performed the experiments and analysed the data, JK performed the experiments and analysed the data, RL and PA performed the experiments JG and JOG designed the study and analysed the data. US, as an employee of the sponsor, was involved in the determination of the study design, but had no control over the study conduct, or the collection and analysis of data. All authors have been involved in drafting the manuscript or revising it critically for important intellectual content; All authors have given final approval of the version to be published and take public responsibility for appropriate portions of the content. All authors have agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The authors have read and approve of the final version. It remains that physiological and pharmacological stu- dies on the urinary bladder are mainly conducted in ro- dent models [29–32]. These animals are cheap, easy to handle, and a large number of individuals can be investi- gated. However, data generated in rodents may not be fully comparable to the situation in humans, as the ana- tomy, physiology and the day-night-rhythm of these animals is somewhat different. Therefore, pigs were ana- lysed in the present study as these show a more com- parable bladder physiology to humans [4]. Initially the study was intended only with GL pigs as these are cheaper to obtain and therefore used more commonly in animal studies. Additionally, it is common practice to perform whole bladder experiments and drug studies using pig bladders from an abattoir [1–3, 33]. However, the GM has proven itself to be a comparable animal model for urological in vivo long-term studies, not only on the physiological [4] but also on the molecu- lar level, as was also demonstrated in the present study. Based on the data presented here, both pig races are appropriate as a pharmacological animal model concer- ning the investigated targets. Competing interests DL, JK, RL, PA, JG, and JOG declare that they have no competing interests. US is an employee of Dr. R. Pfleger GmbH, Bamberg. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Author details 1 f 1Department of Urology, RWTH Aachen University Hospital, Pauwelsstraße 30, 52074 Aachen, Germany. 2Institute of Pharmacology and Toxicology, Biomedical Research Center BFS, Justus Liebig University Giessen, Giessen, Germany. 3Dr. R. Pfleger GmbH, Bamberg, Germany. Biomedical Research Center BFS, Justus Liebig University Giessen, Giessen, Germany. 3Dr. R. Pfleger GmbH, Bamberg, Germany. Received: 15 February 2018 Accepted: 4 July 2019 Ethics approval and consent to participate pp p p All projects for which our study group received subordinate urinary bladder material were either notified or approved by our licensing authority, the LANUV (Landesamt für Natur-, Umwelt und Verbraucherschutz in Recklinghausen). The secondary use of tissues or organs from dead animals does not require approval by the authorities according to animal welfare regulations in Germany. Publisher’s Note 10. Goepel M, Gronewald A, Krege S, Michel MC. Muscarinic receptor subtypes in porcine detrusor: comparison with humans and regulation by bladder augmentation. Urol Res. 1998;26(2):149–54. 10. Goepel M, Gronewald A, Krege S, Michel MC. Muscarinic receptor subtypes in porcine detrusor: comparison with humans and regulation by bladder augmentation. Urol Res. 1998;26(2):149–54. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 11. Sellers DJ, Yamanishi T, Chapple CR, Couldwell C, Yasuda K, Chess-Williams R. M-3 muscarinic receptors but not M-2 mediate contraction of the porcine detrusor muscle in vitro. J Auton Pharmacol. 2000;20(3):171–6. 12. Mansfield KJ, Liu L, Mitchelson FJ, Moore KH, Millard RJ, Burcher E. Muscarinic receptor subtypes in human bladder detrusor and mucosa, studied by radioligand binding and quantitative competitive RT-PCR: changes in ageing. Br J Pharmacol. 2005;144(8):1089–99. 12. Mansfield KJ, Liu L, Mitchelson FJ, Moore KH, Millard RJ, Burcher E. Muscarinic receptor subtypes in human bladder detrusor and mucosa, studied by radioligand binding and quantitative competitive RT-PCR: changes in ageing. Br J Pharmacol. 2005;144(8):1089–99. 13. Bschleipfer T, Schukowski K, Weidner W, Grando SA, Schwantes U, Kummer W, et al. Expression and distribution of cholinergic receptors in the human urothelium. Life Sci. 2007;80(24–25):2303–7. 13. Bschleipfer T, Schukowski K, Weidner W, Grando SA, Schwantes U, Kummer W, et al. Expression and distribution of cholinergic receptors in the human urothelium. Life Sci. 2007;80(24–25):2303–7. 14. Tyagi S, Tyagi P, Van-le S, Yoshimura N, Chancellor MB, de Miguel F. Qualitative and quantitative expression profile of muscarinic receptors in human urothelium and detrusor. J Urol. 2006;176(4). 14. Tyagi S, Tyagi P, Van-le S, Yoshimura N, Chancellor MB, de Miguel F. Qualitative and quantitative expression profile of muscarinic receptors in human urothelium and detrusor. J Urol. 2006;176(4). 15. Lips KS, Wunsch J, Zarghooni S, Bschleipfer T, Schukowski K, Weidner W, et al. Acetylcholine and molecular components of its synthesis and release machinery in the urothelium. Eur Urol. 2007;51(4):1042–53. 15. Lips KS, Wunsch J, Zarghooni S, Bschleipfer T, Schukowski K, Weidner W, et al. Acetylcholine and molecular components of its synthesis and release machinery in the urothelium. Eur Urol. 2007;51(4):1042–53. 16. Wenge B, Geyer J, Bönisch H. The anticholinergic drugs oxybutynin and trospium chloride are substrates of the human organic cation transporters. In: Naunyn-Schmiedeberg’s archives of pharmacology, vol. 383. Berlin Heidelberg: Springer; 2010. p. 203–8. 17. Franke RM, Scherkenbach LA, Sparreboom A. Acknowledgements The authors thank the research group of the Department of Urology, especially Katja Stollenwerk and Mykhailo Reidman for technical support, and the Institute of Laboratory Animal Sciences at RWTH Aachen University Hospital especially Taddhäus Stopinski for excellent animal care. Furthermore, we thank Stephanie Schmidt of the Institute of Pharmacology and Toxicology, Giessen for her scientific input. This work was also supported by the Cell Culture Facility, a Core Facility of the Interdisciplinary Center for Clinical Research (IZKF) Aachen within the Faculty of Medicine at RWTH Aachen University. 5. Bode G, Clausing P, Gervais F, Loegsted J, Luft J, Nogues V, et al. The utility of the minipig as an animal model in regulatory toxicology. J Pharmacol Toxicol Methods. 2010;62(3):196–220. Page 8 of 8 Page 8 of 8 Page 8 of 8 Leonhäuser et al. BMC Urology (2019) 19:62 (2019) 19:62 Leonhäuser et al. BMC Urology (2019) 19:62 33. 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Antimuscarinic drugs in detrusor overactivity and the overactive bladder syndrome: motor or sensory actions? BJU Int. 2006;98(3):503–7. Publisher’s Note Pharmacogenetics of the organic anion transporting polypeptide 1A2. Pharmacogenomics. 2009;10(3):339–44. 18. Clifford SC, Neal DE, Lunec J. High level expression of the multidrug resistance (MDRI) gene in the normal bladder urothelium: a potential involvement in protection against carcinogens? Carcinogenesis. 1996;17(3):601–4. 19. Bexten M, Oswald S, Grube M, Jia J, Graf T, Zimmermann U, et al. Expression of drug transporters and drug metabolizing enzymes in the bladder Urothelium in man and affinity of the bladder spasmolytic Trospium chloride to transporters likely involved in its pharmacokinetics. Mol Pharm. 2015;12(1):171–8. 20. Leonhäuser D, Vogt M, Tolba RH, Grosse JO. Potential in two types of collagen scaffolds for urological tissue engineering applications - are there differrences in growth behaviour of juvenile and adult vesical cells? J Biomater Appl. 2016;30(7):961–73. 21. Birder LA. More than just a barrier: urothelium as a drug target for urinary bladder pain. Am J Physiol Renal Physiol. 2005;289(3):F489–F95. 22. Apodaca G. The uroepithelium: not just a passive barrier. Traffic. 2004;5(3):117–28. 23. Geyer J, Gavrilova O, Petzinger E. The role of P-glycoprotein in limiting brain penetration of the peripherally acting anticholinergic overactive bladder drug Trospium chloride. Drug Metab Dispos. 2009;37(7):1371–4. 24. Oswald S, Grube M, Siegmund W, Kroemer HK. Transporter-mediated uptake into cellular compartments. Xenobiotica. 2007;37(10–11):1171–95. 24. Oswald S, Grube M, Siegmund W, Kroemer HK. Transporter-mediated uptake into cellular compartments. Xenobiotica. 2007;37(10–11):1171–95. 25. 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Phasic contractions in urinary bladder from juvenile versus adult pigs. Plos One. 2013;8(3). 28. Bollen P, Ellegaard L. The Gottingen minipig in pharmacology and toxicology. Pharmacol Toxicol. 1997;80:3–4. 28. Publisher’s Note Bollen P, Ellegaard L. The Gottingen minipig in pharmacology and toxicology. Pharmacol Toxicol. 1997;80:3–4. 29. Zarghooni S, Wunsch J, Bodenbenner M, Brueggmann D, Grando SA, Schwantes U, et al. Expression of muscarinic and nicotinic acetylcholine receptors in the mouse urothelium. Life Sci. 2007;80(24–25). 29. Zarghooni S, Wunsch J, Bodenbenner M, Brueggmann D, Grando SA, Schwantes U, et al. Expression of muscarinic and nicotinic acetylcholine receptors in the mouse urothelium. Life Sci. 2007;80(24–25). 30. Hanna-Mitchell AT, Beckel JM, Barbadora S, Kanai AJ, de Groat WC, Birder LA. Non-neuronal acetylcholine and urinary bladder urothelium. Life Sci. 2007;80(24–25):2298–302. 31. Matsumoto Y, Miyazato M, Furuta A, Torimoto K, Hirao Y, Chancellor MB, et al. 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Application of a Protocol Based on Trap-Neuter-Return (TNR) to Manage Unowned Urban Cats on an Australian University Campus
Animals
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Received: 16 April 2018; Accepted: 11 May 2018; Published: 17 May 2018 Simple Summary: In Australia, management of the unowned urban cat population is a continuing challenge. This is because the numbers of cats culled in trap-and-kill programs are inadequate to balance the breeding rate of the remaining cats, and also because of immigration of sexually active cats from surrounding areas in a “vacuum” effect into areas where culling has been applied. In contrast, programs based on management of cat reproduction, such as trap-neuter-return (TNR), supplemented by rehoming of socializable cats and kittens, have been shown to produce significant reductions in free-living cat populations. However, evidence is lacking that these approaches are effective in an Australian context. In this paper, we document a nine-year TNR program on an Australian university campus, supplemented by rehoming, that reduced a free-living cat population from 69 to 15 cats, while also rehoming 19 campus-born kittens and managing a further 34 immigrant cats that either joined the resident colony (n = 16), were rehomed (n = 15), or died/disappeared (n = 3). Subsequent institutional support for the program was strong because of a reduction in complaints from campus staff and students, the minimal institutional costs, and the improved health status of the remaining cats, all of which are desexed, microchipped, registered and fed on a daily basis. Abstract: In August 2008, the University of New South Wales (UNSW) in Sydney, Australia, commenced a trap-neuter-return (TNR) program to manage the population of approximately 69 free-roaming unowned urban cats on its Kensington campus. The goals of the program included an ongoing audit of cats on campus, stabilization of cat numbers through TNR, and a subsequent reduction in cat numbers over time while maintaining the health of remaining campus cats. Continuation of the TNR program over nine years resulted in a current population, as of September 2017, of 15 cats, all desexed (78% reduction). Regular monitoring of the cats through a daily feeding program identified a further 34 cats that immigrated on to campus since initiation of the program; these comprised 28 adult cats (16 unsocialized, 12 socialized) and six solitary kittens. In addition, 19 kittens were born on campus, 14 of which were born to immigrant pregnant females. Unsocialized adult immigrants were absorbed into the resident campus population. Where possible, socialized adult immigrants, solitary kittens, and campus-born kittens were removed from campus through rehoming. animals animals animals animals animals Article Helen Swarbrick 1,2,3,* ID and Jacquie Rand 1,4 1 Australian Pet Welfare Foundation, Kenmore, QLD 4069, Australia; jacquie@petwelfare.org.au 2 Campus Cats NSW, Kensington, NSW 2033, Australia; unswcampuscats@yahoo.com 3 School of Optometry and Vision Science, The University of New South Wales, Sydney, NSW 2052, Australia 4 School of Veterinary Science, The University of Queensland, Gatton, QLD 4343, Australia; j.rand@uq.edu.au * Correspondence: h.swarbrick@unsw.edu.au; Tel.: +61-427-992-048 Helen Swarbrick 1,2,3,* ID and Jacquie Rand 1,4 1 Australian Pet Welfare Foundation, Kenmore, QLD 4069, Australia; jacquie@petwelfare.org.au 2 Campus Cats NSW, Kensington, NSW 2033, Australia; unswcampuscats@yahoo.com 3 School of Optometry and Vision Science, The University of New South Wales, Sydney, NSW 2052, Australia 4 School of Veterinary Science, The University of Queensland, Gatton, QLD 4343, Australia; j.rand@uq.edu.au * Correspondence: h.swarbrick@unsw.edu.au; Tel.: +61-427-992-048 1. Introduction Under Australian legislation, cat populations are often categorized as domestic (owned) or non-domestic (feral), with unowned urban cats occupying a grey area between these two categories. Unowned urban cats may be considered as domestic or non-domestic depending on individual state legislation. If classed as non-domestic they are subject to legislation relating to feral pests, and if domestic, they are subject to animal welfare legislation including laws relating to animal abandonment. Unowned urban cats are found in every urban and peri-urban area in Australia. They live close to or around human structures such as houses, factories, schools and shopping strips [1], and survive primarily through casual or regular feeding by the local community, by scavenging human rubbish and by catching prey, mainly introduced mice and rats. Such cats often congregate in colonies and thus are sometimes termed “colony cats”. Other terms often used to refer to unowned urban cats include “free-living”, “free-roaming”, “stray”, “street” or “community” cats. Although they are of the same species (Felis catus), unowned urban cats are usually considered to be distinct from truly feral cats, which do not depend on humans for shelter or food, and are usually found in rural or forested areas some distance from human habitation [2,3]. The primary food source for feral cats is through predation, and thus they may impact significantly on Australian native wildlife. Management of feral cats is a significant challenge [2] and is beyond the scope of this paper. Unowned urban cats largely comprise semi-owned or unowned domestic cats that may receive some care from persons who do not perceive themselves as owning the cat. In an Australian internet survey, 9% of respondents fed a cat daily they did not perceive that they owned [4], and 22% of households provided occasional care to an unowned cat [5]. Some apparently unowned urban cats are lost or wandering owned cats. The distinctions between unowned and owned domestic cats can become blurred and cats may move across categories in certain circumstances [4,6,7]. For example, an owned domestic cat that is lost or abandoned may join a colony of unowned urban cats. Similarly, unowned urban cats may be adopted to become an owned domestic cat. To add to the complexity, some semi-owned cats are desexed and microchipped and some owned domestic cats are not [4]. Unowned and semi-owned domestic cats constitute a large proportion of animal shelter admissions [8,9]. Received: 16 April 2018; Accepted: 11 May 2018; Published: 17 May 2018 Overall, reasons for reductions in the cat population (original residents, immigrants, campus-born kittens; n = 122) included rehoming or return to owner (30%), death/euthanasia (30%) and disappearance (29%). This successful animal management program received some initial funding from the university to support desexing, but was subsequently funded through donations, and continues with the university’s approval and support. Keywords: unowned urban cat; cat management; trap-neuter-return; TNR www.mdpi.com/journal/animals www.mdpi.com/journal/animals Animals 2018, 8, 77; doi:10.3390/ani8050077 2 of 23 Animals 2018, 8, 77 1. Introduction The approach to management of unowned urban cats varies considerably around Australia depending on state laws, council by-laws and practices, and local community attitudes. Perceived problems with unowned urban cat populations include uncontrolled breeding and population growth, nuisance behaviors such as fighting, spraying and roaming, the potential for cats to harbor diseases such as toxoplasmosis, and the perceived threat to native wildlife [3,10–15]. On the other hand, many individuals and community groups, either openly or covertly, support and feed colonies of unowned cats, often combining this with rescue and adoption of young cats and kittens, and management of reproduction through desexing [1,3,6,16]. These people are motivated by compassionate reasons, including a desire to stop cats breeding and to make their life better and safer, positive perceptions of themselves and from others for feeding a stray cat, and also the role of free-roaming cats in rodent control [1,4,6]. The principal official approach taken to manage unwanted urban cat populations is to trap and kill (cull) those colonies that give rise to persistent complaints from the public. This approach to cat population management has not been demonstrated to be effective in reducing cat numbers in the medium to long term [17,18], mainly because the number of cats that can reasonably be culled is an insufficient proportion of the population to exceed the replacement rate through breeding and immigration [10,19,20]. In particular, culling creates a “vacuum” effect that encourages immigration of other cats from surrounding areas [2,12–14]. This management strategy also places major stresses on shelters and municipal pounds (local government animal facilities) and veterinary clinics that must cope with euthanasia of otherwise healthy cats and kittens, not only in terms of resources, but also in emotional and ethical costs for staff involved, including post-traumatic stress which can lead to depression, substance abuse, high blood pressure and, tragically, suicide [21–24]. There is currently significant interest in Australia, and elsewhere, in reducing this burden on pounds and shelters, such as 3 of 23 Animals 2018, 8, 77 demonstrated by the activities of the G2Z organization (www.g2z.org.au), and the Australian Pet Welfare Foundation (www.petwelfare.org.au). demonstrated by the activities of the G2Z organization (www.g2z.org.au), and the Australian Pet Welfare Foundation (www.petwelfare.org.au). An alternative management strategy, which is being used increasingly in other countries such as the USA and in Europe, is trap-neuter-return (TNR) [7,12–14,16–18]. 1. Introduction This strategy involves trapping unowned cats, desexing them, and returning them to the location where they were trapped to resume their life with their colony. This management strategy frequently also involves removing young kittens and socialized adult cats for rehoming, contributing to a rapid reduction in colony size, and this is considered best practice [1]. In TNR programs, associated animal management at the time of desexing typically includes vaccination and parasite treatment, treatment for minor health issues, and in some cases microchipping, or ear-tipping or tattooing for later visual identification. Rarely, a cat cannot be returned to the location where it was trapped because of safety, environmental or practical reasons—in this situation, an alternative location must be found for release [25]. Cats considered too unhealthy to be returned to their home location are humanely euthanized, typically fewer than 1% of cats [26]. A number of published studies, including from the USA, UK and Italy, have demonstrated that TNR in a targeted area can be successful in reducing a cat population over time, particularly when augmented with rehoming of kittens and socialized adults. For example, Levy and co-workers [7] reported a reduction in free-roaming cat numbers on a Florida university campus from 155 to 23 over a 11-year period under a TNR program. Natoli and colleagues described a reduction in free-roaming cat numbers in Rome after a period of TNR management [16]. In a recent paper, Spehar and Wolf [17] described a TNR program conducted in Massachusetts, which reduced free-roaming cat numbers from approximately 300 to zero over a 17-year period. In a separate paper, Spehar and Wolf [18] also detailed a reduction in cat numbers in an area of Chicago managed over a 10-year period by a neighborhood TNR program. Earlier studies by Neville and Remfry [12], Neville [13] and Zaunbrecher and Smith [14] also detailed reduction or stabilization of small cat colonies using TNR. Recently, a survey of TNR practices in Australia reported that this management approach resulted in a median reduction in colony numbers from 12 to 7 cats over a median period of two years [1]. Although this study was uncontrolled and relied on self-reports of people engaged in TNR, it does suggest that this management approach may be successful in an Australian context for reducing urban stray cat numbers. Campus Background The University of New South Wales (UNSW) campus at Kensington is an urban university campus of approximately 38 hectares in the eastern suburbs of Sydney, which was established in 1949 on the site of previous racing stables and racecourse. Its immediate environs include a major racecourse and stables to the north (Royal Randwick racecourse), a suburban shopping strip to the south (Kingsford shops), a large hospital complex to the east (Prince of Wales hospital), and surrounding suburban residential areas (Kensington, Randwick and Kingsford). The campus currently has a human population of approximately 53,000 students and 6000 academic and support staff. The campus has probably always carried a resident unowned cat population from its days as a horse racing property, when cats may well have been encouraged to control vermin. Since its establishment as a university, the cats on campus have been sporadically fed by students, staff and community volunteers, and occasional attempts were made to manage the increasing population through trapping and desexing. However, there was minimal involvement of university management in these activities, and no financial support. Consequently, attempts to limit and manage the university cat population had little success, and the population was largely uncontrolled. In 2000, an informal organization called the Campus Cat Coalition (CCC) was formed to apply TNR principles for management of the cat population, with regular cat feeding by university staff. There was some consultation with university management but no formal support. Indeed, strong opposition to the presence of any cats on the campus was voiced in some parts of the university, mainly in relation to potential impacts on campus wildlife and the threat of disease to campus users. Because of a lack of university support or funding, the CCC program began to falter. In January 2008, some kittens were found dead in the basement of a university building, concomitant with a flea infestation in that building which adversely affected the health of some university staff. A decision was made by university management that the presence of cats on the campus constituted a work health and safety (WHS) issue, and a cat trapping and eradication program was instigated by the university. A pest management company was contracted to remove the cats. In February 2008, four campus cats were trapped over a five-day period. 1. Introduction In studies involving larger populations rather than well-characterized colonies, it is challenging to enumerate the cat population accurately to document effectiveness of management approaches over time, although methods are available to monitor change in cat density in a subset of the population [27]. Changes in rates of shelter impoundment are therefore often used as a surrogate for reductions in the overall unowned cat population. Thus, when TNR was applied rigorously in a Florida zip code, shelter impoundment of unowned cats decreased by 66% over a 2-year period, representing a decrease in admissions from 13 to 4 cats per 1000 residents, compared to a 12% reduction in non-target areas [26]. On the other hand, there have been reports of TNR programs where cat numbers have not reduced or have even increased [28–30]. In these reports, the reasons for failure to reduce overall cat numbers included not desexing a significant proportion of the female cats [28,29], immigration of cats into the colony from surrounding areas and failure to manage these adults and resulting kittens by desexing or adoption [29], and dumping by humans of unwanted animals [30]. These effects, which counteract the impact of TNR in managing populations, have also been noted as a factor reducing the effectiveness of population control even in reports of successful TNR programs [14,16,17]. In any non-lethal cat management program, it must therefore be recognized that the TNR process acts to reduce or limit the growth rate of the colony through reproduction, but may not in itself reduce cat numbers. Any population reduction occurs primarily through adoption of socialized cats and kittens, natural death or euthanasia of sick animals, and disappearance or emigration of cats [6,7,12–14,16–18,31]. TNR works together with these factors to reduce reproduction and thus to minimize replacement of animals lost from the colony. Nevertheless, other factors such as immigration of cats from surrounding areas can counteract this effect. Thus, the impact of TNR interventions on 4 of 23 Animals 2018, 8, 77 unowned urban cat populations can be complex, and ongoing management of colonies becomes an important component in optimizing reductions in the cat population [6,7,12,28]. The purpose of this paper is to report outcomes of a carefully-managed and rigorously-applied TNR program conducted over nine years on an Australian university campus. 1. Introduction This study provides strong evidence for the effectiveness of targeted TNR, supplemented by rehoming and management of immigrant cats, to humanely reduce the numbers of unowned urban cats in an Australian context. 2.1. Data Collection and Management From the beginning of the TNR program in August 2008, an ongoing careful audit of campus cats was conducted on the basis of daily observations of cats seen at feeding sites and other campus locations, by members of the CCC and other interested members of university staff and students. Cats were given names for easier identification, and photographs and descriptions were recorded and cross-referenced amongst observers. Later in the program, outdoor battery-operated motion-activated infrared trail cameras were used to document and identify campus cats. A database of cats observed on campus was established, including names, approximate ages, descriptions of markings and behavioral characteristics, sex and desexed status, vaccination status and microchip numbers. This database was updated on a regular and ongoing basis. Input from members of the CCC who had cared for the campus cats prior to initiation of the TNR program was also incorporated. Over the course of the nine-year program, the CCC team was able to estimate the campus cat population on an ongoing basis with a high level of confidence. For this study, details of the cats on campus commencing in August 2008 were retrieved from database records retained by the CCC, email correspondence, annual reports to the university management, and reports to Annual General Meetings of Campus Cats NSW, the registered animal welfare charity overseeing the campus cat management program. Extensive cross-referencing was employed to maximize data accuracy and to resolve inconsistencies in cat statistics where possible. Data were summarized at three-monthly and annual intervals commencing in September 2008 until September 2017. Data were stratified in terms of sex of cats and desexed status, where possible. Records had not been kept on whether campus cats were entire or desexed before the start of the program. Much of this information was determined from anecdotal evidence, based on the memories of those who had been caring for the cats previously. In other cases, the status of campus cats was determined over time based on observations of physical characteristics and behavior (spraying, fighting, testicles for male cats; pregnancies for female cats), or when cats were trapped for desexing. Data for ages of cats were not readily available because of the absence of records prior to the start of the TNR program. Campus Background Discussions with university management resulted in approval, in August 2008, for a one-year trial of TNR with the following defined goals and targets: • Immediate and ongoing audit of cat numbers on campus • Stabilize current cat numbers through a TNR program, prioritizing desexing of females, then males; 95% desexing target by the end of 2009 • Stabilize current cat numbers through a TNR program, prioritizing desexing of females, then males; 95% desexing target by the end of 2009 • Reduce cat numbers over time through rehoming, humane euthanasia of sick animals, and natural attrition; target 20% reduction by the end of 2009, eventual target 50% reduction in five years 5 of 23 Animals 2018, 8, 77 • Maintain the health of the campus cat population by regular feeding, routine vaccination and parasite management, and veterinary care where necessary • Maintain the health of the campus cat population by regular feeding, routine vaccination and parasite management, and veterinary care where necessary • Maintain the health of the campus cat population by regular feeding, routine vaccination and parasite management, and veterinary care where necessary • Educate and inform the university community about the cat management program • Educate and inform the university community about the cat management program • Facilitate ongoing oversight of the campus cat program through regular meetings with university • Educate and inform the university community about the cat management program • Facilitate ongoing oversight of the campus cat program through regular meetings with university management, and effective rapid response to emerging problems • Facilitate ongoing oversight of the campus cat program through regular meetings with t d ff ti id t i bl g g g p p g g g g y management, and effective rapid response to emerging problems management, and effective rapid response to emerging problems • Comprehensive review of program outcomes and progress towards goals at the end o Comprehensive review of program outcomes and progress towards goals at the end of 2009. Although some funding (approximately $9000 AUD) was provided by the university to support initial desexing and microchipping, the program was intended subsequently to become financially sustainable through donations and fundraising by the CCC. The TNR program achieved its initial goals set with the university by the end of December 2009, and has continued with the approval of university management over subsequent years. Campus Background This paper reports on the outcomes of the program over the nine-year period from its establishment at the end of August 2008 when a formal agreement was signed with university management, up until the most recent audit of cats at the end of September 2017. Campus Background One cat was euthanized, one kitten was rehomed from the local council pound (local government animal control facility), and two cats were released from the council pound to CCC members for rehoming. In April 2008, over a ten-day period a further 12 cats were trapped and all were euthanized. Because it was subsequently established that some of the euthanized cats were already microchipped and desexed, and also as a result of opposition from some university staff and others to this culling operation, trapping ceased late in April 2008. In June 2008, the CCC made a formal approach to the peak university WHS committee, proposing an alternative, more effective and humane approach to management of the campus cat population based on rigorous application of TNR principles. 2.2. Desexing, Feeding, Monitoring and Health Management Cats undergoing TNR were desexed at two local private veterinary clinics. At the time of desexing, all cats were vaccinated, treated for parasites, and received minor health care if needed. Identification was by microchipping and registration on a state database through the local government agency, as mandated by state law. Cats were registered as owned by Campus Cats NSW, and one of the office-bearers of the organization was named as primary carer. Daily feeding of all cats on campus was achieved through the establishment of a cat feeding roster that involved volunteer students and staff of the university and some local community volunteers associated with animal welfare agencies. Cats were routinely fed once a day at around dusk at a number of locations around the campus, discretely hidden from passersby in gardens or behind hedges, and behind or under buildings or external staircases. Cats fed at these sites ranged from approximately one to eight cats per site. Both wet canned food and dry kibble were provided, and some feeders occasionally included raw meat or raw chicken wings as a supplement. Fresh water was also always available at all feeding sites and replenished daily. Some feeding stations incorporated small feeding shelters to protect food from the elements and scavenging birds. As well as helping to maintain the health of the cats, the regular feeding routine provided an opportunity for volunteers to check cats for signs of ill health, identify new (immigrant) cats that appeared on campus, and alert CCC management if any cats were not seen for a few days so that searches for missing cats could be instigated. Further monitoring of cats was achieved later in the program through the occasional use of motion-detection trail cameras placed at feeding stations, and these were particularly useful to locate apparently missing cats that had relocated to different sites, and to monitor the return of cats to their usual feeding sites after TNR or veterinary treatment. Where indicated, veterinary care was provided for cats. In some cases, this meant that cats needed to be trapped for transport to the veterinary practice. Because of the time required to trap sometimes trap-shy individuals, and because of the stress this could cause these cats, the initiation of veterinary care was only considered once other options, such as on-site provision of medication under veterinary supervision, had been attempted. 2.1. Data Collection and Management Approximate ages of cats were recorded on the CCC database based on estimates from veterinarians at the time of desexing after the program had commenced, whereas the ages of cats that were already desexed were based on anecdote and the memories of those who had participated in campus cat care prior to August 2008. The university campus is not an isolated tract of land or an island, and it was inevitable that cats from the surrounding areas immigrated on to the campus during the course of the nine-year TNR 6 of 23 Animals 2018, 8, 77 program. Immigrant cats were typically detected by cat feeders when they appeared at cat feeding sites, and this was confirmed where possible by the use of trail cameras. In some cases, particularly when young kittens were found, these were reported to the CCC directly by members of the university staff who had noticed their presence. Data on immigrant cats that were discovered on campus during the program were gathered with as much pertinent information as possible about age, sex and desexed status. These immigrant cats were categorized as either unsocialized (based on behavioral characteristics such as extreme wariness, avoiding interaction with humans, hissing and aggressive vocalizations when approached) or socialized (approachable and able to be handled). Solitary kittens that were discovered on campus were placed in this latter category, whereas kittens born on campus were recorded separately. Unsocialized immigrant cats were absorbed permanently into the campus cat population (and were targeted for desexing), whereas socialized immigrant cats and kittens, and kittens born on campus, typically remained only temporarily on campus before transfer to other agencies for rehoming. Unsocialized (permanent) immigrants, socialized (temporary) immigrants, and kittens born on campus, were reviewed separately in this paper. Because unsocialized cats were absorbed into the campus cat population, global summaries of resident cat numbers over time included these cats as part of the population under ongoing management on the campus. 2.2. Desexing, Feeding, Monitoring and Health Management 3.1. Audit of Campus Cat Numbers over Time 3.1. Audit of Campus Cat Numbers over Time At the start of the program in August 2008, the number of cats on campus was uncertain and uncontrolled breeding made this a moving target. Records had not been kept previously on cat numbers, sex, description, age, or whether cats had been desexed or microchipped. The initial CCC estimate was between 75 and 90 campus cats at the time of preliminary negotiations with university management, but this was subsequently modified as numbers became clearer through an intensive ongoing audit of cats around the university campus by CCC members and other interested campus users. In particular, cats were observed and counted during daily feedings, which were conducted at a regular time of day to encourage cats to come to the feeding stations. On the basis of these careful observations, a total of 74 cats was thus recorded in August 2008. At the start of the program in August 2008, the number of cats on campus was uncertain and uncontrolled breeding made this a moving target. Records had not been kept previously on cat numbers, sex, description, age, or whether cats had been desexed or microchipped. The initial CCC estimate was between 75 and 90 campus cats at the time of preliminary negotiations with university management, but this was subsequently modified as numbers became clearer through an intensive ongoing audit of cats around the university campus by CCC members and other interested campus users. In particular, cats were observed and counted during daily feedings, which were conducted at a regular time of day to encourage cats to come to the feeding stations. On the basis of these careful observations, a total of 74 cats was thus recorded in August 2008. O th CCC b f ili ith d fid t b t th t l ti Once the CCC became more familiar with, and confident about, the campus cat population through the careful ongoing audit process, it became apparent that five cats (5/74; 7% of resident population) either had been double-counted (n = 3) or had been local wandering domestic cats (n = 2) at the time of the initial August 2008 population audit. The “overcount” reflects early uncertainty about cat numbers, and similar issues are likely to occur early in any TNR program. 3.1. Audit of Campus Cat Numbers over Time 3.1. Audit of Campus Cat Numbers over Time As a result, the overall baseline population count was adjusted to 69 cats in CCC records at December 2008, and for all summaries in this paper a baseline population of 69 cats was used. Once the CCC became more familiar with, and confident about, the campus cat population through the careful ongoing audit process, it became apparent that five cats (5/74; 7% of resident population) either had been double-counted (n = 3) or had been local wandering domestic cats (n = 2) at the time of the initial August 2008 population audit. The ‘overcount’ reflects early uncertainty about cat numbers, and similar issues are likely to occur early in any TNR program. As a result, the overall baseline population count was adjusted to 69 cats in CCC records at December 2008, and for all summaries in this paper a baseline population of 69 cats was used. I dditi t th i i l 69 t t t th b i i f th TNR In addition to the original 69 cats present on campus at the beginning of the TNR program, a total of 34 immigrant cats or solitary kittens were identified over the nine-year program, and 19 kittens were born on campus to resident or immigrant cats. In addition to the original 69 cats present on campus at the beginning of the TNR program, a total of 34 immigrant cats or solitary kittens were identified over the nine-year program, and 19 kittens were born on campus to resident or immigrant cats. 2.2. Desexing, Feeding, Monitoring and Health Management In more recent years, as cats became more socialized to their feeders, it became increasingly possible to place certain cats in carry cages for transport to the veterinary practice and, in some cases, for full veterinary examination to be achieved in unsedated campus cats. Usually, however, sedation was required to allow full examination. 7 of 23 7 of 23 Animals 2018, 8, 77 Animals 2018, 8, x 3.1.1. Unsocialized Immigrant Cats 3.1.1. Unsocialized Immigrant Cats Of the 34 immigrant cats, 16 cats (47%) were categorized as unsocialized based on their behavior (wariness, fear of humans, aggression when approached) and were deemed unsuitable for rehoming. These cats were consequently managed through TNR and absorbed into the campus cat resident population. Thus, the total resident cat population managed on the campus during the nine-year TNR program was 85 cats (69 originals + 16 unsocialized immigrants) (Figure 1 and Appendix A). Of the 34 immigrant cats, 16 cats (47%) were categorized as unsocialized based on their behavior (wariness, fear of humans, aggression when approached) and were deemed unsuitable for rehoming. These cats were consequently managed through TNR and absorbed into the campus cat resident population. Thus, the total resident cat population managed on the campus during the nine-year TNR program was 85 cats (69 originals + 16 unsocialized immigrants) (Figure 1 and Appendix A). 0 5 10 15 20 25 30 35 Aug-08 Sep-08 Sep-09 Sep-10 Sep-11 Sep-12 Sep-13 Sep-14 Sep-15 Sep-16 Sep-17 Cat numbers Year Male Female Unsure Figure 1. Resident campus male and female cats (including 69 original cats and 16 unsocialized immigrants) at commencement of the program (August 2008) and at annual intervals from end of September 2008 to end of September 2017. 0 5 10 15 20 25 30 35 Aug-08 Sep-08 Sep-09 Sep-10 Sep-11 Sep-12 Sep-13 Sep-14 Sep-15 Sep-16 Sep-17 Cat numbers Year Male Female Unsure Figure 1. Resident campus male and female cats (including 69 original cats and 16 unsocialized immigrants) at commencement of the program (August 2008) and at annual intervals from end of September 2008 to end of September 2017. Figure 1. Resident campus male and female cats (including 69 original cats and 16 unsocialized immigrants) at commencement of the program (August 2008) and at annual intervals from end of September 2008 to end of September 2017. Figure 1. Resident campus male and female cats (including 69 original cats and 16 unsocialized immigrants) at commencement of the program (August 2008) and at annual intervals from end of September 2008 to end of September 2017. 8 of 23 Animals 2018, 8, 77 3.1.2. Socialized Immigrant Cats and Kittens 3.1.3. Kittens Born on Campus A total of 19 kittens were born on campus during the nine-year program. Five of these kittens were born to two resident cats—the last resident kitten was born in December 2009. Both resident mothers were trapped and desexed. In addition, a total of 14 kittens were born to three newly arrived immigrant female cats. A litter of six kittens was discovered in March 2009 with a friendly adult female. In December 2014, an unsocialized immigrant female cat (seen occasionally over the previous two months) had a litter of four kittens, and again in January 2017 an unsocialized immigrant female cat (noted sporadically over the previous 2–3 months) was found with a litter of four kittens. These 14 kittens were fostered and rehomed. The socialized mother was rehomed, and both unsocialized mothers were desexed and returned to campus. 3.1.2. Socialized Immigrant Cats and Kittens In addition to 16 unsocialized immigrant cats absorbed permanently into the campus cat population, a further 18 socialized immigrant adult cats or solitary kittens were present temporarily on the campus for short periods, usually between one day and two weeks (mode 1–3 days), before their permanent removal, typically for fostering and adoption. Socialized immigrant adult cats (n = 12) were thought to either be lost or wandering owned or semi-owned cats, or have been abandoned by students or nearby residents, and were identified as socialized by their approachability and friendliness to humans. Six immigrant kittens simply appeared on campus, including two kittens found in the engine bay of a student’s car parked on the campus, three kittens found wandering by themselves on campus, and one injured kitten brought on to campus by a student. Solitary kittens were typically fostered and rehomed as soon as possible after their discovery. 3.2. Desexing of Campus Cats At the beginning of the program (August 2008), 10 (14%) of the 69 resident cats on campus were of undetermined sex (Figure 1 and Appendix A). Sex and desexed status were subsequently clarified at the time of TNR, except for one original resident that disappeared before its status could be confirmed. Based on current knowledge of the sex and desexed status of resident cats at the start of the program, there were 33 (49%) males and 35 (51%) females, of which 48% of males (16/33) and 26% of females (9/35) were entire (Figure 2). Socialized immigrant cats and kittens were not included in this summary of sexual status of campus cats, because these cats and kittens were rapidly removed from the campus and thus did not form part of the permanent resident population, and also because the majority of the kittens were of uncertain or unrecorded sex. Cats whose status remained unknown (n = 4 through the nine-year program), primarily because they disappeared before they could be trapped, were also not included. Immediately prior to, and on commencing the agreed management program in August 2008, the CCC targeted fertile females for desexing (Figure 2A). By December 2009, all but one of the 26 female cats remaining on campus had been desexed—this last fertile female proved elusive but was finally trapped and desexed in December 2010. Since then, all resident female cats on campus were desexed, and no kittens have been born to resident cats since December 2009. Male cats were also trapped and desexed from the beginning of the program, although this had a lower priority than female cats. By December 2009, 16 of the remaining 22 male cats (73%) were desexed (Figure 2B). At the most recent audit, all of the remaining male cats on campus were desexed, although the status of one long-term immigrant visitor (assumed male and desexed) has not been confirmed. Unsocialized immigrant cats that were absorbed into the program after 2008 were immediately targeted for TNR and 13 of these 16 cats were successfully desexed (four females, nine males). One male 9 of 23 Animals 2018, 8, 77 cat arrived on campus with an ear tip, and was assumed to have been previously desexed. Another undesexed male disappeared before he could be trapped for desexing, and one unsocialized immigrant visitor remains of unconfirmed status. Animals 2018, 8, x 9 of 23 Figure 2. 3.2. Desexing of Campus Cats Percentage of resident cats (including 69 originals and 16 unsocialized immigrants) desexed versus entire (undesexed) at annual intervals during the 9-year program—(A) female; (B) male. Cats of unknown status (n = 4) were not included 0 10 20 30 40 50 60 70 80 90 100 Aug-08 Sep-08 Sep-09 Sep-10 Sep-11 Sep-12 Sep-13 Sep-14 Sep-15 Sep-16 Sep-17 Percentage of cats Female desexed Female entire A 0 10 20 30 40 50 60 70 80 90 100 Aug-08 Sep-08 Sep-09 Sep-10 Sep-11 Sep-12 Sep-13 Sep-14 Sep-15 Sep-16 Sep-17 Percentage of cats Male desexed Male entire B Figure 2. Percentage of resident cats (including 69 originals and 16 unsocialized immigrants) desexed versus entire (undesexed) at annual intervals during the nine-year program—(A) female; (B) male. Cats of unknown status (n = 4) were not included. 0 10 20 30 40 50 60 70 80 90 100 Aug-08 Sep-08 Sep-09 Sep-10 Sep-11 Sep-12 Sep-13 Sep-14 Sep-15 Sep-16 Sep-17 Percentage of cats Female desexed Female entire A Female desexed Female entire 0 10 20 30 40 50 60 70 80 90 100 Aug-08 Sep-08 Sep-09 Sep-10 Sep-11 Sep-12 Sep-13 Sep-14 Sep-15 Sep-16 Sep-17 Percentage of cats Male desexed Male entire B Figure 2. Percentage of resident cats (including 69 originals and 16 unsocialized immigrants) desexed versus entire (undesexed) at annual intervals during the 9-year program—(A) female; (B) male. Cats Figure 2. Percentage of resident cats (including 69 originals and 16 unsocialized immigrants) desexed versus entire (undesexed) at annual intervals during the nine-year program—(A) female; (B) male. Cats of unknown status (n = 4) were not included. In terms of desexing, socialized adult immigrant cats (n = 12) were dealt with on a case-by-case basis. These cats were initially scanned for a microchip, and this resulted in the return of three cats (25%) to their owners. The owners of two other microchipped cats were not able to be contacted, or did not want the cat returned. Cats were then checked by a veterinary surgeon for general health, and where indicated were desexed (n = 2). All cats received vaccinations and parasite treatment, before placement in foster care prior to rehoming. All adult socialized cats managed by this route (n = 7) were successfully placed in adoptive homes. 3.2. Desexing of Campus Cats Two of the 12 socialized adult immigrant cats disappeared before their status could be verified, and may in fact have been wandering local domestic d h d h i b h In terms of desexing, socialized adult immigrant cats (n = 12) were dealt with on a case-by-case basis. These cats were initially scanned for a microchip, and this resulted in the return of three cats (25%) to their owners. The owners of two other microchipped cats were not able to be contacted, or did not want the cat returned. Cats were then checked by a veterinary surgeon for general health, and where indicated were desexed (n = 2). All cats received vaccinations and parasite treatment, before placement in foster care prior to rehoming. All adult socialized cats managed by this route (n = 7) were successfully placed in adoptive homes. Two of the 12 socialized adult immigrant cats disappeared before their status could be verified, and may in fact have been wandering local domestic owned cats that returned to their nearby homes. 10 of 23 10 of 23 Animals 2018, 8, 77 3.3. Health Management Health of cats was monitored by the cat feeders, facilitated by daily feeding and, where necessary, motion-detection trail cameras at feeding stations. The most common reasons for veterinary treatment among the campus cat population were eye infections, minor injuries, abscesses, skin conditions and dental disease. Various medications in oral form were successfully administered in food usually as crushed tablets, including antibiotics, steroids and other treatments such as medication for hyperthyroidism. 3.4. Colony Size over Time By December 2009, when the program was due for initial review, the number of cats on campus had reduced from 69 to 51 cats, which represented a 26% (n = 18/69) reduction, meeting the 20% target agreed with the university. At the 5-year time point (September 2013), there were 30 cats remaining on campus, representing a 57% (n = 39/69) reduction, again meeting the agreed 50% target. At the time of preparing this paper (September 2017), 15 cats remain on campus, representing a 78% (n = 54/69) reduction in the resident cat population since the TNR program was initiated in August 2008. Case studies of some of the campus cats are included in Appendix B. Nine of the remaining cats at September 2017 were present on campus at the start of the program in August 2008, and six were cats that immigrated on to the campus during the program. Four of these latter cats were younger than those that remained from the original cohort, most of which were now quite elderly (estimated age of original resident cats >10 years). However, two of the remaining six unsocialized immigrant cats were estimated to be older than 10 years when they first appeared on campus. An overall summary of the fates of all cats managed in the program over the period August 2008 to September 2017 is presented in Table 1. An overall summary of the fates of all cats managed in the program over the period August 2008 to September 2017 is presented in Table 1. Table 1. Summary of outcomes for all cats managed in the nine-year program. Total Rehomed Returned to Owner Euthanized Died Disappeared Remaining RESIDENT CATS Original cats 69 7 0 20 12 21 9 Unsocialized immigrant adults 16 0 0 0 2 8 6 Total n (%) 85 7 (8%) 0 (0%) 20 (24%) 14 (16%) 29 (34%) 15 (18%) NON-RESIDENT CATS Socialized immigrant adults 12 7 3 0 0 2 0 Solitary immigrant kittens 6 5 0 0 1 0 0 Kittens born on campus 19 14 0 1 0 4 0 Total n (%) 37 26 (70%) 3 (8%) 1 (3%) 1 (3%) 6 (16%) 0 (0%) OVERALL TOTAL CATS Total n (%) 122 33 (27%) 3 (2%) 21 (17%) 15 (12%) 35 (29%) 15 (12%) 3.5.1. Fates of Resident Cats Table 1. Summary of outcomes for all cats managed in the nine-year program. 3.5.1. Fates of Resident Cats 3.5.1. Fates of Resident Cats Figure 3 summarizes the fates of cats from the resident (original and unsocialized immigrant) campus cat population during the nine-year TNR program. Because many resident socialized cats on the campus had already been rehomed prior to August 2008 (in some cases to protect them from the university’s culling program), few resident campus cats were successfully rehomed after the start of the TNR program. Over the nine-year program, seven cats (7/85; 8% of resident population) were adopted, including two older cats (estimated 15 years old) that were placed in permanent homes in mid-2017. These latter adoptions became possible because of socialization of these cats through their regular daily interactions with volunteer feeders. 11 of 23 11 of 23 Animals 2018, 8, 77 Animals 2018 8 x Figure 3. Fates of resident cats (69 original and 16 unsocialized immigrant cats). 0 5 10 15 20 25 30 35 Rehomed Euthanized Died Disappeared Remaining Number of cats Figure 3. Fates of resident cats (69 original and 16 unsocialized immigrant cats). Figure 3. Fates of resident cats (69 original and 16 unsocialized immigrant cats). Figure 3. Fates of resident cats (69 original and 16 unsocialized immigrant cats). A total of 20 cats (20/85; 24% of resident population) were humanely euthanized during the nine- year program (mean 2.2 resident cats per year). The primary reason for euthanasia was severe ill health (n = 16/20, 80% of euthanized cats; Table 2). A total of 20 cats (20/85; 24% of resident population) were humanely euthanized during the nine-year program (mean 2.2 resident cats per year). The primary reason for euthanasia was severe ill health (n = 16/20, 80% of euthanized cats; Table 2). Table 2. Reasons for euthanasia, categorized by estimated age groups. Table 2. Reasons for euthanasia, categorized by estimated age groups. Table 2. Reasons for euthanasia, categorized by estimated age groups. Age Group < 5 Years * Age Group 5–10 Years * Age Group > 10 Years * Acute renal disease 1 Intestinal sarcoma 2 Renal failure 3 Tumorous cancer on chest 1 Saddle thrombus 1 Intestinal sarcoma 1 FIV positive ** 2 Feline hypertrophic cardiomyopathy 1 Complications of hyperthyroidism 1 Excessive aggression 1 Aggressive oral squamous cell carcinoma 1 Old age (estimated 20 years)—debilitation 1 Undiagnosed illness 3 Other 1 Total 9 Total 5 Total 6 Table 2. Reasons for euthanasia, categorized by estimated age groups. 3.5.1. Fates of Resident Cats Age Group <5 Years * Age Group 5–10 Years * Age Group >10 Years * Hit by car 2 Heart failure ** 1 Hit by car 1 Accident (caught in machinery) 1 Severe chronic respiratory disease ** 1 Accident (trapped in demolished building) 1 Died during surgery 1 FIV+ ***—unknown cause 1 Elderly—unknown cause 2 Multiple organ lesions ** 2 Unknown cause 1 Total 7 Total 3 Total 4 * Age at time of death, based on estimates—see text; ** based on necropsy; *** positive for feline immunodeficiency virus. which three were hit by cars on roads surrounding the campus. Based on our data on deaths and euthanasia in the resident cat population (n = 34/85), and the approximate number of cat-years represented in this dataset (n = 419 cat-years), we estimate an average mortality rate in our campus cat population (including adults and kittens) of approximately 8.1% per annum over the nine-year program. Table 3. Causes of death (other than euthanasia), categorized by estimated age groups. Age Group <5 Years * Age Group 5–10 Years * Age Group >10 Years * Hit by car 2 Heart failure ** 1 Hit by car 1 Accident (caught in machinery) 1 Severe chronic respiratory disease ** 1 Accident (trapped in demolished building) 1 Table 3. Causes of death (other than euthanasia), categorized by estimated age groups. Age Group <5 Years * Age Group 5–10 Years * Age Group >10 Years * Hit by car 2 Heart failure ** 1 Hit by car 1 Accident (caught in machinery) 1 Severe chronic respiratory disease ** 1 Accident (trapped in demolished building) 1 Died during surgery 1 FIV+ ***—unknown cause 1 Elderly—unknown cause 2 Multiple organ lesions ** 2 Unknown cause 1 Total 7 Total 3 Total 4 * Age at time of death, based on estimates—see text; ** based on necropsy; *** positive for feline immunodeficiency virus. 3.5.1. Fates of Resident Cats Age Group < 5 Years * Age Group 5–10 Years * Age Group > 10 Years * Acute renal disease 1 Intestinal sarcoma 2 Renal failure 3 Tumorous cancer on chest 1 Saddle thrombus 1 Intestinal sarcoma 1 FIV positive ** 2 Feline hypertrophic cardiomyopathy 1 Complications of hyperthyroidism 1 Excessive aggression 1 Aggressive oral squamous cell carcinoma 1 Old age (estimated 20 years)—debilitation 1 Undiagnosed illness 3 Other 1 Total 9 Total 5 Total 6 * Age at time of death, based on estimates—see text; ** positive for feline immunodeficiency virus. * Age at time of death, based on estimates—see text; ** positive for feline immunodeficiency virus. Another 14 cats (14/85; 16% of resident population; mean 1.6 resident cats per year) died or were found dead during the 9-year program (Table 3). Five of these cats met with accidental deaths, of Another 14 cats (14/85; 16% of resident population; mean 1.6 resident cats per year) died or were found dead during the nine-year program (Table 3). Five of these cats met with accidental deaths, of which three were hit by cars on roads surrounding the campus. ee were hit by cars on roads surrounding the campus. d on our data on deaths and euthanasia in the resident cat population (n = 34/85) Table 3. Causes of death (other than euthanasia), categorized by estimated age groups. e were hit by cars on roads surrounding the campus. on our data on deaths and euthanasia in the resident cat population (n = 34/85 Table 3. Causes of death (other than euthanasia), categorized by estimated age groups. which three were hit by cars on roads surrounding the campus. Based on our data on deaths and euthanasia in the resident cat population (n = 34/85), and the approximate number of cat-years represented in this dataset (n = 419 cat-years), we estimate an average mortality rate in our campus cat population (including adults and kittens) of approximately 8.1% per annum over the nine-year program. Table 3. Causes of death (other than euthanasia), categorized by estimated age groups. Age Group <5 Years * Age Group 5–10 Years * Age Group >10 Years * Hit by car 2 Heart failure ** 1 Hit by car 1 Accident (caught in machinery) 1 Severe chronic respiratory disease ** 1 Accident (trapped in demolished building) 1 Table 3. Causes of death (other than euthanasia), categorized by estimated age groups. 3.5.1. Fates of Resident Cats 0 5 10 15 20 25 30 Rehomed Returned to owner Euthanized Died Disappeared Number of cats Figure 4. Fates of socialized immigrant adult cats (n = 12), solitary kittens (n = 6) and kittens born on campus (n = 19). 3.5.2. Fates of Socialized Immigrant Cats, and Kittens Born on Campus 3.5.2. Fates of Socialized Immigrant Cats, and Kittens Born on Campus 2. Fates of Socialized Immigrant Cats, and Kittens Born on Campus Fates of Socialized Immigrant Cats, and Kittens Born on Campus Most (70%; 26/37) of the socialized immigrant adult cats (7/12), solitary kittens (5/6), and kittens born on campus (14/19) were managed rapidly through fostering followed by rehoming (Figure 4). Most (70%; 26/37) of the socialized immigrant adult cats (7/12), solitary kittens (5/6), and kittens born on campus (14/19) were managed rapidly through fostering followed by rehoming (Figure 4). Figure 4. Fates of socialized immigrant adult cats (n = 12), solitary kittens (n = 6) and kittens born on campus (n = 19). 0 5 10 15 20 25 30 Rehomed Returned to owner Euthanized Died Disappeared Number of cats Figure 4. Fates of socialized immigrant adult cats (n = 12), solitary kittens (n = 6) and kittens born on campus (n = 19). Figure 4. Fates of socialized immigrant adult cats (n = 12), solitary kittens (n = 6) and kittens born on campus (n = 19). Figure 4. Fates of socialized immigrant adult cats (n = 12), solitary kittens (n = 6) and kittens born on campus (n = 19). Five of the seven rehomed adults were removed from the campus on the same day they were discovered, one cat took a week to capture for foster care, and another cat was rehomed after almost three months on campus. A further three adult domestic cats with microchips were reunited on the day of their discovery with their owners, all of whom lived within half a kilometer of the university campus. Two socialized immigrant adult cats were briefly seen on campus on several occasions over a 3–5 months period, then disappeared. Five of the seven rehomed adults were removed from the campus on the same day they were discovered, one cat took a week to capture for foster care, and another cat was rehomed after almost three months on campus. 3.5.1. Fates of Resident Cats A further three adult domestic cats with microchips were reunited on the day of their discovery with their owners, all of whom lived within half a kilometer of the university campus. Two socialized immigrant adult cats were briefly seen on campus on several occasions over a 3–5 months period, then disappeared. Of the six solitary immigrant kittens, five were removed and placed in adoptive homes. Three were removed within three days of their discovery, whereas two took up to two weeks to capture before removal for rehoming. One kitten died within three days from severe trauma sustained prior to being brought on to campus by a concerned student. Of the six solitary immigrant kittens, five were removed and placed in adoptive homes. Three were removed within three days of their discovery, whereas two took up to two weeks to capture before removal for rehoming. One kitten died within three days from severe trauma sustained prior to being brought on to campus by a concerned student. Of the 19 kittens born on campus, five kittens were born to resident cats early in the program. Four of these kittens disappeared and are thought to have died, and one kitten was euthanized due to inoperable hernia. Of the 19 kittens born on campus, five kittens were born to resident cats early in the program. Four of these kittens disappeared and are thought to have died, and one kitten was euthanized due to inoperable hernia. The other 14 campus-born kittens (all of which were born to immigrant pregnant cats) were successfully placed in adoptive homes. Where necessary, kittens remained with their mothers on campus until weaned, and then were separated at about 4–6 weeks of age for socialization in foster care. Kittens were then transferred to a rescue/adoption agency either prior to or immediately after early age desexing (at approximately 1 kg in weight), for further socialization and rehoming. The other 14 campus-born kittens (all of which were born to immigrant pregnant cats) were successfully placed in adoptive homes. Where necessary, kittens remained with their mothers on campus until weaned, and then were separated at about 4–6 weeks of age for socialization in foster care. Kittens were then transferred to a rescue/adoption agency either prior to or immediately after early age desexing (at approximately 1 kg in weight), for further socialization and rehoming. 3.5.1. Fates of Resident Cats g g y y Multiple organ lesions ** 2 Unknown cause 1 Total 7 Total 3 Total 4 * Age at time of death, based on estimates—see text; ** based on necropsy; *** positive for feline i d fi i i Based on our data on deaths and euthanasia in the resident cat population (n = 34/85), and the approximate number of cat-years represented in this dataset (n = 419 cat-years), we estimate an average mortality rate in our campus cat population (including adults and kittens) of approximately 8.1% per annum over the nine-year program. immunodeficiency virus. A total of 29 cats (29/85; 34% of resident population; mean 3.2 animals per year) simply y p g A total of 29 cats (29/85; 34% of resident population; mean 3.2 animals per year) simply disappeared and their fates are unknown. Although some of these cats appeared to be ill or injured 12 of 23 12 of 23 Animals 2018, 8, 77 Animals 2018, 8, x prior to their disappearances (n = 4), most appeared in good health at the time they disappeared and the reasons for their disappearances remain speculative. prior to their disappearances (n = 4), most appeared in good health at the time they disappeared and the reasons for their disappearances remain speculative. prior to their disappearances (n = 4), most appeared in good health at the time they disappeared and the reasons for their disappearances remain speculative. prior to their disappearances (n = 4), most appeared in good health at the time they disappeared and the reasons for their disappearances remain speculative. p pp pp g y pp the reasons for their disappearances remain speculative. 3.5.2. Fates of Socialized Immigrant Cats, and Kittens Born on Campus Most (70%; 26/37) of the socialized immigrant adult cats (7/12), solitary kittens (5/6), and kittens born on campus (14/19) were managed rapidly through fostering followed by rehoming (Figure 4). he reasons for their disappearances remain speculative. 3.5.2. Fates of Socialized Immigrant Cats, and Kittens Born on Campus Most (70%; 26/37) of the socialized immigrant adult cats (7/12), solitary kittens (5/6), and kittens born on campus (14/19) were managed rapidly through fostering followed by rehoming (Figure 4). Figure 4. Fates of socialized immigrant adult cats (n = 12), solitary kittens (n = 6) and kittens born on campus (n = 19). 4. Discussion 4. Discussion This study reports in detail the results obtained over a nine-year cat management program conducted on the UNSW campus using a TNR protocol to manage cat numbers, combined with adoption of socialized cats and kittens, natural attrition, and active management of immigrant cats. Over the nine-year period, 122 cats in total were managed as part of the program. These comprised 69 original residents (57%), 16 unsocialized immigrants incorporated into the resident population (13%), 18 socialized immigrant adults and kittens (15%), and 19 kittens born on campus (16%). Socialized immigrant adults and kittens, and kittens born on campus, were targeted for rapid removal for rehoming or return to their owners. This left 70% of the total 122 cats (69 original residents and 16 unsocialized immigrants; total 85 cats) to be actively managed on an ongoing basis h This study reports in detail the results obtained over a nine-year cat management program conducted on the UNSW campus using a TNR protocol to manage cat numbers, combined with adoption of socialized cats and kittens, natural attrition, and active management of immigrant cats. Over the nine-year period, 122 cats in total were managed as part of the program. These comprised 69 original residents (57%), 16 unsocialized immigrants incorporated into the resident population (13%), 18 socialized immigrant adults and kittens (15%), and 19 kittens born on campus (16%). Socialized immigrant adults and kittens, and kittens born on campus, were targeted for rapid removal for rehoming or return to their owners. This left 70% of the total 122 cats (69 original residents and 16 unsocialized immigrants; total 85 cats) to be actively managed on an ongoing basis on the campus. 13 of 23 13 of 23 Animals 2018, 8, 77 The current campus cat population (at September 2017) comprises 15 cats, representing a 78% reduction in resident cat numbers from the original population of 69 cats in August 2008. Therefore, only 12% of the 122 cats managed through the course of the nine-year program remain on campus. 4.1.1. Rehoming The rehoming rate of 8% (n = 7) for the resident cat population of 85 in this program (Table 1) was low compared to previous studies of TNR [7,18,31]. Many adult socialized resident cats had been adopted into permanent homes prior to initiation of the TNR program, and this early phase of adoption is not reflected in our numbers. In comparison, Levy et al. reported that 47% of cats in their TNR program were rehomed [7]. This may reflect the much higher proportion in their population of kittens and younger cats, which typically form the majority of cats rehomed early in a TNR program, compared to our older campus population in which most of the resident female cats were already desexed. However, in our study, 83% of socialized immigrant cats and kittens, and 74% of kittens born on campus, were successfully rehomed, including three cats returned to their owner. When the overall rehoming/return-to-owner rate in our program is considered (36/122; 30%), this is comparable to other TNR programs that have documented rehoming rates from 21% to 32% [18,31] (Table 4). Approximately 8% of all immigrant cats (25% of socialized immigrants) were returned to their owners who lived within 500 m of the campus. Owner contact details from microchip registration were used to locate the owners. This wandering behavior of owned cats is consistent with GPS tracking data for South Australian pet cats, where the average daytime range was 1 hectare, and 2 hectares at night [32]. It is also consistent with research demonstrating that 75% of lost cats when found were located within 500 m of their home [33]. 4.1. Reduction in Cat Population over Time For the 122 cats managed during the program, the overall reduction in cat numbers over nine years resulted from rehoming/return to owner (36; 30%), euthanasia (21; 17%), death (15; 12%), and unexplained disappearance (35; 29%) (Table 1). Although comparisons of these statistics with other studies of TNR in similar populations are difficult because of differences in duration and methodology of study, and ambiguous data recording, an attempt to compare outcome data is presented in Table 4. Table 4. Comparisons of outcome data from similar TNR studies. Table 4. Comparisons of outcome data from similar TNR studies. p Study (1◦Author, Ref) This Study Levy [7] Spehar [18] Zaunbrecher [14] Hughes [31] Centonze [6] * Duration (years) 9 11 9 3 2 1.5 ** Original cohort (n) 69 155 195 41 158 920 Immigration (n) 34 NR NR 6 NR (103) Born on site (n) 19 NR NR NR NR (498) Total managed (n) 122 155 195 47 158 920 Remaining (n) 15 23 44 36 95 678 Remaining (%) 12 15 23 77 60 74 Rehomed *** (%) 30 47 32 0 21 (26) Euthanasia (%) 17 11 3 2 15 (0) Death (%) 12 6 7 11 4 (16) Disappeared (%) 29 21 34 11 0 (16) Other (%) 0 0 2 0 0 (0) NR = not reported. * ambiguous numbers (note that numbers in brackets from the Centonze and Levy paper [6] are unreliable by authors’ admission). ** median—range from two weeks to 15 years. *** includes “returned to owner”. NR = not reported. * ambiguous numbers (note that numbers in brackets from the Centonze and Levy paper [6] are unreliable by authors’ admission). ** median—range from two weeks to 15 years. *** includes “returned to owner”. 4.1.2. Euthanasia and Death The overall euthanasia rate amongst the resident cat population was 24% (20/85) over a nine-year period, or 17% (21/122) for the total cat population (Table 1). These cases were primarily due to severe and terminal illnesses in the original adult resident cat population (Table 2). There was a higher rate of euthanasia earlier in the program. Overall, one-third of cases of euthanasia occurred during the 14 of 23 Animals 2018, 8, 77 first 18 months of the program, primarily involving younger cats. Later cases of euthanasia related to terminal but relatively common cat diseases such as renal disease and neoplasia, involving older cats. The overall euthanasia rate is higher than Levy’s study [7] of 11% over 11 years, but differences may reflect that Levy’s study population comprised younger cats compared to our older campus cat population. Our euthanasia rate is also significantly higher than the 3% reported by Spehar and Wolf [18], but comparable to other reports [31]. This variability in euthanasia rates may reflect differences in program duration, and methodological and demographic differences between studies. It could also be argued that the close monitoring of our campus population meant that sick cats were identified quickly and euthanized following terminal diagnosis, rather than disappearing or being found dead later due to their illness. Furthermore, in most calculations of overall euthanasia rate, rehomed cats are assumed to remain alive, further confounding comparisons between studies with different rehoming rates. Our philosophy relating to euthanasia developed during the course of the program as a result of early experiences. For example, early in the program, some younger cats were euthanized because they were found to be FIV-positive, but this practice subsequently ceased as the CCC became more knowledgeable about this condition. Specifically, the risk of false positive test results, the high cost of testing, and the low incidence and late occurrence of clinical disease were felt to argue against testing and euthanasia. Furthermore, advice from other organizations such as Alley Cat Allies [34] suggested that a better way to limit FIV in the campus population was through desexing of male cats to reduce fighting and bite wounds, the main route of transmission of this virus. A further 16% of the resident cats, or 12% of the overall cat population in our program, died or were found dead (Tables 1 and 3). 4.1.2. Euthanasia and Death Reported percentages of cats found dead in other TNR studies vary from 4% to 16% (Table 4), but differences in study duration and methodology make comparisons difficult. Rehoming of cats may also confound these estimates as these cats are effectively lost to follow up and are assumed to remain alive in calculations of overall death rates. The average mortality rate based on deaths and euthanasia in our campus cat population (including adults and kittens) of approximately 8.1% per annum is consistent with the calculated death rate for pet cats of 7% per annum, based on the reported average age at death of 14 years [35]. Our rates of euthanasia and death are also comparable with those that have been reported in pet cat populations; for example, the estimated annual pet cat mortality rate was 8.3% based on a telephone survey of North American cat owners [36]. The main causes for death or euthanasia in our population, where this could be determined with reasonable confidence, included trauma, neoplasia and renal disease—the latter two causes were predominant in cats over five years of age (Tables 2 and 3). Because of the campus-based rather than veterinary practice-based nature of this program, it is not surprising that some deaths were of unknown cause or for unspecified reasons. Studies in several countries have reported the most common diseases leading to death or euthanasia in pet cats. Cause of death/euthanasia in cats younger than five years are predominantly due to trauma or viral and respiratory disease, whereas death/euthanasia in older cats, such as our cohort, are mainly related to renal disease, neoplasia, cardiovascular disorders, or non-specific illness [37,38]. Thus, our findings of rate and causes of cat mortality in our unowned urban cat population managed with TNR differ little from those commonly cited in relation to owned cat populations. This is consistent with previous findings that TNR programs may be associated with comparable or even increased longevity compared with owned cats [39], and that unowned urban cats in TNR programs show similar levels of health and profiles of disease as owned pet cats [40,41]. 4.1.4. Summary of Reasons for Reduction in Cat Population 4.1.4. Summary of Reasons for Reduction in Cat Population In summary, in common with other studies of TNR in targeted populations, the main reasons for reductions in the campus cat population in our program were rehoming, death, euthanasia and disappearance. Although concerns relating to the welfare of released cats are often cited by opponents of TNR programs, the death, euthanasia and disappearance rates in our campus cat population were similar to those reported for pet cats. Active identification and management of immigrant cats also played an essential role in control of the campus population. Immigrant cats (n = 34) represented a potential increase of 50% on the original resident population of 69 cats during the nine-year program and, if not appropriately managed, could have contributed significantly to population growth by approximately 4% per year. The birth of kittens to immigrant cats further increased the potential for population growth. At the same time, it must be acknowledged that our program reduced overall cat numbers (see Figure 1 and Appendix A), potentially creating the circumstances in which a “vacuum effect” may have encouraged the influx of immigrant cats. We cannot know what would have happened had the resident population remained stable and resources to support the cat population had remained constant. Nevertheless, failure of some TNR programs to control cat populations because of uncontrolled immigration of sexually intact animals from surrounding areas, and the dumping of unwanted cats or kittens, has been reported by others [29,30]. Furthermore, our program did not have the capacity to address management of the source of immigrant cats through community desexing programs for owned cats and promotion of responsible pet ownership in the areas surrounding the campus. The potential impact of such strategies on immigration of owned cats on to the campus is thus unclear. Nevertheless, a critical factor responsible for the gradual reduction in resident cat numbers during the nine-year program was the rigorous application of desexing to limit cat reproduction, which otherwise may have maintained or even increased the cat population. Cats have a large reproductive potential. A single queen may produce an average of ten kittens per year, and female kittens can become pregnant at four months of age [43,44]. Failure to desex all (or nearly all) female cats in a colony is one of the reasons that some TNR programs have failed to reduce cat numbers [28,29]. 4.1.3. Disappearance The most common cause for loss of cats in our study population of 122 cats was unexplained disappearance, accounting for 29% of cats and representing a mean of 3.9 disappearances per year across the overall population (Table 1). Reported disappearance rates from similar TNR studies 15 of 23 Animals 2018, 8, 77 range from 11% to 34% (Table 4), but as discussed previously, these comparisons are confounded by differences in study duration and methodology [6,7,14,18,31]. Furthermore, these statistics assume that adopted cats do not disappear after rehoming. However, 15% of pet cat owners lose their cat at least once in a 5-year period, and 30% of pet cats go missing at least once in their lifetime [42]. The fates of cats in our program that disappeared are unknown. Likely reasons include relocation (emigration), adoption, or death (although the majority were apparently healthy at the time of disappearance). Because some cats were becoming increasingly socialized due to their regular interactions with campus cat feeding volunteers, it is certainly possible that some of these more socialized cats were simply adopted informally by students, staff or campus visitors without the knowledge of the CCC team. Others may have left the campus to join nearby urban cat colonies at the racing stables or local shopping strip, possibly in response to competitive behavior from fellow campus cats, or because they were undesexed males looking for mates. In our study, the total number of cats that disappeared (or emigrated, 35) almost exactly balanced the number of cats that appeared (immigrated, 34). This is in contrast to the findings from a one-year Israeli study that compared numbers in desexed and entire colonies of unowned urban cats. In that study, despite high desexing rates for females in the desexed colonies, overall numbers increased compared to the entire cat colonies. Sexually intact cats more readily immigrated into the desexed colonies, whereas desexed cats were less likely to emigrate [29]. 4.1.4. Summary of Reasons for Reduction in Cat Population In our campus population, 95% of females were desexed within the first 15 months of the program, and no kittens were born to resident cats thereafter. However, despite this early application of TNR, three pregnant females immigrated on to campus and gave birth to kittens, highlighting the ongoing need to monitor immigrants, manage new kittens born, and desex immigrant cats as quickly as possible. 16 of 23 Animals 2018, 8, 77 Without active management of immigrant cats, combined with effective suppression of reproduction through TNR, it is likely that the campus cat population would have increased, as was apparent before initiation of the TNR program in August 2008. This emphasizes our view that effective TNR management cannot occur as a “one-off” intervention, but necessitates ongoing management of colonies to minimize the impacts of immigration. It should also be noted that ongoing management is also essential when culling is used to reduce cat populations, because the birth of new kittens and immigration into the culling area due to a “vacuum” effect will act to increase the cat population [2,12–14]. 4.2. Health Management and Maintenance One of the key elements of the university-approved TNR trial reported here was the maintenance of health of the cat population through daily feeding and regular monitoring of cat welfare and health status. Under the original university agreement, veterinary care was also to be provided where this was indicated. Best practice for daily feeding of unowned urban cats usually involves the removal of any left-over food after about 30 min, to reduce attraction of vermin and also to minimize complaints [1]. Because we relied heavily on student volunteers to place food for our resident cats, this approach was not logistically feasible because of students’ other commitments. Furthermore, because of the high volume of campus foot traffic at the end of each day as students left classes, cats frequently remained hidden until later in the evening, after student volunteer feeders had left campus themselves. Our approach therefore was to prescribe carefully the amount of food left per cat, and in recent years premeasured bags of dry kibble were supplied for student feeders to emphasize limitations on the amounts of food provided per cat. The most common reasons for veterinary treatment among the campus cat population such as dental disease, eye infections, minor injuries, abscesses, and skin conditions, differed little from the spectrum of minor illnesses seen in the domestic cat population. In our experience, tablet medications including antibiotics, steroids and even treatment for hyperthyroidism (maintained over a 12-month period) could be successfully administered crushed in the food of the affected cats. Various strategies to ensure that medications were targeted to the cats receiving treatment were adopted, including staged and separate feeding to prevent other cats in the colony from accessing the medication. 4.3. Arguments for and against TNR Management of Cats on Campus 4.3. Arguments for and against TNR Management of Cats on Campus 4.3. Arguments for and against TNR Management of Cats on Campus The two major arguments against maintaining a population of cats on the campus are the potential threat to campus wildlife, and the potential spread of disease from cats to humans. The UNSW campus is an urban campus surrounded by residential housing, other institutions, a shopping precinct and a racecourse. The campus environment could be described as very highly disturbed, with many buildings, paved and concreted areas to expedite student traffic, some large spaces of lawn for recreation, and some garden plantings. The wildlife on the campus consists of some native and introduced bird life, mammals such as rodents (in particular non-native rats), possums and fruit bats, and some populations of small lizards and frogs, although we are not aware of any census of campus wildlife. It is beyond the scope of this study to estimate with any certainty the potential impact of the campus cats on these animal populations. Nevertheless, an argument can be made that by reducing the resident cat population through TNR, and through regular feeding of resident cats, the impact of predation by campus cats is likely to be substantially mitigated. Although it is clear that feral cat populations have impacted on native wildlife in rural and wild areas of Australia [15], there is no published study that establishes that cats have a significant negative impact on native wildlife populations in an Australian urban environment. A study in Perth, Western Australia [45], found that medium-sized native mammals such as possums and bandicoots were not affected by the unregulated presence of cats compared to areas where cats were prohibited or kept indoors at night. The small mardo Antechinus flavipes, which is considered to be highly susceptible to 17 of 23 Animals 2018, 8, 77 cat predation, was actually found in higher numbers in areas where cats were unregulated. Another Perth study [46] concluded that urbanization with associated habitat degradation had a significant negative influence on passerine bird populations, whereas there was no measurable effect of cats. In a study of 24 forest patches in the Sydney metropolitan area, black rats, possums and other birds were found to be the main predators of bird nests, and nest predation reduced when cats were present [47]. 4.3. Arguments for and against TNR Management of Cats on Campus In an Australian study, prey items caught by pet cats were, in decreasing order, mice, rats, small lizards, and then common species of birds [48]. These studies indicate that our population of campus cats probably had minimal effect on the population of native campus wildlife. Opponents of TNR typically raise concerns about released cats killing wildlife, but there is little recognition or acknowledgement that killing healthy cats and kittens damages the health of the humans involved. Research in Australia and elsewhere has found that many shelter staff directly involved with euthanasia of animals develop perpetration-induced post-traumatic stress, and other shelter staff frequently develop compassion fatigue [22–24,49]. These conditions damage mental health and can lead to depression and increase the risk of suicide. In the US, the suicide rate for animal rescue workers is amongst the highest in American workers, with a rate equal to firefighters and police officers [50]. These adverse effects on human mental health deserve to be considered when choosing urban cat management strategies, because traditional complaint-based programs involving trapping and killing cats, as occurred initially on the UNSW campus, result in more cats and kittens entering shelters and their resultant euthanasia compared to TNR programs [25]. A number of diseases are spread from cats to humans, but most are spread by direct contact or by fleas, and therefore pet cats pose a greater risk to humans [51]. Toxoplasmosis is known to be carried by some cats, and can cause disease in humans, wildlife and cats, but is not spread by direct contact [52]. There is evidence that, compared to trap and cull management programs, TNR may reduce environmental contamination by toxoplasmosis oocysts, thus mitigating risks of transmission to other species. In trap and cull programs, new susceptible kittens are in higher proportions due to unchecked reproduction, and these immunologically naïve kittens are more likely to become infected by the Toxoplasma parasite and to shed oocysts in the environment [53,54]. On the other hand, TNR programs, which achieve high desexing rates and actively rehome young kittens, result over time in a population of more mature cats that have a higher probability of being immune to toxoplasmosis and are not shedding oocysts. Furthermore, adult cats that are not yet immune shed fewer cysts than younger cats when infected with Toxoplasma [52,54]. The reduction in overall numbers of free-living cats through TNR also minimizes risks of Toxoplasma infection [55]. 4.3. Arguments for and against TNR Management of Cats on Campus Nevertheless, the CCC was conscious of the potential for disease transmission to our volunteer feeders. Although infection occurs through ingestion of oocysts which only become infectious several days after being shed in cat feces, to mitigate the potential risk of toxoplasmosis infection during management of campus cats we recommended the use of plastic gloves when feeding or handling the cats, and emphasized the importance of personal hygiene after engaging in feeding activities. We also discussed this potential risk with university management, so that they could appropriately inform and instruct ground staff who may work in areas frequented by the cats. 4.5. Institutional Attitudes University management was initially skeptical about the potential of a TNR program to successfully manage and reduce cat numbers, but the rapid reduction in the resident cat population and the significantly reduced level of complaints from the campus community, with no formal complaints recorded after 2009, quickly changed institutional attitudes towards the program. This was enhanced by eliminating costs sustained by the university in managing the resident cats, such as costs associated with previous culling of cats, and with complaint management and resolution. Furthermore, the ongoing direct costs relating to feeding, veterinary care and desexing were borne by the animal welfare charity and volunteers. Regular meetings between CCC and university management expedited increased cooperation. There was a noticeable difference in practical support from university divisions such as facilities/estate management, security and grounds/landscaping teams, as a result of the clear and apparent improvement in the management of the campus cat population, the improved health of the resident cats, and the manifest reduction in numbers. These advantages of TNR programs are also likely to be apparent in community-based TNR programs to manage unowned urban cats. Much of the cost to local councils and governments of trapping and holding cats for minimum periods mandated by Australian state legislation, prior to adoption or killing, can be minimized not only through reduction in the unowned cat population through TNR, but also because of volunteer support and input from local community groups [1]. Furthermore, based on our campus experience, a significantly reduced level of complaints and enhanced local community support for more humane management strategies for unowned urban cats are likely to result from the application of TNR principles. 4.4. Financial Support An estimate of the annual costs associated with this TNR program is not possible because records of expenditure were not kept until later years of the program, and because a majority of the costs associated with the program, in particular food, were met by informal out-of-pocket contributions from feeders and other members of the CCC. Food for the campus cats was supplied as a donation by those feeders who were in full-time or part-time employment, whereas food was supplied on a complimentary basis by the CCC to enrolled university student feeders, from a central campus location. This latter food was funded through donations. Similarly, all veterinary care including desexing, examination and treatment of ill cats, and costs of euthanasia, was funded through donations, supplemented by generous discounts from local veterinary 18 of 23 Animals 2018, 8, 77 practices. Early in the program, most veterinary costs were related to desexing, whereas later in the program most of these costs were associated with health maintenance and management of an aging cat population. In the event of substantial individual veterinary care bills, email appeals to CCC supporters were usually successful in raising the necessary funding to cover costs. Although the university initially provided some limited funds (approximately $9000 AUD) for cat desexing at the start of the program, the CCC was subsequently self-supporting as a registered animal welfare charity since 2009, through regular donations from supporters and fundraising activities. 4.6. Limitations This study has a number of limitations, as would be expected from a retrospective study. The TNR program on the university campus was not initiated as a formal scientific study, but to provide humane management of a free-living unowned urban cat population. At the outset of the program, there was no intention that program outcomes might be formally reported in this paper. No scientific protocol was developed before commencing the program in terms of cat management, nor was there a predetermined schedule of checking or trapping the cats. Management of the cats was developed informally by a small group of untrained cat lovers and citizen scientists, rather than by veterinary scientists. Although the enumeration of cats on the campus during the nine-year program was carried out with diligence and care, it is possible that some cats, particularly if present only transiently on campus, may not have been recorded. No clear data on age of cats was available, because of the absence of records before initiation of the TNR program in August 2008. Records of costs of the TNR program were not maintained, and in any case many of the costs were “hidden”, having been met informally by donations from members of the CCC and other supporters of the program. In-kind contributions, in particular discounts from veterinary practices, were also not readily quantifiable. These factors precluded comparisons with the costs that might have been incurred if a trap and cull approach, as initially pursued by the university, had been continued. The applicability of our findings outside the limited and defined range of a university campus or similar site is unclear. Immigration might constitute a larger component where colonies are 19 of 23 Animals 2018, 8, 77 immediately adjacent to houses with free-roaming owned cats, given the limited range of most pet cats [32]. Furthermore, in common with many previous reports of TNR programs, no control group was included to document and compare changes in cat numbers in an unmanaged population. Further prospective research is therefore recommended to confirm outcomes of this TNR program. 5. Conclusions In summary, a nine-year cat management program conducted on the UNSW Kensington university campus in Sydney, Australia, successfully reduced the campus cat population through a combination of TNR and rehoming. At the beginning of the program, there were 69 resident cats. A further 34 immigrant cats (16 unsocialized and 18 socialized), and 19 kittens born on campus, were documented on the campus during the program. At the time of reporting (end of September 2017), the campus has a resident population of 15 cats. All current resident cats are desexed, clinically healthy and managed on a daily basis by volunteer feeders. Many of these cats are now elderly (>10 years of age), and it is anticipated that resident cat numbers will decline over the next few years due to natural attrition. At the same time, an inevitable continuing influx of immigrant cats is expected to supplement current population levels, necessitating continuing active management through ongoing TNR and rehoming. Author Contributions: H.S. collected and summarized the data presented in the paper, and wrote the paper. J.R. contributed substantially to the preparation and writing of the paper. Funding: No external funding was provided for the study presented here. A minimal contribution to support the initiation of the TNR program ($9000 AUD) was received from the University of New South Wales, but otherwise the program was supported by donations to Campus Cats NSW. Acknowledgments: The authors sincerely acknowledge the significant practical support for the UNSW TNR program from Colleen Ringe, Lisa Lifman and Beverley Lawless, and from the many other volunteer supporters and cat feeders. The veterinary support of Struggletown Veterinary Hospital and Maroubra Junction Veterinary Clinic is also gratefully acknowledged. The contributions of Maddie's Fund®, Found Animals, and The Humane Society of the United States (HSUS) towards processing charges for this paper are gratefully acknowledged. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. Appendix A Appendix A Table A1. Numbers of cats on campus at commencement of the program in August 2008, and in three-monthly steps from September 2008 to September 2017, stratified by sex and desexed status. Table A1. Numbers of cats on campus at commencement of the program in August 2008, and in three-monthly steps from September 2008 to September 2017, stratified by sex and desexed status. Table A1. Numbers of cats on campus at commencement of the program in August 2008, and in three-monthly steps from September 2008 to September 2017, stratified by sex and desexed status. End of Month Male Total Male Desexed Male Entire Female Total Female Desexed Female Entire Sex/Status Uncertain Total Cats August 2008 27 15 12 32 24 8 10 69 September 2008 30 17 13 30 24 6 6 66 December 2008 31 18 13 27 24 3 3 61 March 2009 28 19 9 26 25 1 3 57 June 2009 26 17 9 26 25 1 3 55 September 2009 23 16 7 26 25 1 3 52 December 2009 22 16 6 26 25 1 3 51 March 2010 19 15 4 25 24 1 3 47 June 2010 19 15 4 25 24 1 3 47 September 2010 19 16 3 24 23 1 2 45 December 2010 19 16 3 24 24 0 2 45 March 2011 17 15 2 21 21 0 2 40 June 2011 16 14 2 20 20 0 2 38 September 2011 15 13 2 18 18 0 2 35 December 2011 16 13 3 18 18 0 2 36 March 2012 16 13 3 18 18 0 2 36 June 2012 15 12 3 18 18 0 2 35 September 2012 15 13 2 17 17 0 2 34 December 2012 15 13 2 14 14 0 2 31 March 2013 13 11 2 13 13 0 2 28 June 2013 12 10 2 13 13 0 2 27 20 of 23 Animals 2018, 8, 77 Table A1. Cont. 1. Tan, K.; Rand, J.; Morton, J. Trap-neuter-return activities in urban stray cat colonies in Australia. Animals 2017, 7, 46. [CrossRef] [PubMed] 2. Doherty, T.S.; Dickman, C.R.; Johnson, C.N.; Legge, S.M.; Ritchie, E.G.; Woinarski, J.C.Z. Impacts and management of feral cats Felis catus in Australia. Mamm. Rev. 2017, 47, 83–97. [CrossRef] Appendix B Case studies: The following case studies are included to illustrate the variety of campus cat histories. Pearl was one of the original campus cats at the start of the TNR program. She is a ginger desexed female cat, born in approximately 2002. Over time, she became socialized due to regular contact with her feeders, and enjoyed increasing interactions with students and staff on campus. She appeared regularly in Facebook posts around the world as a result of her friendliness and approachability, and was featured by Alley Cat Allies in one of their annual Feral Cat Day programs. In July 2017, at an estimated age of 15 years, Pearl was successfully rehomed to an indoor living situation with two other rescue cats, and has settled well into this new domestic environment. Basil, an undesexed male black cat, appeared on campus in December 2014, but was initially extremely wary of humans so was categorized as an unsocialized immigrant. In January 2016, he was eventually trapped, desexed and returned to his campus home. During surgery, he was found to be microchipped, and his original owner was contacted. She had given him away as a kitten in 2010, and had lost track of his whereabouts. She did not want him returned to her, but has continued to financially support his on-campus care. Soon after desexing, Basil began to interact more with his feeders, and has become well-socialized to certain carers. Kisa was a young undesexed female tabby when the TNR program began, and was a regular breeding cat. In December 2008, she was trapped in advanced pregnancy, and gave birth to five kittens in an off-campus foster placement in January 2009. Because of her unsocialized nature, she was desexed soon after the birth of her kittens and was returned to campus, and the kittens were successfully adopted. Five years later in 2014, Kisa inexplicably disappeared from campus. Early in 2017, the CCC was contacted by a suburban veterinary practice, which advised that Kisa had been brought into the practice, several kilometers distant, after having been hit by a car. She had been identified from her microchip. Unfortunately, she required euthanasia because of the extent of her injuries. We have no explanation why she was found so far from her original campus home, or how she survived during the 3 years following her disappearance from campus. 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The impact of an integrated program of return-to-field and targeted trap-neuter-return on feline intake and euthanasia at a municipal animal shelter. Animals 2018, 8, 55. [CrossRef] [PubMed] 25. Spehar, D.D.; Wolf, P.J. The impact of an integrated program of return-to-field and targeted trap-neuter-return on feline intake and euthanasia at a municipal animal shelter. Animals 2018, 8, 55. [CrossRef] [PubMed] p , , [ ] [ ] 26. Levy, J.K.; Isaza, N.M.; Scott, K.C. Effect of high-impact targeted trap-neuter-return and adoption of community cats on cat intake to a shelter. Vet. J. 2014, 21, 269–274. [CrossRef] [PubMed] 26. Levy, J.K.; Isaza, N.M.; Scott, K.C. Effect of high-impact targeted trap-neuter-return and adoption of community cats on cat intake to a shelter. Vet. J. 2014, 21, 269–274. [CrossRef] [PubMed] 22 of 23 Animals 2018, 8, 77 27. Boone, J.D.; Slater, M. A Generalized Population Monitoring Program to Inform the Management of Free-Roaming Cats. 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Roetman, P.; Tindle, H.; Litchfield, C.; Chiera, B.; Quinton, G.; Kikillus, H.; Bruce, D.; Kays, R. References Cat Tracker South Australia: Understanding Pet Cats through Citizen Science; Discovery Circle Initiative, University of South Australia: Adelaide, Australia, 2017. 33. Huang, L.; Coradini, M.; Rand, J.; Morton, J.; Albrecht, K.; Wasson, B.; Robertson, D. Search methods used to locate missing cats and locations where missing cats are found. Animals 2018, 8, 5. [CrossRef] [PubMed] 34. Alley Cat Allies. Feline Immunodeficiency Virus. Available online: https://www.alleycat.org/resources/ feline-immunodeficiency-virus-fiv/ (accessed on 16 May 2018). 35. O’Neill, D.G.; Church, D.B.; McGreevy, P.D.; Thomson, P.C.; Brodbelt, D.C. Longevity and mortality of cats attending primary care veterinary practices in England. J. Feline Med. Surg. 2015, 17, 125–133. [CrossRef] [PubMed] 36. New, J.C., Jr.; Kelch, W.J.; Hutchinson, J.M.; Salman, M.D.; King, M.; Scarlett, J.M.; Kass, P.H. 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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 [CrossRef] 46. Grayson, J.; Calver, M.; Lymbery, A. Species richness and community composition of passerine birds in suburban Perth: Is predation by pet cats the most important factor? In Pest or Guest: The Zoology of Overabundance; Lunney, D., Eby, P., Hutchings, P., Burgin, S., Eds.; Royal Zoological Society of New South Wales: Mosman, Australia, 2007; pp. 195–207. 47. Matthews, A.; Dickman, C.R.; Major, R.E. The influence of fragment size and edge on nest predation in urban bushland. Ecography 1999, 22, 349–356. [CrossRef] 48. Franklin, M.; Rand, J.; Marston, L.; Morton, J. Prey captured by owned cats and dogs in Australia. Animals 2018, submitted for publication. 23 of 23 Animals 2018, 8, 77 49. Anderson, K.A.; Brandt, J.C.; Lord, L.K.; Miles, E.A. 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Duration of immunity of shedding of Toxoplasma gondii oocysts by cats. J. Parasitol. 1995, 81, 410–415. [CrossRef] [PubMed] 55. Torrey, E.F.; Yolken, R.H. Toxoplasma oocysts as a public health problem. Trends Parasitol. 2013, 29, 380–384. [CrossRef] [PubMed] © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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The Structure of Podocarpus spinulosus, (Smith) R. Br.
Annals of botany
1,910
public-domain
7,327
The Structure of Podocarpus spinulosus, (Smith) R. Br. The Structure of Podocarpus spinulosus, (Smith) R. Br. BY F. T. BROOKS, M.A. Senior Demonstrator of Botany, Cambridge University. BY F. T. BROOKS, M.A. Senior Demonstrator of Botany, Cambridge University. AND WALTER STILES, B.A. A ssistant- Lecturer in Botany in the University of Leeds. A ssistant- Lecturer in Botany in the University of Leeds. [Annals of Botany, Vol. XXIV. No. XCIV. April, 1910.] Introduction. JL HE following account of the structure of Podocarpus is based mainly JL upon material of Podocarpus spinulosus , (Smith) R. Br., which was obtained from the Botanic Gardens, Sydney, New South Wales. The material was sent to Professor Seward by Mr. Maiden, and was handed over to us for investigation. Material of Podocarpus alpimts obtained from the Royal Gardens, Kew, and of one or two other species of which only a limited stock was available, were examined at the same time for the sake of comparison. Pilger (13) in his monograph of the Taxaceae places Podocarptts and four other genera — Pherosphaera , Microcachrys , Saxegothaea , Dacrydium — in the sub-family Podocarpoideae, the other sub-families of the group being the Phyllocladoideae, containing the single genus Phyllocladtts , and the Taxoideae, comprising Taxus , Torreya , Cephalotaxus . Pilger evidently then considers the Podocarpoideae to be very closely related to the Taxoideae. Later work, however, would appear to necessitate a modifica¬ tion of this view. The systematic position of P odocarpus and closely allied genera is discussed in the latter part of this paper. The genus Podocarpus , as described in Pilger’ s monograph, contains no less than sixty-three species, the large majority of which are found in the Southern Hemisphere. New Zealand, Australia, and the Malayan islands are at present the home par excellence of the genus, while smaller centres of distribution are such different regions as East Africa (Sao Thome), Cape Colony and Natal, Assam, Japan, China, Central America, West Indies, and Chili. Where the genus is present within the tropics it is generally [Annals of Botany, Vol. XXIV. No. XCIV. April, 1910.] and Stiles. — The Structure of 306 found on mountain ranges at a considerable elevation. Just as Pinus may be considered the dominant Coniferous genus of the Northern Hemisphere, so Podocarpus occupies a similar position among the Conifers of the Southern regions. In regard to the distribution of the genus in geological times, most of the specimens referred to Podocarpus or closely allied genera have been determined from leaves only, and it is well known that leaf determinations are frequently untrustworthy. In this connexion mention may be made of the striking similarity in external features between the leaves of some species of Agathis and those of the Nageia section of Podocarpus . Introduction. Seward and Ford (16) found, on cutting sections of a species originally referred to Dammar a Motleyi , that the plant so named is really a species of Podocarpus. Assuming that some at least of the specimens are correctly deter¬ mined, then Podocarpus , like Araucaria, must have been widely distributed in the North temperate zone during the later Mesozoic and Tertiary times. Pilger states that the leaves from the Potomac or later Mesozoic flora of the United States are to be referred to the Nageia section of the genus on account of the plurinerved form of venation, while leaves from the Eocene and Miocene strata of Europe belong to the uninerved sections of the genus. Podocarpus shows a considerable range of habit among its many species. Several of them are large trees ; e. g. P. amarus attains a height of sixty metres in Java, and P. dacrydioides forms practically pure forests in New Zealand. On the other hand, P. alpinus , found on mountains in Tasmania, is a low shrub, and P. nivalis , on exposed places in the alpine regions of New Zealand, becomes a dwarf shrub with prostrate branches. Stem. The structure of the wood of P. spimdosus is very similar to that of P. macrophyllus described by Penhallow. Resin canals are absent in the wood, but there is a ring of them present outside the phloem. Their place in the wood is taken by resin cells, which are present in considerable numbers. The bordered pits of the secondary tracheides are uniseriate ; no trace of the Araucarian type of pitting could be discerned, such as has been described by Gothan (8) for Dacrydium spp. and for Saxegothaea by Beust (3), Gothan (8), and Stiles (17). The medullary rays are invariably uniseriate, and are from one to three cells deep. The cells of the rays contiguous to the pith frequently contain resinous material, but apart from this all the cells of the rays are alike, there being no tracheidal cells such as are met with in the rays of Pinus and some other Conifers. There is a small central pith which consists mainly of resin cells and groups of stone cells ; the latter are very conspicuous. Resin cells are also extremely abundant in the cortex. Podocarpus spinulosus , (Smith) R. 307 Reproductive Organs. With regard to the reproductive organs Podocarpus is generally dioecious, though very rarely monoecious. This distribution of the sexes is the same as that in Dacrydium and in Microcachrys , but Saxegothaea is monoecious. The reproductive organs in Taxus are also dioecious, and in Araucaria and Agathis they are generally dioecious. Leaf. The leaves of P. spinulosus , as indeed is the case in the large majority of the species, are arranged spirally on the stem axis. It is interesting to note, however, that in the Nageia section of the genus the leaves have a decussate or subdecussate arrangement. The leaf of P. spinulosus is about 4-6 cm. long by 3-4 mm. broad, and ends in an acute point. The anatomy of the leaf of P. spinulosus is very similar to that of P. chilinus described by Worsdell (29), and that of P. neriifolius and P. falcaUts examined by Bernard (1). As in all the narrow-leaved species of the genus, there is a single vascular bundle ; this is accompanied on its phloem side by a resin canal. Placed laterally to the vascular bundle is the transfusion tissue, which is extremely well developed. The transfusion tracheides show numerous reticulations on their walls when the leaf is cut at right angles to the midrib. On the side of the transfusion tissue remote from the bundle, there is a layer of parenchymatous cells, beyond which is the ‘ accessory transfusion tissue’, such as is described by Worsdell for P. chilinus . This tissue runs outwards almost to the margin of the leaf. This ‘ accessory transfusion tissue ’ of Worsdell corresponds to the ‘ hydrostereome transversal ’ of Bernard. The cells composing this tissue are long and lignified, and, as Worsdell points out, they have the appearance of stone cells rather than of tracheides. Their function presumably is primarily that of strengthening the lamina of the leaf. and Stiles . — The Structure of and Stiles . — The Structure of The axis of the cone, as seen in transverse section, contains a ring of collateral vascular bundles of the usual structure. No trace of centripetal xylem could be found, such as is present in the axis of the male cone of Saxegothaea (17). Each vascular bundle is accompanied by a single resin / canal outside the phloem, but it seems to be the general rule that these resin canals are not functional, for in most of them no distinct epithelial layer could be seen. On the other hand, resin cells are frequent throughout the fundamental tissue. A single vascular bundle, consisting of a few tracheides and phloem elements, passes out to each sporophyll. It arises by division of one of the axial bundles, the plane of division being radial ; the two derived bundles lie side by side before one of them passes out to the sporophyll. The tracheides in the sporophyll are somewhat scattered, and some of them may represent centripetal xylem. The phloem dies out before the xylem, as it ordinarily does in the termination of the vascular bundles of the leaves. A small resin canal, which appears to be non¬ functional, accompanies the single vascular bundle in its passage from the cone axis, but this canal dies out very quickly. In Saxegothaea , on the other hand, the resin canal of the sporophyll is a much more conspicuous structure, and passes out practically as far as the xylem elements extend. The microsporangia of our material were either ready for dehiscence or else dehiscence had already taken place. Remains of two layers of cells within the sporangium wall could be made out. The wall of the sporangium is very much like that of Saxegothaea (10, 17) and Araucaria (16). It is one cell wide except on the outside, and the vertical cell-walls are very thick, and have numerous strengthening bands on them running at right angles to the surface (cf. PI. XXI, Fig. i). The line of dehiscence of the sporangium is oblique, as can be seen by an examination of PI. XXI, Fig. 2. Pilger (13) states that the dehiscence of the sporangium in the Taxaceae is longitudinal, but this is without doubt incorrect for P. spinu- tosus. Thibout (20) correctly figures the dehiscence of the microsporangia of several species of Podocarpus as being oblique. and Stiles . — The Structure of In Saxegothaea (17) the line of dehiscence is transverse. (1) Male Cone. The male cones are borne either singly on a very short axis which arises in the axil of an ordinary foliage leaf, or more often they are arranged in clusters of two to four which are borne on a similar abbreviated axis, each cone being attached by means of a short stalk. The male cone is from 1 cm. to 1*5 cm. long and about 2*5 mm. in diameter. At the base of each male cone are a few tiny bracts which are spirally arranged along the short stalk of the cone. The axis of the cone bears a number of spirally arranged sporophylls, each one of which consists of a pedicel which becomes flattened distally to a lamina on either side. Each sporophyll bears on its under surface two ovoid microsporangia. 308 (2) Pollen-grains. As is well known, the pollen-grains of Podocarpus are winged like those of Pinus. The material of P. spinulosus available contained only pollen-grains which were mature. The material had been fixed in a dilute solution of formalin, and apparently had all been collected at one time. Several observers have recently related the occurrence of as many as eight prothallial cells in the pollen-grains of different species of Podocaipiis. Thibout (20) describes the presence of several prothallial cells in P. poly - stachyus. Coker (5) mentions the division in P. coriacea of the two original prothallial cells, but he considers that these divisions are abnormal. Podocarpus spinulosus , {Smith) R. 309 Jeffrey and Chrysler (9) found that in two other species, P. fernigineiis and P. dacrydioides , there were as many as eight prothallial cells present, and that the generative cell also had divided. They and Miss Young (31) also record several prothallial cells in species of Dacrydium. Jeffrey and Chrysler incline to the view that these divisions of the prothallial cells and the generative cell cannot be regarded as a primitive feature. Burlingame (4) reports the occurrence of a similar group of prothallial cells in P. totarra Hallii. Noren (10) records a similar complex of prothallial cells in Saxegothaea , and Thomson (23) describes the occurrence of the same in Microcachrys. The latter (21) has also found that in Agathis as many as thirty to forty prothallial cells may be present. It was thought advisable to compare the structure of the male gametophyte of P. spinulosus with that of other species of Podocarpus previously described. Microtome sections of male cones were cut whose microsporangia had not dehisced, and were stained with Heidenhain’s Iron Alum Haematoxylin with a background stain of Orange G. It will be seen on examination of PI. XXI, Figs. 3-8, that the contents of the pollen- grains are almost exactly the same as those figured by Jeffrey and Chrysler for P. ferrugineus and P. dacrydioides. In Figs. 3 and 4 both prothallial cells have divided in a plane at right angles to that of the section, but in Figs. 5 and 6 divisions in the first and second prothallial cells respectively have not occurred in this plane. From an examination of Fig. (2) Pollen-grains. 7, which is a section of a pollen-grain cut tangentially at its prothallial end, it is apparent that there may be another plane of division of the prothallial cells at right angles to that indicated in Figs. 3 and 4, &c. Thus as many as eight prothallial cells may be present. The generative cell is generally conspicuous with its aggregation of protoplasm around the central nucleus. It is evident from Fig. 5 that the generative cell sometimes divides before the liberation of the pollen in a manner which is probably the same as that described for the first time by Jeffrey and Chrysler (9) as occurring in the pollen of P. ferrugineus and P. dacrydioides. The tube- nucleus is the largest nucleus present in the pollen-grain. All the nuclei of our material were in the resting condition, as indicated by the large nucleolus and absence of a reticulum. After a time the walls of the prothallial cells break down, and the nuclei come to lie free in the cytoplasm, as will be seen on examination of Fig. 8. Jeffrey and Chrysler record a similar phenomenon in P. polystachyus. It is apparent then from this examination of P. spinulosus , and from the resume of work done on other species given above, that the multi¬ cellular condition of the male gametophyte of Podocarpus must be con¬ sidered a characteristic of the genus, especially as the species investigated come from widely different regions (New Zealand, Australia, Cuba, Java). The fact that a similar condition of the male gametophyte exists in and Stiles. — The Structure of 310 Dacrydium, Microcachrys , and Saxegothaea is another indication that the Podocarpeae are a natural group. It is impossible, with our present meagre knowledge of the inter-relation-* ships of the Conifers, to say whether this multicellular condition of the male gametophyte of Podocarpus is primitive or derived. Jeffrey and Chrysler (9) consider that it is not a primitive feature, but it seems to us that data for deciding the question are not at present available. The fact that a similar prothallial complex is present in Saxegothaea and Microcachrys— two genera which on other grounds are considered to be relatively primitive-^ is a point in favour of considering the prothallial complex of Podocarpus primitive. (2) Pollen-grains. The fact that the divisions of the nuclei of the prothallial cells are karyokinetic is in keeping with the interpretation that the occurrence of several such cells is a normal feature for the genus. The presence of wings in the pollen-grains of Podocarpus does not point to any necessary relationship with the Abietineae, a group from which we consider the Podocarpeae to be considerably removed, for in the Podocarpeae the wings on the pollen-grains appear to have been evolved within the group.1 Jeffrey and Chrysler (9) consider, on the other hand, that the ground plan of the male gametophyte of Podocarpus is very similar to that of the Abietineae, but we cannot gather from their statements what adequate grounds they have for this conclusion. Jeffrey and Chrysler bring the proliferation of the prothallial cells in Araucarieae into line with the peculiar protosiphonogamic method of fertilization in this group described by Thomson (22). But this does not account for the occurrence of several prothallial cells in Podocarpus .2 On examining our material it was seen that the pollen-grains alight in the micropyle, and so presumably the growth of the pollen-tubes is a normal one through the nucellus. (3) Female Fructification. Of the female fructification we had material of three species, Podo - carpus spimdosus , P. alpinus , and a third species. We had the first in largest quantity, so this species will be described more fully and the others compared with it. The female fructification is borne laterally on the stem in the axil of a bract about six millimetres long, resembling a small foliage leaf. In the axil of the bract is the peduncle, about six or seven millimetres long and nearly a millimetre wide, and somewhat flattened in the vertical plane at right angles to that containing the main axis and the peduncle. At its upper end it bears three pairs of bracts decussately arranged (cf. PI. XXI, Fig. 9). The lowest pair are small, about one and a half milli¬ metres long, and are recurved and more or less leaf-like. They lie in the plane containing both stem and pedicel. The other two pairs of bracts* Burlingame (4). 1 Cf. Thomson Podocarpus spinulosus , {Smith) R. 1 1 which are inserted close together, appear more or less swollen and fleshy ; they are about five or six millimetres long, and fused together except at the tip. The two lower bracts are about a millimetre longer than the upper pair; each bears, just above the point where the four bracts become free from one another, a single stalked anatropous ovule. Occasionally one of the two lower bracts is sterile, while the upper pair of bracts in all cases examined were both sterile. Generally one of these is developed more strongly than the other. Pilger (13) notes a good deal of variation in the bracts of P. spinulosus. Sometimes the lowest pair may be missing, while the other four may vary a great deal in size relatively to one another. The following account of the internal structure is of the normal case where the two lowest bracts are present, and the next two bracts are fertile and of approximately equal size, while the uppermost pair are also fairly well developed. The peduncle contains in transverse section a somewhat elliptical ring of endarch collateral bundles which anastomose somewhat. Each bundle is accompanied on its phloem side by a single resin canal. A section through the base of the fused bracts shows a similar structure (PI. XXI, Fig. 10). (3) Female Fructification. At the chalazal end of the ovule the two lateral bundles bend round through about a right angle and descend, one on each side of the ovule, for a short distance towards the micropyle ; as seen in transverse section they lie on the diameter of the ovule (PL XXI, Fig. 17). The central bundle continues its course over the top of the ovule, and in so doing divides into two bundles (PL XXI, Fig. 18) which descend a short distance towards the micropyle (PL XXI, Fig. 17). All these descending bundles soon die out, and all show a tendency to amphivasal structure (PL XXI, Fig. 20). The nucellus of the ovule is surrounded by two integuments. The inner is fused for about half its length with the nucellus. The outer integument or epimatium is free from the inner for a short distance at the micropyle end, and on the side remote from the ovular stalk; on the other side it is either not present or is completely fused with the stalk.1 Where free, the innermost layer of the inner integument often con¬ sists largely of resin cells. These are apparently continued into the part of the ovule where the inner integument and nucellus become fused, and so apparently mark the line of division between these. A zone containing larger and more numerous resin cells also seems to mark the line of division between the inner and outer integuments, but these do not extend far enough towards the chalaza, and are not definite enough to enable one to state whether the descending bundles belong to any particular integument or to the nucellus. In a species of Podocarpus grown in the Cambridge Botanic Garden, the structure was found, on the whole, to be similar. The peduncle is borne in the axil of an ordinary foliage leaf. In the stage examined the peduncle is about six millimetres long and nearly a millimetre broad, and, as in P. spinulosus , is flattened in a vertical plane at right angles to that con¬ taining the peduncle and the branch bearing it. In the few specimens available for examination the lowest pair of leaf-like bracts were not present, and the whole fructification consisted of two pairs of bracts fused together, of which one or both of the lower pair bore ovules. The arrangement of the bundles is practically the same as in P. spinulosus. (3) Female Fructification. The bundles divide occasionally and reunite, the resin canals sometimes dividing as well as the bundle ; in other cases the canal arises de novo outside one of the bundles, and occasionally it may be absent altogether from one of the bundles. This behaviour points to the canals being functionless, and there is no epithelium properly developed. Resin cells are extremely abundant throughout the whole of the parenchyma of the bracts. At a higher level the ring of bundles widens out in one plane, and the bundles then come to arrange themselves in two groups of three at opposite sides of the wider diameter with two bundles between them (PI. XXI, Figs. 11 and 13). The two groups at each end belong to the fertile scales and ovules, the bundles between them belonging one to each of the sterile scales. This state of affairs is intermediate between that figured by Van Tieghem (27) for Podocarpus sinensis , where the sterile bracts also contain each a group of three bundles, and that figured by Strasburger (18) for Podocarpiis chiueusisy Wall., apparently the same species as that described by Van Tieghem, where the sterile bracts have no vascular supply. In one place the stronger of two sterile bract bundles had two small groups of xylem elements some little distance away from it on the xylem side (PI. XXI, Figs. 12 and 19) ; these apparently correspond to the two inner bundles of one of the groups in Van Tieghem’s figures. As the bundles of the sterile bracts die out, some transfusion tracheides appear. Of the group of three bundles serving the fertile scale, the middle one, passes out into the bract subtending the ovule, the other two approach and Stiles. — The S 'true hire of 312 one another and fuse by their margins to form a V-shaped bundle with its xylem and phloem inversely orientated with regard to the xylem and phloem of the subtending bract bundle (PI. XXI, Fig. 13). This V-shaped bundle next straightens itself out (PI. XXI, Fig. 14) and begins to divide into three (PI. XXI, Fig. 15), so that as the chalazal end of the ovule is approached there are found three bundles, each, as a rule, with a small non-functional resin canal (PI. XXI, Fig. 16). (3) Female Fructification. One bundle of the stem passes into the subtending leaf, while the two bundles on each side of it pass into the peduncle. These almost imme¬ diately divide up into a ring of bundles. At the base of the fused bracts 1 See Postscript. Podocarpus spinulosus , ( Smith ) R. 313 two sets of three bundles are present; these belong to the fertile scales. One of these bundles on each side divides, and so gives rise to the bundles of the two sterile scales. Additional small bundles may be present on the inside of the sterile scale bundles as in P. spinulosus . The xylem of the bundles tends to be semicircular in shape, more or less enclosing the phloem on three sides. The resin canals, of which there is one on the phloem side of each bundle, are much larger than in P. spinulosus , and as a rule the resin duct divides after the division of the bundle to which it belongs. The ovular bundle, as in P. spinulosus , divides into three ; when on a level with the nucellus, transfusion tissue begins to appear both centripetally and laterally, and the division into three bundles is no longer obvious. These bundles die out at the chalaza, and do not descend on the sides of the ovule as in P. spinulosus . For the last part of their course they are composed of transfusion-like tracheides only. In Podocarpus alpinus , material of which was obtained from the Royal Gardens, Kew, the peduncle is very short, being less than a milli¬ metre long. The lowest pair of bracts are extremely small and scale¬ like, although Pilger (13) states they are absent altogether; of the next pair only one bears an ovule, while of the upper sterile pair one is often aborted altogether, and the other is small compared with those of the lower pair. The vascular system at the base of the fused bracts consists of four bundles arranged along the sides of a square ; of these one serves the fertile scale and two the ovule it subtends, the fourth serving the opposite sterile scale. The upper sterile scale has usually no vascular supply. (3) Female Fructification. The two ovular bundles fuse as in the other species described, but at first almost the whole of the xylems of the two bundles come in contact, so that the resulting bundle is almost concentric ; further up, however, it straightens itself out. Nearer the chalaza it divides into two bundles which curve over the top of the ovule and descend a little way towards the micro- pyle on the sides of the ovule in much the same way as the lateral bundles do in P. spinulosus. In P. alpinus the resin canal accompanying each of these two bundles extends down to the level of the nucellus where it occurs on the inner margin of the outer integument, to which these bundles would appear to belong. Other canals also appear on the inner margin of the outer integument ; these presumably correspond with the middle bundle of P. spinulosus, which bends over the ovule and then divides into two. On the whole, it would seem from their behaviour in P. alpinus that these bundles all belong to the outer integument, but the evidence at present is insufficient to decide this point. As far as the female fructification is concerned, the Podocarpeae obviously fall into two divisions ; one in which the megasporophylls are aggregated into cones, and which comprises the monotypic genera Saxe- got haea and Microcachrys } the other in which the fertile megasporophylls and Stiles. — The Structure of 314 are mostly either solitary or associated in pairs, and which includes the genera Dacrydimn and Podocarpits. A remarkably different condition exists in two species of Podocarpus, P. andinus and P. spicatus , in which the megasporophylls bearing ovules occur at intervals along the fertile branch. In all a single ovule is present on the upper surface of the megasporophyll, and is borne in a more or less reversed position. Assuming that the Podocarpeae form a natural group, a conclusion which from general considerations seems inevitable, there appears to be every reason to suppose that the state found in Dacrydium and Podo¬ carpus is derived from that in Saxegothaea and Microcachrys by reduc¬ tion in the number of scales in the cone, from an indefinite number to two or three pairs decussately arranged in Podocarpus. 1 But Thomson (24) states that the sporangium bundle arises in the same way as that of Saxegothaea, by branching from the sporophyll bundle. (3) Female Fructification. Here very occasionally both scales of two pairs may contain ovular bundles (27) j in some species normally both bracts of the lower pair are fertile, while in many species, e. g. P. alpimis and P. elatus , only one scale of the whole fructification is fertile. The female fructification is then to be regarded as a very reduced cone. In P. andinus and P. spicatus it would appear that the axis, instead of becoming very much shortened, has lengthened out a good deal, and the sporophylls are now separated by comparatively long internodes. The insertion of the ovule differs markedly from that found in Saxegothaea and Microcachrys. In these genera the single ovule is sessile on the upper surface of the megasporophyll, whereas in Podo¬ carpus it is borne on a stalk which apparently arises from the upper surface of the bract which bears it. Dacrydium exhibits transitions between; the two cases, for in some species the ovule is borne on the upper surface of the scale, in others it is more or less as in Podocarpus. The internal structure of the megasporophylls of the Podocarpeae also shows a similar series of stages. In Saxegothaea one bundle leaves the axis of the. cone and gives off the ovular supply by branches arising from it ; in Microcachrys the two bundles are presumably separate from the cone axis, but very close together (30) ; 1 in Podocarpus one bundle of the peduncle passes up into the megasporophyll, while two others, one on each side of the sporophyll, join up and supply the ovule. It seems fairly clear that Saxegothaea is primitive for the Podocarpeae, and the state of things found in Podocarptts is derived from that found in Saxegothaea. The much smaller amount of centripetal xylem and the loss of function of the resin canals in some species support this view. The independence of the vascular supply of the ovule from the vascular bundle of the subtending bract can be easily, and it seems to us reasonably, explained as due to basipetal evolution of the vascular system, as the ovule has become more and more important in relation to the subtending bract., Podocarpus spinul-osus , (Smith) R. (3) Female Fructification. 315 In Saxegothaea , where the ovule is a small appendage of the bract, the bundles supplying it arise by branching from the bundle of the sporophylh As the ovule has become relatively of more importance than the bract, its vascular supply has also become of more importance, until in Podocarpus the vascular supply of the ovule is carried down into the peduncle indepen¬ dently of the supply of the megasporophyll. This point of view has also been emphasized by Thomson (24), in regard to the megasporophyll of Dacrydium , in some species of which the behaviour of the bundles is very similar to that of Podocarpus. With regard to the branching of the ovular bundles, Favre (7) has rather vaguely described their behaviour in Podocarpus sinensis , where he says the ovular bundle divides a little below the summit of the organ into a large number of branches which extend downwards in the plane of separation of the integuments and so form a vascular cupule which is prolonged up to the point where the nucellus becomes free. In P. spinulosus and P. alpinus the (presumably) integumentary bundles are much less developed, and in Podocarpus sp. they are practically not developed at all. How the ovular bundles are to be homologized with those found in the ovules of other Gymnosperms (11) there are not at present sufficient data to decide. Havre’s species would apparently throw light on this question, but his description is too vague to be of much help. Moreover Strasburger’s description of P . chinensis (19), which is apparently the same species as P. sinensis, is almost identical with our description of P. spinulosus. In P. spinulosus and P. alpinus there seems to be an outer integu¬ mentary system arising from the chalazal bundles. Whether there is an inner integumentary system as well cannot yet be decided, but what facts there are available point against it. General Considerations. The relationships of Podocarpus have already been discussed at some length in the course of this paper. The genus seems to be a recently, modified type with close relationships to Dacrydium, Micro each rys, and Saxegothaea. Its present wide geographical range, the wings on the pollen -grains, the frequently functionless nature of the resin canals, and the peculiar structure of the female fructification are all considerations which point to this conclusion. Microcachrys and Saxegothaea are un¬ doubtedly more primitive than Dacrydium and Podocarpus. Saxegothaea would appear to be a connecting link with the Araucarieae, and the complex of prothallial cells in the pollen-grains of the Podocarpeae generally can be brought into relation to the numerous prothallial cells described for the pollen-grains of the Araucarieae. Miss Robertson (14) has pointed out that the somewhat isolated genus Phyllocladus has definite points of resemblance to Podocarpus, on the one and Stiles. — The Structure of 316 hand, and relationships less marked, on the other, to the Taxeae. It would seem to us that the differences between Phyllocladus and the Taxeae are greater than Miss Robertson supposes — two of her four points of resemblance between them being of an unsatisfactory nature. Thus the markings of the centripetal wood of Phyllocladus are practically the same as those in the transfusion tracheides of Podocarpus , and the presence of a certain amount of centripetal wood in Phyllocladus is not sufficient evidence of phylogenetic relationship. There are considerable differences in the two groups between the structure of the male cones, and these differences become no less apparent when we consider the male gametophytes. The only point of resemblance between the female fructifications of the two groups is the generally fleshy consistency of the ripe seeds. Phyllocladus is certainly near the Taxeae in its female fructification, because its symmetrical arillus, like that of the latter group, only develops at a late stage. There seem to be no striking resem¬ blances in anatomical characters between the Podocarpeae and the Taxeae. Thus the points of similarity between the Podocarpeae and the Taxeae do not seem to us to be very striking, though, as has been supposed in the past, the two groups may perhaps be connected by Phyllocladus. With regard to the relationships of the Podocarpeae with the Abietineae, there appears to be no evidence of any recent phylogenetic connexion between the two groups. General Considerations. The structure of the male gameto¬ phytes in the two groups has already been discussed, and the anatomical characters both of the female reproductive structures and of the vegetative organs are very different in these two orders of Coniferae. Summary. 1. The structure of the stem and leaf of Podocarpus spinulosus is similar to that of other species described respectively by Penhallow and Worsdell. 1. The structure of the stem and leaf of Podocarpus spinulosus is similar to that of other species described respectively by Penhallow and Worsdell. 2. The structure of the microsporangium is similar to that of Saxe - to that of other species described respectively by Penhallow and Worsdell. 2. The structure of the microsporangium is similar to that of Saxe - gothaea and Araucaria , but the line of dehiscence is oblique. 2. The structure of the microsporangium is similar to that of Saxe - gothaea and Araucaria , but the line of dehiscence is oblique. 3. Several prothallial cells are present in the pollen-grains of P. spinu- losus , as in other species of the Podocarpeae investigated. The walls of the prothallial cells break down at a later stage and their nuclei come to be free in the cytoplasm. 4. The generative cell of the pollen-grain sometimes divides in the same way as that described by Jeffrey and Chrysler as occurring in P. ferrugineus and P. dacrydioides. 5. The course of the vascular bundles in the female fructification is described in detail. One bundle from the peduncle passes into the mega- sporophyll, while a pair of bundles, one on each side of the sporophyll trace, unite and serve the ovule. The bundle thus formed has its xylem and phloem inversely orientated as compared with the megasporophyll d l { h) Podocarpus spinulosus , {Smith) R . 317 bundle. At the chalazal end of the ovule it divides to form ultimately about four bundles which soon die out. 6. Podocarpus is regarded as a modified genus derived from a type similar to Saxegothaea. 7. The Podocarpeae are a natural group with no very definite con¬ nexions with the Taxeae, though Phyllocladus may form a connecting link. There is no evident relationship between the Podocarpeae and the Abie- tineae. It is likely that the former are connected with the Araucarieae by means of Saxegothaea. p 8. Gothan, W. : Zur Anatomie lebender und fossiler Gymnospermen- Holzer. Berlin, 1905. Jeffrey, E. C., and Chrysler, M. A.: The Microgametophyte of the Podocarpineae. Amer. Nat., xli, p. 355, 1907. 7. Favre, E. : Recherches sur la fleur femelle du Podocarpus sinensis. Ann. des Sci. Nat., Bot., s^r. 5, iii, p. 379, 1865. h l b d df l l l Brooks and Stiles. — The Structure of Podocarpus. 3 1 10. Noren, C. O. : Zur Kenntnis der Entwicklung von Saxegothaea conspicua, Lindl. Sven Botanisk Tidskrift, ii, p. ioi, 1908. p 11. Oliver, F. W. : The Ovules of the Older Gymnosperms. Ann. Bot., xvii, p. 451, 1903. 12. Penhallow, D. P. : The Anatomy of the Coniferales. Amer. Nat., xxxviii, 1904. y A. Engler, Das Pflanzenreich, iv, 5. Taxaceae, p. 13. Pilger, R. : In A. Engler, Das Pflanzenreich, iv, 5. Taxaceae, p. 54, 1903. 14. Robertson, A. : Some Points in the Morphology of Phyllocladus alpinus, Hook. Ann. Bot., xx, p. 259, 1906. p 15. - : The Taxoideae: a Phylogenetic Study. New Phytologist, vi, p. 92, 1907. 16. Seward, A. C. and Ford, Miss S. O. : The Araucarieae, Recent and Extinct. Phil. Trans. Roy. Soc., ser. B, cxcviii, p. 305, 1906. 17. Stiles, W. : The Anatomy of Saxegothaea conspicua , Lindl. New Phytologist, vii, p. 209, 1 90S. 18. Strasburger, E. : Die Coniferen und die Gnetaceen. Jena, 1872. 19. - : Die Angiospermen und die Gymnospermen. Jena, 1879. ’ 20. Thibout, E. : Recherches sur l’appareil male des Gymnospermes. Lille, 1896. 21. Thomson, R. B. : Preliminary note on the Araucarieae. Science, N.S., xxii, p. 88, 1905. 22. - : The Araucarieae — a Protosiphonogamic Method of Fertilization. Science, N.S., xxv, p. 271, 1907. p 23. - : On the Pollen of Microcachrys tetragona. Bot. Gaz., xlvii, p. 26, 1909. 24. - : The Megasporophyll of Saxegothaea and Microcachrys. Bot. Gaz., xlvii, P- 345* i9°9- 25. Tison, A. : Lanucelle stigmatifere et la pollinisation chez le Saxe-Gothaea conspicua. Compt Rendus, cxlvii, p. 137, 1908. p 26. - : Sur le Saxe-Gothaea conspicua , Lindl. Mem. de la Soc. Linn, de Normandie, xxiii, p. 139, 1909. p 27. Van Tieghem, P. : Anatomie comparee de la fleur femelle et du fruit des Cycadees, des Coniferes et des Gnetacees. Ann. des Sci. Nat., s^r. 5, x, p. 269, 1869. p 28. Veitch : Manual of the Coniferae. Edited by A. Kent, London, 1900. 29. Worsdell, W. C. : On Transfusion Tissue. Trans. Linn. Soc., ser. 2, v, p. 301, 1897. , , , , p , 30. - : Observations on the Vascular System of the Female Flowers of Coniferae. Ann. Bot., xviii, p. 527, 1899. 31. Young, Miss M. S. : The Male Gametophyte of Dacrydium. Bot. Gaz., xliv, p. 189, 1907. Botany School, Cambridge, October , 1809. Postscript. — Since this paper was sent in for publication we have received Tison’s second paper on Saxegothaea (26). This author considers the epimatium and ovular stalk in Podocarpus as together equivalent to the epimatium in Saxegothaea. He also refers to two figures of the ovule of Saxegothaea published by one of us (17), which both show an epimatium ‘ qui enveloppe completement l’ovule et son tegument a la fagon de celui des Dacrydium et des Podocarpus .’ One of these figures was a diagram to show the ovular supply arising as a branch from the sporophyll bundle and was not intended to show details of ovular structure. Never¬ theless, inasmuch as it represented the epimatium as present on the under side of the ovule, it was incorrect. The other figure showed a section of the ovule cut more or less parallel to the cone scale, and so showed the epimatium present on both sides ; it did not show an epimatium completely surrounding the ovule. Moreover, only on Tison’s view can it be con¬ sidered as proved that the epimatium completely surrounds the ovule in Podocarpus. 1. Bernard, C. : Le Bois centripete dans les feuilles des Coniferes. Beihefte zum Botanischen Centralblatt, xvii, p. 293, 1904. p 2. Bertrand, C. E. : Anatomie des Gn^tac^es et des Coniferes. Ann. des Sci. Nat., Bot., ser. 5, xx, p. 5, 1874. 3. Beust. : Untersuchungen iiber fossile Holzer aus Gronland. Allgem. Schweiz. Gesellschaft, neue Denkschrift, xxix, 1884. 4. Burlingame, L. L. : The Staminate Cone and Male Gametophyte of Podocarpus. Bot. Gaz., xlvi, p. 161, 1908. p 5. Coker, W. C. : Notes on the Gametophytes and Embryo of Podocarpus. Bot. Gaz., xxxiii, p. 89, 1902. 6. Eichler, A. W. : Excursus morphologicus de formatione florum Gymnospermarum. Ann. des Sci. Nat., Bot., sdr. 4, xix, p. 257, 1863. 7. Favre, E. : Recherches sur la fleur femelle du Podocarpus sinensis. Ann. des Sci. Nat., Bot., s^r. 5, iii, p. 379, 1865. p 8. Gothan, W. : Zur Anatomie lebender und fossiler Gymnospermen- Holzer. Berlin, 1905 9. Jeffrey, E. C., and Chrysler, M. A.: The Microgametophyte of the Podocarpineae. Amer. Nat., xli, p. 355, 1907. EXPLANATION OF PLATE XXI. Illustrating Messrs. Brooks and Stiles’s Paper on Podocarpus. Illustrating Messrs. Brooks and Stiles’s Paper on Podocarpus. All the figures are of Podocarpus spinulosus. All the figures are of Podocarpus spinulosus. Brooks, F. T. and Stiles, Walter. 1910. "The structure of Podocarpus spinulosus, (Smith) R. Br." Annals of botany 24, 305–318. https://doi.org/10.1093/oxfordjournals.aob.a089268. View This Item Online: https://www.biodiversitylibrary.org/item/262605 DOI: https://doi.org/10.1093/oxfordjournals.aob.a089268 Permalink: https://www.biodiversitylibrary.org/partpdf/319776 All the figures are of Podocarpus spinulosus. Fig. 1. Microsporophyll as seen in radial section through the cone, x Fig. 2. Microsporophyll showing oblique dehiscence of sporangia, x 63. Fig. 2. Microsporophyll showing oblique dehiscence of sporangia, x 63. Figs. 3-8. Sections of pollen-grains showing contents. Stained with Heidenhain’s Iron Alum aematoxylin and Orange G. x 725. Haematoxylin and Orange G. x 725. Fig. 9. Diagram of female fructification indicating the position of the sections shown in the ollowing diagrams. Figs. 10-18. Diagrams representing sections through the female fructification at different levels. Figs. 10-18. Diagrams representing sections through the female fructification at different levels. Fig. 19. Transverse section of the vascular bundle of a sterile scale of the female fructification showing two small isolated groups of xylem outside the main bundles. x 300. Fig. 19. Transverse section of the vascular bundle of a sterile scale of the female fructification showing two small isolated groups of xylem outside the main bundles. x 300. Fig. 20. Transverse section of, presumably, an integumentary vascular bundle showing a tendency to amphivasal structure, x 300. Fig. 20. Transverse section of, presumably, an integumentary vascular bundle showing a tendency to amphivasal structure, x 300. c/huvaZs of Botany, c/huvaZs of Botany, Y. T.B. esn d W.S. del. BROOKS Y. T.B. esn d W.S. del. Y. T.B. esn d W.S. del. BROOKS & STILES - PODOCARPU5 SPINULOSUS. Vol. XXIV. PL. XXI. Vol. XXIV. PL. XXI. Hirth litih et imp. Hirth litih et imp. Holding Institution New York Botanical Garden, LuEsther T. Mertz Library This file was generated 30 March 2024 at 18:48 UTC Copyright & Reuse Copyright & Reuse Copyright Status: Public domain. The BHL considers that this work is no longer under copyright protection. This document was created from content at the Biodiversity Heritage Library, the world's largest open access digital library for biodiversity literature and archives. Visit BHL at https://www.biodiversitylibrary.org. This file was generated 30 March 2024 at 18:48 UTC
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Two new freshwater fish species of the genus Telestes (Actinopterygii, Cyprinidae) from karst poljes in Eastern Herzegovina and Dubrovnik littoral (Bosnia and Herzegovina and Croatia)
ZooKeys
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Two new freshw ZooKeys 180: 53–80 (2012) doi: 10.3897/zookeys.180.2127 www.zookeys.org Two new freshw ZooKeys 180: 53–80 (2012) doi: 10.3897/zookeys.180.2127 www.zookeys.org Two new freshw ZooKeys 180: 53–80 (2012) doi: 10.3897/zookeys.180.2127 www.zookeys.org s of the genus Telestes Research article Two new freshwater fish species of the genus Telestes (Actinopterygii, Cyprinidae) from karst poljes in Eastern Herzegovina and Dubrovnik littoral (Bosnia and Herzegovina and Croatia) G. Bogutskaya1,†, Primož Zupančič2,‡, Ivan Bogut3,§, Alexander M. Naseka1,| 1 Zoological Institute, Russian Academy of Sciences, 1 Universitetskaya Emb., St Petersburg 199034, Russia 2 Dolsko 14, 1262 Slovenia 3 Agronomski fakultet u Sveučilišta u Mostaru, Zavod za ribarstvo, zoologiju i zaštitu voda, Biskupa Čule 10, 88000 Mostar, Bosnia and Hercegovina † urn:lsid:zoobank.org:author:F6957EF1-366E-484E-BF40-211E17A93A1B ‡ urn:lsid:zoobank.org:author:0DC4A949-8AF7-4E80-8305-FDF271318A36 § urn:lsid:zoobank.org:author:248F52CE-3AC6-4AA4-9EA2-FB5BCD95D7EF | urn:lsid:zoobank.org:pub:D89D42DE-5B28-48B7-9431-2D603C985CAF Corresponding author: Nina G. Bogutskaya (nbogutskaya@rambler.ru) Academic editor: C. Baldwin  |  Received 23 September 2011  |  Accepted 19 March 2012  |  Published 5 April 2012 urn:lsid:zoobank.org:pub:D89D42DE-5B28-48B7-9431-2D603C985CAF Citation: Bogutskaya NG, Zupančič P, Bogut I, Naseka AM (2012) Two new freshwater fish species of the genus Telestes (Actinopterygii, Cyprinidae) from karst poljes in Eastern Herzegovina and Dubrovnik littoral (Bosnia and Herzegovina and Croatia). ZooKeys 180: 53–80. doi: 10.3897/zookeys.180.2127 Copyright Nina G. Bogutskaya et al. This is an open access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Keywords Cypriniformes, West Balkans, Alpha Taxonomy and new taxa Abstract Two new species, Telestes dabar and T. miloradi, are described on the basis of morphological comparisons of isolated geographical populations of fishes identified earlier as T. metohiensis. A lectotype is designated for T. metohiensis, whose range is shown to include waters of Gatačko, Cerničko, and Nevesinjsko poljes in Eastern Herzegovina. Telestes dabar from Dabarsko Polje (Eastern Herzegovina) and T. miloradi from Konavosko Polje (south Croatia) share with T. metohiensis the following combination of characters that distinguish them from the rest of the genus Telestes: pharyngeal teeth in one row, usually 5–4; preoperculo- mandibular canal not communicating with the infraorbital canal; mouth subterminal, the tip of the mouth cleft on or below the level of the ventral margin of the eye; postcleithrum minute or absent; ventral por- tion of the trunk with a dark stripe on a pale background; and dorsal portion of trunk uniformly dark and bordered ventrally by a dark midlateral stripe. Telestes dabar and T. miloradi are distinguishable from T. metohiensis in usually having 8½ branched dorsal-fin rays (vs. usually 7½), 9 or 10 gill rakers (vs. 7–10, Nina G. Bogutskaya et al. / ZooKeys 180: 53–80 (2012) 54 usually 8), and the dark stripe on the ventral portion of the trunk below the main pigmented area of the back narrow and usually not reaching posteriorly to the caudal peduncle (vs. dark stripe wide and extending posteriorly to the caudal peduncle). Telestes dabar is distinguished from T. miloradi by having scales on most of the body situated close to one another and overlapping in a region behind the pectoral girdle and usually on the caudal peduncle (vs. overlapping scales on most of the body); the lateral line usually incomplete and interrupted, with 24–69, usually 54–65, total scales (vs. lateral line usually complete, with 55–67 total scales); scales above and below the lateral line slightly smaller than lateral-line scales (vs. of about equal size); head width 43–52% HL (vs. 48–58% HL); and lower jaw length 10–12% SL or 36–41% HL (vs. 8–10% SL or 33–38% HL). Telestes miloradi, a very local endemic species, is known only by historical sam- ples. Telestes dabar is an abundant fish in Dabarsko Polje, but its range is critically restricted during the dry season by a few permanent sources. Nothing is known about its occurrence in underground karst waters. Introduction Karst basins in Croatia and Bosnia and Herzegovina are populated by many local en- demic species most of them belonging to the cyprinid subfamily Leuciscinae (Mrakovčić et al. 2006; Kottelat and Freyhof 2007; Jelić et al. 2008; Zupančič 2008). Among leu- ciscine cyprinids of the Dinaric karst, most numerous is a group of species that was formerly assigned to the genus Phoxinellus Heckel, 1843. In a revision of this group (Zupančič and Bogutskaya 2002; Bogutskaya and Zupančič 2003) ten Phoxinellus spe- cies were divided into two groups, one with two subgroups. Later (Freyhof et al. 2006), based on nuclear and mtDNA sequences, Phoxinellus was found to be paraphyletic because it includes three unrelated monophyletic units. The scientific name Phoxinellus was therefore restricted to species having plain colouration, a small or absent post- cleithrum, no genital papilla, and an almost entirely naked body (species included: type species P. alepidotus Heckel, 1843, P. dalmaticus Zupančič & Bogutskaya, 2000, and P. pseudalepidotus Bogutskaya & Zupančič, 2003). Species having an irregularly spotted colour pattern, a large postcleithrum, an increased number of precaudal anal-fin ptery- giophores, and a large female genital papilla were assigned to a new genus, Delminichthys Freyhof, Lieckfeldt, Bogutskaya, Pitra & Ludwig, 2006 (species included: type species D. adspersus (Heckel, 1843), D. ghetaldii (Steindachner, 1882), D. jadovensis Zupančič & Bogutskaya, 2002, and D. krbavensis Zupančič & Bogutskaya, 2002). Finally, species having a small or absent postcleithrum, no genital papilla, a dark midlateral stripe from the head to the caudal peduncle, and non-overlapping scales were assigned to Telestes (T. croaticus (Steindachner, 1866), T. fontinalis (Karaman, 1972), and T. metohiensis (Stein- dachner, 1901)), bringing the total number of species of Telestes to 11. These generic assignments were later supported by a molecular phylogenetic study based on the same mitochondrial but different nuclear markers (Perea et al. 2010). Two new freshwater fish species of the genus Telestes (Actinopterygii, Cyprinidae)... 55 Zupančič and Bogutskaya (2002) and Bogutskaya and Zupančič (2003) rede- scribed T. metohiensis based on 251 specimens (including syntypes) from Nevesinjsko Polje, Gatačko Polje, Cerničko Polje, and Dabarsko Polje karsts in Bosnia and Her- zegovina, and from Ljuta River in Konavle Region [Konavosko Polje] in southern Croatia. Bogutskaya and Zupančič (2003: 375) mentioned that the syntypes from the Ljuta River were distinguished by having 8½ branched anal-fin rays (vs. usually 7½ in most other syntypes). Introduction Specimens from Dabarsko Polje (Vrijeka and Opačica rivers) were also only tentatively identified as metohiensis because of having overlap- ping scales (vs. non-overlapping, narrowly to widely spaced) and usually 8½ branched anal-fin rays (Zupančič and Bogutskaya 2002: 417). Since 2003, we have examined additional material of the entire T. metohiensis species complex, and two species – one from Ljuta River in Konavosko Polje and one from Dabarsko Polje – are described here as new. Methods Measurements were made point to point to the nearest 0.1 mm and follow methods used by Bogutskaya and Zupančič (2010) for Squalius species. In most aspects, they coincide with the scheme by Kottelat and Freyhof (2007: fig. 1), but a few clarifica- tions of the latter are as follows. Standard length (SL) was measured from the anteri- ormost point of the upper lip (not of the snout) to the end of the hypural complex. Head length (HL) was measured from the anteriormost point of the upper lip to the posteriormost point of the opercular membrane. Interorbital width was measured including the skin fold. The term ‘length of dorsal fin’ is used for the length of the dorsal-fin base, and the term ‘depth of dorsal fin’ is used for the length of the longest ray of the dorsal fin. Respective terms are used for the measurements of the anal fin. The last two branched rays articulating on a single pterygiophore in the dorsal and anal fins are noted as “1½”. Total lateral-line scale count includes all pored scales, from the first one just behind the posttemporal bone to the posteriormost one located on the bases of the caudal-fin rays. Total number of lateral-row scales includes all scales, pored and non-pored, from the first one just behind the posttemporal bone to the posteriormost one located on the bases of the caudal-fin rays. Osteological char- acters were examined from dissections and from radiographs. Statistical analyses were performed with Microsoft Excel and Statistica 6.0 packages. Abbreviations used: NMW, Naturhistorisches Museum, Wien; SMNH, Slovenian Museum of Natural History; PZC, Collection of P. Zupančič, Dolsko (Slovenia); SMF, Senckenberg Museum, Frankfurt a. Main; ZISP, Zoological Institute, Russian Acad- emy of Sciences, St Petersburg; ZMH, Zoologisches Museum und Institut, Universität Hamburg; (cephalic sensory canals) CIO, infraorbital canal; CPM, preoperculo-man- dibular canal; CSO, supraorbital canal; and CST, supratemporal canal. Nina G. Bogutskaya et al. / ZooKeys 180: 53–80 (2012) 56 Results Telestes dabar sp. n. urn:lsid:zoobank.org:act:4A50A07B-57BD-472B-9B00-6941D8C6779A http://species-id.net/wiki/Telestes_dabar Figs 1a, 2a, 3a Telestes dabar sp. n. urn:lsid:zoobank.org:act:4A50A07B-57BD-472B-9B00-6941D8C6779A http://species-id.net/wiki/Telestes_dabar Figs 1a, 2a, 3a Holotype. NMW 95295, 79.1 mm SL, BOSNIA & HERZEGOVINA: Dabarsko Polje, Opačica River at Potkom, 43°5.9'N, 18°7.6'E, 15 Sept. 2006, coll. Zupančič. Paratypes. NMW 95300, 6, 55.2–71.1 mm SL, same data as holotype; PZC 525, 72, 32.5–51.3 mm SL, same data as holotype; PZC 526, 13, 48.0–73.8 mm SL, same data as holotype; PZC 565, 21, 35.5–56.7 mm SL, same locality, 8 July 2011; ZISP 54995, 15, 38.1–58.9 mm SL, same locality, 8 July 2011; SMNH 444, 35.5–60.8 mm SL, same locality, 8 July 2011; PZC 279, 12, 44.2–71.9 mm SL, BOSNIA & HER- ZEGOVINA: Dabarsko Polje, Vrijeka River, 24 May 2001; PZC 521, 13, 54.0–62.6 mm SL, same locality as 279, 15 Sept. 2004; PZC 575, 18, 40.5–69.8 mm SL, same locality as 279, 15 Sept. 2006. Paratypes. NMW 95300, 6, 55.2–71.1 mm SL, same data as holotype; PZC 525, 72, 32.5–51.3 mm SL, same data as holotype; PZC 526, 13, 48.0–73.8 mm SL, same data as holotype; PZC 565, 21, 35.5–56.7 mm SL, same locality, 8 July 2011; ZISP 54995, 15, 38.1–58.9 mm SL, same locality, 8 July 2011; SMNH 444, 35.5–60.8 mm SL, same locality, 8 July 2011; PZC 279, 12, 44.2–71.9 mm SL, BOSNIA & HER- ZEGOVINA: Dabarsko Polje, Vrijeka River, 24 May 2001; PZC 521, 13, 54.0–62.6 mm SL, same locality as 279, 15 Sept. 2004; PZC 575, 18, 40.5–69.8 mm SL, same locality as 279, 15 Sept. 2006. Diagnosis. Telestes dabar is distinguished from T. metohiensis and T. miloradi by having the following combination of characters: slightly curved dark stripe (obvious in live and preserved specimens) present from just behind operculum to vertical just ante- rior to origin of anal fin, this stripe narrow and separated from dark pigmented area on back along its entire length; scales on most of body not overlapping but situated close to one another; scales overlapping behind pectoral girdle along lateral line and usually Figure 1. a Telestes dabar, holotype, female, 79.1 mm SL, NMW 95295, Bosnia & Herzegovina: Opačica River, Dabarsko Polje b Telestes metohiensis, female, 82.1 mm SL, PZC 293, Bosnia & Herzego- vina: Zovidolka River (Zalomka River system), Nevesinjsko Polje. a b a b Figure 1. a Telestes dabar, holotype, female, 79.1 mm SL, NMW 95295, Bosnia & Herzegovina: Opačica River, Dabarsko Polje b Telestes metohiensis, female, 82.1 mm SL, PZC 293, Bosnia & Herzego- vina: Zovidolka River (Zalomka River system), Nevesinjsko Polje. Telestes dabar sp. n. urn:lsid:zoobank.org:act:4A50A07B-57BD-472B-9B00-6941D8C6779A http://species-id.net/wiki/Telestes_dabar Figs 1a, 2a, 3a Two new freshwater fish species of the genus Telestes (Actinopterygii, Cyprinidae)... 57 Figure 2. a Telestes dabar, male paratype, live specimen, ZISP 54995, 58.9 mm SL, Bosnia & Herzegovina: Opačica River, Dabarsko Polje b Telestes metohiensis, live specimen, male, 86.2 mm SL, PZC 567, Bosnia & Herzegovina: spring Ljeskovik in Zalomka River, Nevesinjsko Polje c Telestes metohiensis, live specimen, male, 84.5 mm SL, PZC 566, Bosnia & Herzegovina: Zovidolka River (Zalomka River system), Nevesinjsko Polje. c a b a a b b c c c Figure 2. a Telestes dabar, male paratype, live specimen, ZISP 54995, 58.9 mm SL, Bosnia & Herzegovina: Opačica River, Dabarsko Polje b Telestes metohiensis, live specimen, male, 86.2 mm SL, PZC 567, Bosnia & Herzegovina: spring Ljeskovik in Zalomka River, Nevesinjsko Polje c Telestes metohiensis, live specimen, male, 84.5 mm SL, PZC 566, Bosnia & Herzegovina: Zovidolka River (Zalomka River system), Nevesinjsko Polje. on caudal peduncle; snout with fleshy tip projecting over upper lip; mouth subtermi- nal with tip of mouth cleft at or below level of ventral margin of eye; lateral line usually interrupted, with 24–69 total lateral-line scales; branched dorsal-fin rays usually 8½; branched anal-fin rays usually 8½; gill rakers 9 or 10; total vertebrae 39–41, mode 40; abdominal vertebrae 22–24, mode 22; caudal vertebrae 16–18, mode 17; head width 43–52% HL; and lower jaw long, length 10–12% SL. Description. Morphometric data are summarised in Table 1a, selected counts in Tables 2–4. General appearance can be seen in Figs 1a and 2a. Body compressed, elon- Nina G. Bogutskaya et al. / ZooKeys 180: 53–80 (2012) 58 Figure 3. a Telestes dabar, alizarin stained specimen, PZC 575, 63.7 mm SL, Bosnia & Herzegovina: Vrijeka River, Dabarsko Polje b Telestes metohiensis, alizarin stained specimen, 63.3 mm SL, PZC 312, Bosnia & Herzegovina: spring Ljeskovik in Zalomka River, Nevesinjsko Polje. a b a b a Figure 3. a Telestes dabar, alizarin stained specimen, PZC 575, 63.7 mm SL, Bosnia & Herzegovina: Vrijeka River, Dabarsko Polje b Telestes metohiensis, alizarin stained specimen, 63.3 mm SL, PZC 312, Bosnia & Herzegovina: spring Ljeskovik in Zalomka River, Nevesinjsko Polje. gate. Caudal-peduncle depth only slightly less than half maximum body depth; head length greater than maximum body depth. Eye small, its diameter smaller than snout length. Snout fleshy, slightly to markedly projecting beyond upper lip (similar to a feature Kottelat and Freyhof [2007: fig. Telestes dabar sp. n. urn:lsid:zoobank.org:act:4A50A07B-57BD-472B-9B00-6941D8C6779A http://species-id.net/wiki/Telestes_dabar Figs 1a, 2a, 3a 39] called the “rostral cap,” which covers all or part of upper lip); snout terminating laterally in prominent crease along anterior edge of first infraorbital. Mouth subterminal, tip of mouth cleft at level of ventral margin of eye or, more frequently, below it. Lower jaw-quadrate junction at vertical through anterior half of eye. Length of lower jaw 10–12% SL or 36–41% HL, or 102–132% depth of operculum. Dorsal fin with 7½ (9 specimens), 8½ (151) or 9½ (1) branched rays, 8½ in holo- type. Dorsal-fin origin above posterior end of pelvic-fin base. Anal fin with 8½ (153) or 9½ (8) branched rays, 8½ in holotype. Outer margin of anal fin slightly concave or almost straight. Caudal fin moderately forked, lobes weakly pointed, with 9+8 princi- pal branched rays. Total gill rakers (Table 2) 9 (20) or 10 (20), 10 in holotype. Pharyngeal teeth 5–4, hooked, slightly serrated (examined in 5 specimens). Scales covering entire body including pre-pectoral area and abdomen, non-over- lapping on most parts of body but overlapping in triangular-shaped area just behind pectoral girdle and usually on caudal peduncle at least behind anal fin (Fig. 3a); lateral- line scales always overlapping, sometimes a few posteriormost scales not overlapping. Scales irregularly set but close to one another. Most flank scales oval, somewhat deeper than long; scales on caudal peduncle more elongated (longer than deep) having promi- nent posterior attenuation. Trunk scales smaller than lateral-line scales but not con- Two new freshwater fish species of the genus Telestes (Actinopterygii, Cyprinidae)... 59 Table 1a. Morphometric data of T. dabar. Table 1a. Morphometric data of T. dabar. T. Telestes dabar sp. n. urn:lsid:zoobank.org:act:4A50A07B-57BD-472B-9B00-6941D8C6779A http://species-id.net/wiki/Telestes_dabar Figs 1a, 2a, 3a Bogutskaya et al. / ZooKeys 180: 53–80 (2012) 60 Table 1b. Morphometric data of T. miloradi. Table 1b. Morphometric data of T. miloradi. T. Telestes dabar sp. n. urn:lsid:zoobank.org:act:4A50A07B-57BD-472B-9B00-6941D8C6779A http://species-id.net/wiki/Telestes_dabar Figs 1a, 2a, 3a dabar Holo- type females, n=26 males, n=13   min max mean SD min max M SD SL, mm 79.1 60.3 81.8 67.8 55.5 69.0 61.1 Maximum body depth (% SL) 21.6 21.6 26.0 23.6 1.3 21.7 25.0 22.6 0.9 Depth of caudal peduncle (% SL) 9.8 9.8 12.4 11.0 0.7 10.0 12.4 11.2 0.7 Depth of caudal peduncle (% length of caudal peduncle) 47.9 47.7 59.6 53.6 4.3 44.6 62.2 52.9 5.2 Maximum body width (% SL) 13.4 11.4 16.0 13.7 1.2 12.0 22.0 14.2 2.6 Predorsal length (% SL) 55.3 53.5 58.7 56.9 1.1 53.8 57.3 55.5 1.2 Postdorsal length (% SL) 36.3 32.8 36.6 35.2 1.0 34.4 37.2 35.8 1.0 Prepelvic length (% SL) 51.6 50.4 55.5 52.7 1.4 50.6 54.4 52.2 1.0 Preanal length (% SL) 71.2 68.3 73.2 70.8 1.3 68.6 71.9 69.9 1.0 Pectoral – pelvic-fin origin length (% SL) 24.5 24.5 27.7 25.6 0.9 22.3 26.5 24.1 1.2 Pelvic – anal-fin origin length (% SL) 20.2 17.2 20.2 19.0 0.6 16.6 20.3 18.4 1.3 Length of caudal peduncle (% SL) 20.6 18.5 21.9 20.5 0.8 19.2 23.5 21.2 1.1 Dorsal-fin base length (% SL) 10.3 9.3 12.3 10.9 0.8 9.9 12.2 11.2 0.7 Dorsal fin depth (% SL) 18.9 16.6 20.2 18.6 1.4 18.5 21.8 19.7 0.9 Anal-fin base length (% SL) 12.1 9.2 12.1 10.6 0.8 10.3 12.4 11.2 0.5 Anal fin depth (% SL) 12.3 11.9 14.8 13.2 0.8 13.4 17.2 15.9 1.1 Pectoral fin length (% SL) 19.9 18.1 21.2 19.9 0.8 22.5 26.7 24.2 1.3 Pelvic fin length (% SL) 14.8 12.9 16.3 14.9 0.9 16.3 17.7 16.9 0.5 Head length (% SL) 27.1 25.6 28.7 27.1 0.9 26.6 29.6 28.1 1.0 Head length (% body depth) 125.1 104.0 125.1 115.3 5.9 110.8 135.1 124.3 7.6 Head depth at nape (% SL) 17.4 17.1 19.3 18.1 0.7 17.0 18.8 18.2 0.5 Head depth at nape (% HL) 64.2 62.4 70.7 66.8 2.3 60.6 69.3 65.0 2.7 Maximum head width (% SL) 13.5 11.6 14.6 13.6 0.7 12.4 14.5 13.6 0.6 Maximum head width (% HL) 50.0 42.8 52.3 50.1 2.4 44.7 52.1 48.3 2.4 Maximum cranial width (% cranium roof length) 68.9 61.9 72.6 65.8 3.1 59.7 68.8 64.4 2.5 Snout length (% SL) 8.5 7.4 9.2 8.1 0.5 7.8 9.0 8.5 0.4 Snout length (% HL) 31.4 27.7 32.8 29.7 1.3 28.5 31.7 30.3 1.1 Eye horizontal diameter (% SL) 6.6 5.9 7.2 6.6 0.4 6.3 7.8 6.8 0.4 Eye horizontal diameter (% HL) 24.4 22.1 26.8 24.5 1.5 22.1 26.8 24.3 1.4 Eye horizontal diameter (% interorbital width) 74.5 66.3 81.9 73.3 4.7 69.5 82.0 74.4 4.7 Postorbital distance (% HL) 53.0 48.8 54.0 52.2 1.4 47.1 53.0 50.2 1.9 Interorbital width (% SL) 8.9 8.2 9.6 9.1 0.4 8.5 9.9 9.2 0.4 Interorbital width (% HL) 32.8 31.3 36.0 33.4 1.2 29.0 35.6 32.7 1.9 Length of upper jaw (% HL) 28.6 24.5 30.5 27.8 1.9 26.2 29.5 27.7 1.1 Length of upper jaw (% SL) 7.7 6.6 8.5 7.5 0.6 7.0 8.6 7.8 0.5 Length of lower jaw (% SL) 10.6 9.5 11.2 10.2 0.5 9.7 11.5 10.9 0.5 Length of lower jaw (% HL) 39.2 35.8 40.7 37.6 1.3 35.6 41.1 38.6 1.6 Length of lower jaw (% interorbital width) 119.7 106.4 123.4 112.6 4.4 110.2 129.8 120.2 6.4 Length of lower jaw (% depth of operculum) 110.2 102.4 120.0 108.2 4.5 105.7 131.7 116.6 8.2 Nina G. Telestes dabar sp. n. urn:lsid:zoobank.org:act:4A50A07B-57BD-472B-9B00-6941D8C6779A http://species-id.net/wiki/Telestes_dabar Figs 1a, 2a, 3a 61 Table 1c. Morphometric data of T. metohiensis. Table 1c. Morphometric data of T. metohiensis. T. Telestes dabar sp. n. urn:lsid:zoobank.org:act:4A50A07B-57BD-472B-9B00-6941D8C6779A http://species-id.net/wiki/Telestes_dabar Figs 1a, 2a, 3a miloradi Holo- type females, n=9 males, n=3   min max mean SD min max Mean SD SL, mm 66.7 34.1 66.7 57.9 61.8 Maximum body depth (% SL) 22.4 21.7 25.8 23.5 1.5 19.6 24.6 23.0 2.9 Depth of caudal peduncle (% SL) 11.4 10.4 11.6 11.2 0.4 9.6 11.3 10.8 1.0 Depth of caudal peduncle (% length of caudal peduncle) 53.8 49.8 54.8 52.4 2.1 41.8 52.8 49.1 6.4 Maximum body width (% SL) 14.3 12.9 15.3 14.6 0.7 11.4 16.8 15.0 3.1 Predorsal length (% SL) 55.9 55.9 57.7 57.0 0.7 54.3 54.9 54.5 0.3 Postdorsal length (% SL) 35.2 32.8 35.3 34.3 1.0 35.4 35.6 35.5 0.1 Prepelvic length (% SL) 51.6 51.5 53.3 52.3 0.9 50.0 50.2 50.1 0.1 Preanal length (% SL) 72.8 68.2 72.8 70.3 1.9 68.2 68.4 68.3 0.1 Pectoral – pelvic-fin origin length (% SL) 25.1 25.1 27.6 26.2 1.1 23.3 25.0 24.4 1.0 Pelvic – anal-fin origin length (% SL) 20.8 16.6 20.8 18.3 1.7 17.6 18.4 18.2 0.5 Length of caudal peduncle (% SL) 21.2 20.5 22.1 21.3 0.7 21.4 23.0 22.0 0.9 Dorsal-fin base length (% SL) 11.6 9.9 11.6 10.9 0.7 11.2 12.6 12.2 0.8 Dorsal fin depth (% SL) 19.6 19.2 20.9 19.8 0.8 20.9 22.1 21.4 0.6 Anal-fin base length (% SL) 12.4 10.7 12.4 11.9 0.6 11.8 11.9 11.8 0.1 Anal fin depth (% SL) 13.2 0.0 15.0 12.5 4.7 13.7 15.5 14.3 1.0 Pectoral fin length (% SL) 21.7 18.5 21.7 20.8 1.0 23.5 25.4 24.8 1.1 Pelvic fin length (% SL) 15.6 13.6 15.6 14.6 0.8 16.9 17.6 17.2 0.4 Head length (% SL) 26.8 25.4 28.0 27.0 0.8 26.5 29.0 28.2 1.4 Head length (% body depth) 119.9 105.0 119.9 115.0 6.0 117.6 135.4 123.5 10.3 Head depth at nape (% SL) 18.3 16.9 18.3 17.6 0.5 16.7 19.0 18.2 1.4 Head depth at nape (% HL) 68.1 62.5 68.1 65.1 2.3 62.9 65.7 64.7 1.6 Maximum head width (% SL) 14.1 12.3 14.5 14.0 0.7 12.6 16.9 15.4 2.5 Maximum head width (% HL) 52.5 48.7 53.6 52.0 1.5 47.5 58.2 54.6 6.2 Maximum cranial width (% cranium roof length) 62.8 59.6 73.9 66.0 5.0 58.3 65.2 62.9 4.0 Snout length (% SL) 9.0 8.0 9.7 8.9 0.5 8.5 8.6 8.6 0.0 Snout length (% HL) 33.7 31.4 34.7 33.0 1.2 29.5 32.4 30.5 1.7 Eye horizontal diameter (% SL) 6.5 6.3 6.9 6.5 0.2 6.6 6.7 6.7 0.0 Eye horizontal diameter (% HL) 24.4 22.5 26.4 24.2 1.4 22.9 25.2 23.7 1.3 Eye horizontal diameter (% interorbital width) 70.2 64.7 83.0 71.3 5.9 69.4 83.4 74.1 8.1 Postorbital distance (% HL) 49.1 48.9 51.6 50.2 1.2 50.2 51.0 50.5 0.5 Interorbital width (% SL) 9.3 8.1 9.7 9.2 0.5 8.0 9.6 9.1 0.9 Interorbital width (% HL) 34.8 31.9 34.8 34.1 0.9 30.2 33.1 32.1 1.7 Length of upper jaw (% HL) 27.9 26.0 28.1 27.2 0.9 23.5 25.1 24.0 0.9 Length of upper jaw (% SL) 7.5 6.6 7.9 7.3 0.4 6.7 6.8 6.8 0.1 Length of lower jaw (% SL) 9.3 8.4 10.4 9.5 0.6 8.7 10.4 9.8 1.0 Length of lower jaw (% HL) 34.7 33.1 38.4 35.3 1.8 32.9 35.9 34.9 1.7 Length of lower jaw (% interorbital width) 99.8 99.8 114.3 103.7 6.1 108.5 109.1 108.7 0.4 Length of lower jaw (% depth of operculum) 104.0 95.6 104.0 101.1 2.9 95.6 107.1 103.3 6.7 Two new freshwater fish species of the genus Telestes (Actinopterygii, Cyprinidae)... Telestes dabar sp. n. urn:lsid:zoobank.org:act:4A50A07B-57BD-472B-9B00-6941D8C6779A http://species-id.net/wiki/Telestes_dabar Figs 1a, 2a, 3a Bogutskaya et al. / ZooKeys 180: 53–80 (2012) 62 Table 1d. Morphometric data of T. metohiensis. Table 1d. Morphometric data of T. metohiensis. T. Telestes dabar sp. n. urn:lsid:zoobank.org:act:4A50A07B-57BD-472B-9B00-6941D8C6779A http://species-id.net/wiki/Telestes_dabar Figs 1a, 2a, 3a metohiensis, Gatačko and Cerničko poljes lectotype females, n=25 males, n=10   min max mean SD min max mean SD SL, mm 87.9 60.26 102.1 74.9 54.4 75.7 63.1 Maximum body depth (% SL) 23.9 19.9 25.1 22.5 1.4 20.7 24.1 22.5 1.0 Depth of caudal peduncle (% SL) 11.3 9.7 11.9 10.7 0.5 10.4 11.8 11.2 0.5 Depth of caudal peduncle (% length of caudal peduncle) 61.5 46.7 61.5 53.0 4.1 46.7 55.7 51.0 3.1 Maximum body width (% SL) 16.0 11.1 16.4 14.3 1.7 14.1 15.6 14.8 0.6 Predorsal length (% SL) 57.6 49.4 60.1 56.5 2.2 53.7 57.7 55.7 1.3 Postdorsal length (% SL) 33.4 31.0 36.5 33.7 1.6 32.5 38.0 35.2 1.6 Prepelvic length (% SL) 52.0 46.0 55.9 52.7 1.9 50.4 53.8 51.9 1.3 Preanal length (% SL) 69.1 65.6 73.6 70.2 1.9 67.1 69.7 68.5 0.9 Pectoral – pelvic-fin origin length (% SL) 27.5 24.7 29.7 27.0 1.3 24.1 27.2 25.5 1.1 Pelvic – anal-fin origin length (% SL) 19.8 16.4 20.1 18.5 1.1 15.6 18.2 17.0 0.8 Length of caudal peduncle (% SL) 18.3 19.1 22.3 20.3 1.2 20.9 22.8 22.0 0.6 Dorsal-fin base length (% SL) 11.1 9.5 11.5 10.8 0.6 10.8 13.6 12.0 1.0 Dorsal fin depth (% SL) 17.6 14.9 18.3 17.2 0.8 16.3 19.6 18.5 1.1 Anal-fin base length (% SL) 11.3 9.1 12.0 10.9 0.7 10.0 13.2 11.9 1.1 Anal fin depth (% SL) 12.3 12.4 15.3 13.7 0.8 12.8 15.4 14.3 1.0 Pectoral fin length (% SL) 18.4 17.0 19.8 18.4 0.8 22.7 25.3 23.6 0.8 Pelvic fin length (% SL) 12.9 12.4 15.3 13.7 0.8 15.4 17.7 16.6 0.7 Head length (% SL) 27.0 23.8 28.2 26.6 1.1 26.1 28.2 27.3 0.6 Head length (% body depth) 112.9 111.0 132.5 120.4 6.6 114.4 131.8 122.8 5.2 Head depth at nape (% SL) 17.4 15.5 18.7 17.3 0.8 15.9 18.1 17.3 0.7 Head depth at nape (% HL) 64.6 62.9 71.8 65.1 2.2 58.5 66.1 63.5 2.7 Maximum head width (% SL) 14.8 12.9 15.7 14.8 0.7 14.5 15.7 14.9 0.4 Maximum head width (% HL) 54.9 50.6 59.3 55.7 2.7 52.8 56.9 54.7 1.6 Maximum cranial width (% cranium roof length) 63.5 62.9 78.6 68.6 4.4 65.4 71.7 68.2 2.7 Snout length (% SL) 8.1 7.3 8.8 8.2 0.4 8.5 9.4 8.9 0.4 Snout length (% HL) 30.0 27.9 33.1 30.8 1.6 31.2 34.2 32.5 1.1 Eye horizontal diameter (% SL) 6.0 4.6 6.9 5.9 0.5 5.3 6.8 6.0 0.4 Eye horizontal diameter (% HL) 22.4 19.4 25.2 22.1 1.5 19.5 24.8 22.1 1.8 Eye horizontal diameter (% interorbital width) 70.7 57.0 80.2 68.2 6.9 60.2 71.8 65.1 4.3 Postorbital distance (% HL) 51.9 47.9 55.6 51.9 2.4 46.2 53.1 50.8 2.3 Interorbital width (% SL) 8.5 7.7 9.2 8.7 0.4 8.4 9.8 9.3 0.5 Interorbital width (% HL) 31.6 29.6 34.8 32.6 1.7 30.8 35.8 34.0 1.5 Length of upper jaw (% HL) 27.8 27.0 31.0 29.2 1.0 27.0 30.9 29.0 1.1 Length of upper jaw (% SL) 7.5 6.4 8.3 7.8 0.4 7.5 8.4 7.9 0.3 Length of lower jaw (% SL) 9.9 8.3 11.3 10.3 0.8 9.5 11.0 10.3 0.6 Length of lower jaw (% HL) 36.7 35.0 42.2 38.7 2.2 34.8 41.2 37.8 2.1 Length of lower jaw (% interorbital width) 116.0 106.5 132.5 120.6 6.4 101.6 121.9 113.7 6.9 Length of lower jaw (% depth of operculum)  107.2 106.5 117.9 112.5 3.0 109.6 119.2 112.7 3.5 Nina G. Telestes dabar sp. n. urn:lsid:zoobank.org:act:4A50A07B-57BD-472B-9B00-6941D8C6779A http://species-id.net/wiki/Telestes_dabar Figs 1a, 2a, 3a 63 Table 2. Gill-raker counts in Telestes dabar, T. miloradi and T. metohiensis. 7 8 9 10 mean SD T. dabar 20 20 9.5 0.51 T. miloradi 1 11 6 9.3 0.57 T. metohiensis, Gatačko Polje 2 33 15 1 8.3 0.58 T. metohiensis, Nevesinjsko Polje 6 28 12 1 8.2 0.67 Table 2. Gill-raker counts in Telestes dabar, T. miloradi and T. metohiensis. siderably so. All scales well ossified, usually visible without staining. In live specimens, scales clearly visible because of some silver highlights (Fig. 2a). Lateral line complete (2 specimens), long but incomplete (5) or interrupted (33) as in specimen in Fig. 3a; if interrupted, gaps typically comprising absence of a few scales in a few places, 24–69 in total (Table 3), 65 with one interruption in holotype. Lateral line making clear curva- ture above anal-fin origin. Number of scales in total lateral series 62–69 (modal range 65–67), 68 in holotype. Parietal segment of CSO lacking. CPM not communicating with CIO, terminat- ing over the upper margin of opercular antedorsal process. CSO complete with 8, rarely 7 or 9, pores. CIO complete with 14–17 pores and with 4 canal openings on first infraorbital. CPM complete or interrupted between the angulo-articular and pre- operculum and/or between preoperculum and operculum, CPM with 14–17 pores (4, rarely 5, canal openings on dentary, and 7–9, usually 8, canal openings on preopercu- lum). CST complete, with 5–7 pores or narrowly interrupted in middle. p p y p Total vertebrae (Table 4) 39 (49), 40 (101) or 41 (11), 40 in holotype; abdominal vertebrae 22 (101), 23 (58) or 24 (2), 22 in holotype; caudal vertebrae 16 (9), 17 (82) or 18 (70), 18 in holotype; predorsal vertebrae 13 (24), 14 (126) or 15 (11), 14 in holotype; intermediate vertebrae 3 (123) or 4 (38), 3 in holotype. Most frequent verte- bral formulae 22+17 (41), 22+18 (60) and 23+17 (40), 22+18 in holotype. p Colouration. In live specimens, dark back contrasting sharply with pale area below lateral midline, even in small specimens. Black midlateral stripe extending from head to caudal peduncle forming ventral border of darkly pigmented area on Table 3. Total lateral-line scale and total lateral-series scales counts in Telestes dabar, T. miloradi and T. metohiensis. Total lateral-line scales Total lateral-series scales 0–53 54–57 58–61 62–65 66–69 70–73 mean SD range mean SD T. Telestes dabar sp. n. urn:lsid:zoobank.org:act:4A50A07B-57BD-472B-9B00-6941D8C6779A http://species-id.net/wiki/Telestes_dabar Figs 1a, 2a, 3a metohiensis (affinis nomen museale), Nevesinjsko Polje lectotype females, n=33 males, n=14   min max mean SD min max M sd SL, mm 87.9 57.8 113.8 79.5 57.7 86.2 70.4 Maximum body depth (% SL) 23.9 19.3 24.1 21.8 1.1 19.8 23.8 21.6 1.4 Depth of caudal peduncle (% SL) 11.3 9.0 12.0 10.7 0.7 9.6 12.3 11.1 0.7 Depth of caudal peduncle (% length of caudal peduncle) 61.5 42.7 60.0 54.2 4.5 43.6 59.9 51.8 4.2 Maximum body width (% SL) 16.0 11.6 17.6 14.5 1.4 11.0 16.6 14.3 1.7 Predorsal length (% SL) 57.6 54.2 59.4 57.1 1.3 54.4 58.4 56.3 1.0 Postdorsal length (% SL) 33.4 29.9 36.4 34.0 1.3 33.8 36.5 35.2 0.7 Prepelvic length (% SL) 52.0 51.6 55.1 53.3 1.0 50.1 54.6 51.6 1.2 Preanal length (% SL) 69.1 67.8 73.4 70.9 1.3 65.9 70.3 68.8 1.3 Pectoral – pelvic-fin origin length (% SL) 27.5 24.2 30.0 25.9 1.2 21.7 25.4 23.8 1.0 Pelvic – anal-fin origin length (% SL) 19.8 16.7 44.1 19.4 4.6 15.6 19.9 17.5 1.3 Length of caudal peduncle (% SL) 18.3 18.0 21.3 19.7 0.9 20.0 23.2 21.4 0.8 Dorsal-fin base length (% SL) 11.1 9.4 12.9 11.1 0.8 8.1 12.1 10.4 1.2 Dorsal fin depth (% SL) 17.6 15.8 20.4 17.9 1.4 15.9 21.8 18.0 1.5 Anal-fin base length (% SL) 11.3 9.4 12.0 10.9 0.6 10.5 13.2 11.4 0.7 Anal fin depth (% SL) 12.3 10.6 15.0 13.1 1.1 12.0 16.2 14.0 1.4 Pectoral fin length (% SL) 18.4 18.0 21.9 20.2 1.0 21.8 26.4 23.8 1.4 Pelvic fin length (% SL) 12.9 12.5 15.6 14.1 0.8 14.2 17.5 15.7 0.9 Head length (% SL) 27.0 26.7 29.4 27.8 0.8 26.3 30.1 28.0 1.1 Head length (% body depth) 112.9 114.5 142.6 127.4 6.6 116.5 142.9 130.0 8.5 Head depth at nape (% SL) 17.4 16.5 18.7 17.6 0.6 15.7 18.8 17.3 0.9 Head depth at nape (% HL) 64.6 60.0 67.9 63.4 1.8 57.1 65.9 62.0 2.9 Maximum head width (% SL) 14.8 13.7 16.6 14.9 0.6 13.3 16.2 14.7 0.8 Maximum head width (% HL) 54.9 50.5 61.2 53.7 2.3 51.0 57.4 53.0 2.4 Maximum cranial width (% cranium roof length) 63.5 64.9 79.6 72.5 4.3 66.3 75.8 69.1 2.8 Snout length (% SL) 8.1 7.9 9.8 8.7 0.5 7.7 9.3 8.6 0.4 Snout length (% HL) 30.0 29.4 33.4 31.3 1.1 27.2 33.0 30.8 1.6 Eye horizontal diameter (% SL) 6.0 4.7 7.7 6.1 0.7 5.4 8.0 6.3 0.7 Eye horizontal diameter (% HL) 22.4 17.3 27.3 21.9 2.5 20.6 26.4 22.5 2.0 Eye horizontal diameter (% interorbital width) 70.7 52.0 86.7 67.5 8.3 59.8 92.5 73.1 11.9 Postorbital distance (% HL) 51.9 49.7 57.2 53.3 1.9 48.1 54.4 51.2 1.8 Interorbital width (% SL) 8.5 8.0 10.0 9.0 0.6 7.5 9.9 8.7 0.8 Interorbital width (% HL) 31.6 29.7 35.9 32.5 1.9 26.2 34.8 31.2 2.7 Length of upper jaw (% HL) 27.8 26.7 30.5 28.7 0.9 27.4 29.8 28.5 0.8 Length of upper jaw (% SL) 7.5 7.2 8.7 8.0 0.4 7.3 8.9 8.0 0.5 Length of lower jaw (% SL) 9.9 10.0 12.2 10.7 0.5 10.0 12.5 11.0 0.9 Length of lower jaw (% HL) 36.7 36.3 43.8 38.5 1.5 36.3 44.0 38.9 2.5 Length of lower jaw (% interorbital width) 116.0 110.0 133.8 121.2 6.0 109.1 138.7 123.0 9.8 Length of lower jaw (% depth of operculum) 116.5 110.8 133.8 116.1 4.9 111.5 132.3 118.5 7.0 Two new freshwater fish species of the genus Telestes (Actinopterygii, Cyprinidae)... Telestes dabar sp. n. urn:lsid:zoobank.org:act:4A50A07B-57BD-472B-9B00-6941D8C6779A http://species-id.net/wiki/Telestes_dabar Figs 1a, 2a, 3a Another black lateral stripe occurring more ventrally, on otherwise pale ventral portion of trunk; this stripe extending from eye or opercle (or just behind oper- cle) to at least vertical through point halfway between origins of pelvic and anal fins, sometimes extending as poorly coalesced spots onto caudal peduncle. Dash-like black marking present along internal procurrent rays of caudal-fin dorsal lobe, and elongate black blotch present at bases of 3rd–7th branched rays of dorsal fin. Black pigment also occurring on rays of dorsal and caudal fins, but its intensity varying among individuals. Peritoneum black. This general pattern of pigmentation retained in formaldehyde-fixed and ethanol-preserved specimens. Live specimens collected from May through September, both males and females, exhibiting yellowish-orange pigment at bases of all fins, especially pectoral and anal fins, and yellowish pigment on iris and along anterior, dorsal and posterior margins of operculum. Colouration of specimens in cold season unknown. back. Another black lateral stripe occurring more ventrally, on otherwise pale ventral portion of trunk; this stripe extending from eye or opercle (or just behind oper- cle) to at least vertical through point halfway between origins of pelvic and anal fins, sometimes extending as poorly coalesced spots onto caudal peduncle. Dash-like black marking present along internal procurrent rays of caudal-fin dorsal lobe, and elongate black blotch present at bases of 3rd–7th branched rays of dorsal fin. Black pigment also occurring on rays of dorsal and caudal fins, but its intensity varying among individuals. Peritoneum black. This general pattern of pigmentation retained in formaldehyde-fixed and ethanol-preserved specimens. Live specimens collected from May through September, both males and females, exhibiting yellowish-orange pigment at bases of all fins, especially pectoral and anal fins, and yellowish pigment on iris and along anterior, dorsal and posterior margins of operculum. Colouration of specimens in cold season unknown. back. Another black lateral stripe occurring more ventrally, on otherwise pale ventral portion of trunk; this stripe extending from eye or opercle (or just behind oper- cle) to at least vertical through point halfway between origins of pelvic and anal fins, sometimes extending as poorly coalesced spots onto caudal peduncle. Dash-like black marking present along internal procurrent rays of caudal-fin dorsal lobe, and elongate black blotch present at bases of 3rd–7th branched rays of dorsal fin. Black pigment also occurring on rays of dorsal and caudal fins, but its intensity varying among individuals. Peritoneum black. Telestes dabar sp. n. urn:lsid:zoobank.org:act:4A50A07B-57BD-472B-9B00-6941D8C6779A http://species-id.net/wiki/Telestes_dabar Figs 1a, 2a, 3a dabar 5 17 8 7 3 54.2 9.58 62–69 66.6 2.41 T. miloradi 6 5 5 2 60.3 3.91 58–69 62.8 2.93 T. metohiensis, Gatačko Polje 11 10 25 5 61.0 3.92 56–71 63.5 3.74 T. metohiensis, Nevesinjsko Polje 1 9 11 19 5 2 61.1 5.32 60–71 65.0 3.07 e 3. Total lateral-line scale and total lateral-series scales counts in Telestes dabar, T. miloradi an etohiensis. Nina G. Bogutskaya et al. / ZooKeys 180: 53–80 (2012) 64 Table 4a. Vertebral counts in Telestes species endemic in Croatia and Bosnia and Herzegovina. Total vertebrae. Table 4a. Vertebral counts in Telestes species endemic in Croatia and Bosnia and Herzegovina. Total vertebrae. 38 39 40 41 42 mean SD T. dabar 49 101 11 39.8 0.56 T. miloradi 1 10 11 40.5 0.60 T. metohiensis, Gatačko Polje 6 33 12 2 39.2 0.68 T. metohiensis, Nevesinjsko Polje 10 112 35 39.2 0.52 T. croaticus 5 21 38.8 0.40 T. fontinalis 1 7 3 39.2 0.60 T. karsticus 2 2 3 41.1 0.89 T. polylepis 2 9 6 41.2 0.66 T. turskyi 2 9 6 1 40.3 0.77 T. ukliva 8 42.0 0 Table 4b. Vertebral counts in Telestes species endemic in Croatia and Bosnia and Herzegovina. Vertebral formulae. 38 39 40 41 42 mean SD T. dabar 49 101 11 39.8 0.56 T. miloradi 1 10 11 40.5 0.60 T. metohiensis, Gatačko Polje 6 33 12 2 39.2 0.68 T. metohiensis, Nevesinjsko Polje 10 112 35 39.2 0.52 T. croaticus 5 21 38.8 0.40 T. fontinalis 1 7 3 39.2 0.60 T. karsticus 2 2 3 41.1 0.89 T. polylepis 2 9 6 41.2 0.66 T. turskyi 2 9 6 1 40.3 0.77 T. ukliva 8 42.0 0 Table 4b. Vertebral counts in Telestes species endemic in Croatia and Bosnia and Herzegovina. Vertebral formulae. formulae. 21+17 22+16 22+17 22+18 22+19 23+16 23+17 23+18 23+19 24+16 24+17 24+18 24+19 T. dabar 41 60 8 40 10 1 1 T. miloradi 1 6 1 4 10 T. metohiensis, Gatačko Polje 6 28 2 5 10 2 T. metohiensis, Nevesinjsko Polje 7 58 12 33 24 T. croaticus 2 3 16 5 T. fontinalis 1 1 7 3 T. karsticus 1 1 2 1 2 T. polylepis 1 1 8 4 1 2 T. turskyi 1 4 1 5 6 1 T. ukliva 3 5 back. Telestes dabar sp. n. urn:lsid:zoobank.org:act:4A50A07B-57BD-472B-9B00-6941D8C6779A http://species-id.net/wiki/Telestes_dabar Figs 1a, 2a, 3a This general pattern of pigmentation retained in formaldehyde-fixed and ethanol-preserved specimens. Live specimens collected from May through September, both males and females, exhibiting yellowish-orange pigment at bases of all fins, especially pectoral and anal fins, and yellowish pigment on iris and along anterior, dorsal and posterior margins of operculum. Colouration of specimens in cold season unknown. Two new freshwater fish species of the genus Telestes (Actinopterygii, Cyprinidae)... 65 Table 4c. Vertebral counts in Telestes species endemic in Croatia and Bosnia and Herzegovina. Abdominal, caudal and predorsal vertebrae in Telestes dabar, T. mi- loradi and T. metohiensis. Abdominal vertebrae Caudal vertebrae Predorsal vertebrae 21 22 23 24 Mean SD 15 16 17 18 19 mean SD 12 13 14 15 mean SD T. dabar 101 58 2 22.4 0.51 9 82 70 17.4 0.59 24 126 11 13.9 0.46 T. miloradi 8 14 22.6 0.49 5 16 1 17.8 0.50 15 7 14.3 0.5 T. metohiensis, Gatačko Polje 36 17 22.3 0.47 7 42 4 16.9 0.46 3 38 12 14.2 0.50 T. metohiensis, Nevesinjsko Polje 1 92 64 22.4 0.51 2 43 105 7 16.7 0.56 2 124 31 14.2 0.42 T. croaticus 2 19 5 22.1 0.52 8 18 16.7 0.47 21 5 14.2 0.40 T. fontinalis 2 9 22.8 0.40 7 4 16.4 0.50 2 9 14.8 0.40 T. karsticus 1 4 2 23.1 0.69 1 5 1 18.0 0.58 4 3 13.4 0.53 T. polylepis 1 13 3 23.1 0.49 2 11 4 18.1 0.60 1 16 13.9 0.24 T. turskyi 5 13 22.7 0.46 1 6 10 1 17.6 0.62 16 2 13.1 0.32 T. ukliva 3 5 23.6 0.52 5 3 18.4 0.52 2 5 1 12.9 0.64 Nina G. Bogutskaya et al. / ZooKeys 180: 53–80 (2012) 66 Figure 4. Map of distribution of Telestes dabar (diamond), T. miloradi (star) and T. metohiensis (square) in karst fields of Eastern Herzegovina and Dubrovnik littoral; white circles show ponors and white circles with black dots - springs. Figure 4. Map of distribution of Telestes dabar (diamond), T. miloradi (star) and T. metohiensis (square) in karst fields of Eastern Herzegovina and Dubrovnik littoral; white circles show ponors and white circles with black dots - springs. Sexual dimorphism. Genital papilla absent in both males and females. Most mor- phometric characters not significantly different between males and females (Table 1a) with five exceptions. Telestes dabar sp. n. urn:lsid:zoobank.org:act:4A50A07B-57BD-472B-9B00-6941D8C6779A http://species-id.net/wiki/Telestes_dabar Figs 1a, 2a, 3a In males, distance between origins of pectoral and pelvic fins long- er than in females (P<0.0001), dorsal fin deeper (P<0.02), anal fin deeper (P<0.0001), pectoral fin longer (P<0.0001), pectoral fin often reaching pelvic-fin origin in males, and pelvic fin longer (P<0.0001), pelvic fin often reaching anal-fin origin in males. Two new freshwater fish species of the genus Telestes (Actinopterygii, Cyprinidae)... 67 Figure 5. a Habitat of Telestes dabar: Opačica River at Potkom, Dabarsko Polje (type locality) b Opačica River 100 m away from the spring (8 July 2011) c–d habitat of Telestes metohiensis: Nevesinjsko Polje; c – spring Ljeskovik in Zalomka River (8 July 2011) d Zovidolka River at Udbine (8 July 2011). All in Bosnia and Herzegovina. a c d b b b d Figure 5. a Habitat of Telestes dabar: Opačica River at Potkom, Dabarsko Polje (type locality) b Opačica River 100 m away from the spring (8 July 2011) c–d habitat of Telestes metohiensis: Nevesinjsko Polje; c – spring Ljeskovik in Zalomka River (8 July 2011) d Zovidolka River at Udbine (8 July 2011). All in Bosnia and Herzegovina. In samples collected in May, ripe males with small but prominent conical breeding tubercles. Tubercles regularly covering entire body, including dorsal and ventral sur- faces of caudal peduncle, except for ventralmost surface of head. Single tubercle located on each scale. On all fins (except for caudal fin), tubercles present on both sides along all rays and on fin membrane, being particularly dense along marginal rays. Tubercles forming rows along outer margins of operculum and pectoral fin; tubercles in those rows larger than others on body. Degree of tubercle development varying between males with regard to both size of tubercles and their location. Tubercles always present on head, back, and pectoral fin. Males retaining tubercles, though reduced in size and density, until September. Distribution. The new species is known from two rivers, Vrijeka and Opačica, in the Dabarsko Polje of Eastern Herzegovina in Bosnia and Herzegovina (Fig. 4). Habitat and biology. From May through September T. dabar is found in shal- low water of those river sections that are adjacent to and filled from underground Nina G. Bogutskaya et al. / ZooKeys 180: 53–80 (2012) 68 springs. There is no current, and the water is clean (Fig. 5a). Telestes miloradi sp. n. l id b k Telestes miloradi sp. n. urn:lsid:zoobank.org:act:9A45C6CE-B7BF-4593-BAF6-0D10C45C78EA http://species-id.net/wiki/Telestes_miloradi Fig. 7 Holotype. NMW 95296 (ex 51169), 66.7 mm SL; CROATIA: stream Ljuta at Gruda [misspelt Grinda in Steindachner (1901: 197)), 1901, coll. Kolombatovič. Holotype. NMW 95296 (ex 51169), 66.7 mm SL; CROATIA: stream Ljuta at Gruda [misspelt Grinda in Steindachner (1901: 197)), 1901, coll. Kolombatovič. Paratypes. NMW 51169, 13 (? syntypes [now paralectotypes] of Paraphoxinus metohiensis), 31.4–62.6 mm SL, same data as holotype; NMW 51170, 4 (syntypes [now paralectotypes] of Paraphoxinus metohiensis), 57.9–66.4 mm SL, same data as holotype; NMW 51171, 3 syntypes [now paralectotypes] of Paraphoxinus metohiensis, 74.6–83.1 mm SL, same data as holotype; NMW 51173, 1 syntype [paralectotype] of Paraphoxinus metohiensis, 119.3 mm SL, same data as holotype. Diagnosis. Telestes miloradi is distinguished from T. metohiensis and T. dabar by having the following combination of characters: slightly curved, relatively nar- row dark stripe (obvious in most preserved specimens) present on ventral portion of trunk from just behind operculum to vertical at or anterior to origin of anal fin, this stripe separated from dark pigmented area on back along its entire length; scales on most of body not overlapping; mouth subterminal with tip of mouth cleft at or be- low level of ventral margin of eye; snout not fleshy; lateral line complete with 55–67 total scales; branched dorsal-fin rays 8½; branched anal-fin rays 8½; gill rakers usu- ally 8–10, mode 9; total vertebrae usually 40 or 41; abdominal vertebrae 22–23, mode 23; caudal vertebrae 16–18, mode 18; head width 48–58% HL, and lower jaw short, length 8–10% SL. Diagnosis. Telestes miloradi is distinguished from T. metohiensis and T. dabar by having the following combination of characters: slightly curved, relatively nar- row dark stripe (obvious in most preserved specimens) present on ventral portion of trunk from just behind operculum to vertical at or anterior to origin of anal fin, this stripe separated from dark pigmented area on back along its entire length; scales on most of body not overlapping; mouth subterminal with tip of mouth cleft at or be- low level of ventral margin of eye; snout not fleshy; lateral line complete with 55–67 total scales; branched dorsal-fin rays 8½; branched anal-fin rays 8½; gill rakers usu- ally 8–10, mode 9; total vertebrae usually 40 or 41; abdominal vertebrae 22–23, mode 23; caudal vertebrae 16–18, mode 18; head width 48–58% HL, and lower jaw short, length 8–10% SL. Description. Telestes dabar sp. n. urn:lsid:zoobank.org:act:4A50A07B-57BD-472B-9B00-6941D8C6779A http://species-id.net/wiki/Telestes_dabar Figs 1a, 2a, 3a Females with eggs and just-spent females were caught on 24 May 2001 in Vrijeka River and mature males and just-spent females on 31 May 2000 in Opačica River. The size of the ripe eggs was 1.3–1.7 mm in diameter. In all examined samples females predominate. The smallest spent female was 45.0 mm SL, and the smallest ripe male 43.7 mm SL. No other fishes were caught in Opačica together with T. dabar while D. ghetaldii were collected in Vrijeka. Etymology. The specific name, dabar, refers to the type locality, Dabarsko, or Da- bar Polje; it is a noun in apposition. Telestes miloradi sp. n. l id b k Morphometric data are summarised in Table 1b, selected counts in Tables 2–4. General appearance can be seen in Figs 7a and 7b. Body compressed, elongate. Caudal peduncle depth equal to or only slightly less than half maximum body depth; head length greater than maximum body depth. Eye small, its diam- eter smaller than snout length. Snout not fleshy, rostral cap covering only part of upper lip, at least in preserved specimens. Mouth subterminal, tip of mouth cleft Two new freshwater fish species of the genus Telestes (Actinopterygii, Cyprinidae)... 69 at level of ventral margin of eye or, more frequently, below it. Lower jaw-quadrate junction at vertical through anterior half of eye. Length of lower jaw 8–10% SL or 33–38% HL, or 96–107% depth of operculum (equal to depth of operculum on average). Dorsal fin with 8½ branched rays. Dorsal-fin origin above posterior end of pelvic- fin base. Anal fin with 8½ branched rays. Outer margin of anal fin slightly concave. Caudal fin moderately forked, lobes weakly pointed, with 9+8 principal branched rays. Total gill rakers (Table 2) 8 (1 specimen), 9 (11) or 10 (6), 10 in holotype. Pharyngeal teeth 5–4, hooked, slightly serrated (examined in 5 specimens). Scales covering entire body including pre-pectoral area and abdomen, overlap- ping on most parts of body. Scales regularly set; lateral-line scales and scales above and below it of about equal size. Lateral line complete (Table 3), 55–67 scales in total, 61 in holotype. Lateral line not curving above anal-fin origin. Number of scales in total lateral series 58–67 (modal range 62–64), 63 in holotype. Parietal segment of CSO lacking. CPM not communicating with CIO, terminat- ing over upper margin of opercular antedorsal process or communicating with CIO (on one side in 3 specimens). CSO complete with 8, rarely 7 or 9, pores. CIO com- plete with 14–16 pores and with 4 canal openings on first infraorbital. CPM complete, with 14–16 pores (4 canal openings on dentary, and 7–9, usually 8, on preoperculum). CST complete, with 5 or 7 pores. Total vertebrae (Table 4) 39 (1), 40 (10) or 41 (11), 41 in holotype; abdominal vertebrae 22 (8) or 23 (14), 23 in holotype; caudal vertebrae 17 (5), 18 (16) or 19 (1), 18 in holotype; predorsal vertebrae 13 (15) or 14 (7), 14 in holotype; intermediate ver- tebrae 3 (12) or 4 (9), 3 in holotype. Comparative remarks Telestes dabar, T. miloradi, T. metohiensis, T. croaticus, and T. fontinalis are distinguished from all congeners by having the pharyngeal teeth in one row, 5–4 or 5–5 (vs. usually 2.5–5.2 or 2.5–4.2), having the preoperculo-mandibular canal terminating in a free pore at the upper margin of the opercular antedorsal process and not communicating with the infraorbital canal (vs. communicating), and in lacking a postcleithrum (vs. postcleithrum present). All five species possess a dark midlateral stripe from the head to the caudal peduncle that forms the ventral border of the darkly pigmented area on the back. This feature is similar to that in some other Telestes species (Kottelat and Freyhof 2007: 282–289). Within this group, T. dabar, T. miloradi, and T. metohiensis differ from T. croaticus and T. fontinalis in having an additional black lateral stripe occurring on the otherwise pale ventral portion of the trunk. This character was con- sidered unique for T. metohiensis (Kottelat and Freyhof 2007: 284). In T. dabar and T. miloradi this ventral stripe (Fig. 1a, 2a, 7) is narrow and extends from just behind the operculum maximally to a vertical through the origin of the anal fin. In T. metohiensis, the stripe (Fig. 1b, 2b–c) is wide and usually extends posteriorly to the caudal peduncle where it merges with the main pigmented area. The pale area between the dark area on the back and the ventral stripe varies in length and depth, being the smallest in females (Fig. 1b, 2b–c). Besides the presence of the ventral stripe, Telestes dabar and T. miloradi are fur- ther distinguishable from T. croaticus and T. fontinalis by usually having 8½ branched dorsal-fin rays (vs. usually 7½). Telestes dabar differs from T. croaticus by usually having 40 total vertebrae (vs. usually 39) (Table 4); a maximum head width of 42–52% HL (averaging 50% HL in females and 48% HL in males), which is considerably smaller than the head depth at nape, 61–71% HL (averaging 67% HL in females and 65% HL in males) (vs. the maximum head width only slightly smaller than the head depth or about equal to it); and a smaller size, up to 82 mm SL (vs. up to 160 mm). Telestes da- bar can be further distinguished from T. fontinalis by having 5–4 pharyngeal teeth (vs. Telestes miloradi sp. n. l id b k Vertebral formulae 22+17 (1), 22+18 (7), 23+17 (5) and 23+18 (10), 23+18 in holotype. Colouration. In preserved specimens, dark back contrasting sharply with pale area below lateral midline. Dark midlateral stripe extending from head to caudal peduncle forming ventral border of darkly pigmented region on back(faded in some specimens). Another, more conspicuous, dark lateral stripe, occurring on ventral portion of trunk, nar- row and not extending posterior to vertical through origin of anal fin. Peritoneum dark. i Sexual dimorphism. Genital papilla absent in both males and females. Most mor- phometric characters not significantly different between males and females (Table 1). In the three male specimens examined, dorsal fin deeper than in females (P=0.0105); pectoral fin longer (P<0.001), pectoral fin nearly reaching pelvic-fin origin in males and well short of pelvic fin in females; and pelvic fin longer (P<0.0001), pelvic fin almost reaching anal-fin origin in males, well short of anal fin in females.h ii Distribution. The new species is known from Ljuta River in Konavosko Polje, also called Konavoska Ljuta, of Dubrovnik littoral (Fig. 4). Only historical NMW samples are known to us. Etymology. The species is named for Milorad Mrakovčić, Zagreb, in recognition of his many contributions to the study of freshwater fishes in the Adriatic basin. Nina G. Bogutskaya et al. / ZooKeys 180: 53–80 (2012) 70 Comparative remarks 5–5); a usually long, though slightly incomplete and narrowly interrupted, lateral line that reaches the posterior half of the caudal peduncle and has 24–69, usually 54–65, total scales (vs. a short, incomplete, and widely interrupted lateral line terminating in the area between the pectoral and anal fins with usually 23–37 total scales); usually 17 or 18 caudal vertebrae (vs. 16); usually 13 or 14 predorsal vertebrae (vs. 15) (Table 4); 3 or 4 intermediate vertebrae (vs. 5); and a moderately compressed body without any ventral keel (vs. a markedly laterally compressed body and a scaled ventral keel in front of the pelvic fins). i Telestes miloradi further differs from T. croaticus and T. fontinalis in usually having a long, complete lateral line with 55–67 scales vs. an often incomplete and interrupted lateral line with (18–45)51–70 and (17)23–37(56) scales, respectively. The new spe- cies further differs from T. croaticus in having 8–10, most frequently 9, gill rakers, (vs. 8–9, most frequently 8); usually 40 or 41 total vertebrae (vs. 38 or 39); 22–23, mode 23, abdominal vertebrae (vs. 21–23, mode 22); and 17–19, mode 18, caudal vertebrae Two new freshwater fish species of the genus Telestes (Actinopterygii, Cyprinidae)... 71 (vs. 16–17, mode 17) (Table 4). Telestes miloradi can be further distinguished from T. fontinalis by having 5–4 pharyngeal teeth (vs. 5–5); usually 40 or 41 total vertebrae (vs. 38–40, usually 39); 17–19, usually 18, caudal vertebrae (vs. 16–17, often 16) (Table 4); 3 or 4 intermediate vertebrae (vs. 5); and a moderately compressed body without a ventral keel (vs. a markedly laterally compressed body and a scaled ventral keel in front of the pelvic fins).f Telestes miloradi differs from T. croaticus, T. fontinalis, T. metohiensis, and T. dabar in having comparatively well-overlapped scales, especially on the anterior part of the flank and on the caudal peduncle; and scales of about the same size in the lateral line and above and below it (vs. scales usually non-overlapping on most of the body, and scales above and below the lateral line smaller than the lateral-line scales). The scale pattern in T. miloradi is very similar to the pattern found in most leuciscine fishes, e.g. T. karsticus Marčić & Mrakovčić, 2011 (Fig. 8), T. turskyi (Heckel, 1843), T. ukliva (Heckel, 1843). Comparative remarks The presence of overlapping scales is a plesiomorphic feature for the Leuciscinae (Bogutskaya 1990, 1991), as is an interconnection of CPM and CIO (e.g., T. karsticus). In addition to overlapping scales, a few specimens of T. miloradi exhibit interconnected CPM and CIO. In this study, we compared specimens of T. metohiensis from Gatačko, Cerničko, and Nevesinjsko poljes and found that specimens from the Nevesinjsko Polje (labeled as affinis by Steindachner although the name affinis was never published), though somewhat different in usually having smaller and more scattered scales, are similar to T. metohiensis from the Gatačko and Cerničko poljes in all other aspects examined by us (Tables 1c–d, 2–4). We suppose that a reason for Steindachner’s opinion could be the differences evident between the NMW specimens of T. metohiensis from the Gatačko Polje (Mušnica River, Gacko) and those from the Nevesinjsko Polje (Zalomka River) (Fig. 6). Additional material examined by us revealed that the differences noted above in scales may be size-dependent: larger specimens of T. metohiensis in both poljes usually have more densely set scales. A specimen of T. metohiensis from Gatačko Polje, NMW 51176:3 (87.9 mm SL) is designated here as lectotype to ensure taxonomic stability in the event that T. metohiensis from the Nevesinjsko Polje is recognised as taxonomically distinct in the future. Both T. dabar and T. miloradi are distinguishable from T. metohiensis by usually having 8½ branched dorsal-fin rays (vs. usually 7½: 7½ found in 192 specimens and 8½ in 22); 39–41, modes 40 and 41, total vertebrae (vs. 38–40, rarely 41, mode 39); 16–19, modes 17 and 18, caudal vertebrae (vs. 15–17, usually 16 or 17) (vs. 15–17, usually 16 or 17) (Table 4; differences are statistically significant at P<0.0001); and more numerous gill rakers, 9 or 10 (vs. (7)8–9(10), most frequently 8) (Table 2). Tel- estes dabar further differs from T. metohiensis in usually having an interrupted lateral line with 24–69, usually 54–65, total scales (vs. usually complete with 58–65 scales) (Table 3; difference is statistically significant at P<0.0001). The scale pattern also dis- tinguishes T. dabar from T. metohiensis. In T. dabar, the scales (Fig. 3a) are densely set, but they do not overlap on most parts of the body except behind the pectoral girdle and on the caudal peduncle; the scales above and below the lateral line are only slightly Nina G. Bogutskaya et al. Comparative remarks metohiensis Th h T l i k f f f h 13 i k lj l d i a b Figure 7. Telestes miloradi, Croatia: Ljuta River at Gruda, Konavosko Polje a Holotype, male, 66.7 mm SL, NMW 95296 b paratype, female, 119.3 mm SL, NMW 51173. Figure 8. Telestes karsticus, PZC 504, 62.6 mm SL, Croatia: Sušik River, Danube drainage. a b Figure 7. Telestes miloradi, Croatia: Ljuta River at Gruda, Konavosko Polje a Holotype, male, 66.7 mm SL, NMW 95296 b paratype, female, 119.3 mm SL, NMW 51173. a b Figure 7. Telestes miloradi, Croatia: Ljuta River at Gruda, Konavosko Polje a Holotype, male, 66.7 mm SL, NMW 95296 b paratype, female, 119.3 mm SL, NMW 51173. a a b Figure 7. Telestes miloradi, Croatia: Ljuta River at Gruda, Konavosko Polje a Holotype, male, 66.7 mm SL, NMW 95296 b paratype, female, 119.3 mm SL, NMW 51173. b Figure 7. Telestes miloradi, Croatia: Ljuta River at Gruda, Konavosko Polje a Holotype, male, 66.7 mm SL, NMW 95296 b paratype, female, 119.3 mm SL, NMW 51173. Figure 8. Telestes karsticus, PZC 504, 62.6 mm SL, Croatia: Sušik River, Danube drainage. Figure 8. Telestes karsticus, PZC 504, 62.6 mm SL, Croatia: Sušik River, Danube drainage. Figure 8. Telestes karsticus, PZC 504, 62.6 mm SL, Croatia: Sušik River, Danube drainage. Comparative remarks / ZooKeys 180: 53–80 (2012) 72 Figure 6. Telestes metohiensis. a NMW 51176:3, lectotype, 87.9 mm SL, ‘Mušica’ [Mušnica] River b NMW 51090 (labelled as affinis nomen museale), 57.9 mm SL, Zalomska [Zalomka River]. a b Figure 6. Telestes metohiensis. a NMW 51176:3, lectotype, 87.9 mm SL, ‘Mušica’ [Mušnica] River b NMW 51090 (labelled as affinis nomen museale), 57.9 mm SL, Zalomska [Zalomka River]. smaller than the lateral-line scales; and the scales are oval, somewhat deeper than long, on the flanks and elongated, longer than deep with a prominent posterior attenuation, on the caudal peduncle. In T. metohiensis the scales are more or less widely spaced and do not overlap on the entire body except for the lateral line; this feature can be seen in live specimens, Fig. 2b); the scales (Fig. 3b) above and below the lateral line usually are considerably smaller than the lateral-line scales; and the scales are almost circular on both the flanks and the caudal peduncle. Telestes dabar is further distinguished from T. metohiensis by having a black-and-white general colouration except for yellowish- orange pigmentation of the fin bases and yellow pigmentation in the iris and along the operculum in adults (vs. yellowish-green or greenish-bronze pigmentation of the whole body and fins in both young and adults, Fig. 2b–c). With regard to the morphometric features, T. dabar is rather similar to T. metohiensis, differing only in having a narrower head, maximum head width 12–15% SL or 43–52% HL and maximum cranial width 60–73% cranium roof length (vs. 13–17% SL or 51–59% HL, and 64–80% cranium roof length) (Table 1; differences are statistically significant at P<0.0001).f fi Besides the characters mentioned above, Telestes dabar differs from T. miloradi in having the lateral-line scales larger than the scales above and below it (vs. of about equal size); usually an interrupted lateral line making a sharp curvature upward above the anal-fin base (vs. complete and making no sharp curvature); the length of lower jaw 10–12% SL, 36–41% HL, 102–132% depth of operculum (vs. 8–10% SL, 33–38% HL, 96–107% depth of operculum) (Table 1; differences are statistically significant at P<0.0001). Two new freshwater fish species of the genus Telestes (Actinopterygii, Cyprinidae)... 73 f fi p f g ( p yg , yp ) 7 Comments on the distribution and conservation of T. dabar, T. miloradi and T. Comments on the distribution and conservation of T. dabar, T. miloradi and T. metohiensis The three Telestes species are known from four of the 13 main karst poljes located in Eastern Herzegovina (Bosnia and Herzegovina) and in Dubrovnik littoral area (Croa- tia) (Fig. 4). These karst poljes are part of a high-karst geotectonic unit known as Di- naric Karst, which consists of Mesozoic carbonate formations. The depth of soluble and highly karstified rocks here exceeds 3000 m (Milanović 1981, 2006). A polje (means “field” in many Slavic languages) is a large closed depression draining underground with a flat floor. Its streams may be permanent, intermittent and perennial, and, in natural conditions, a polje is subject to periodic flooding and becomes a lake. In East- ern Herzegovina and in the Dubrovnik littoral area stepwise poljes are distributed from Nina G. Bogutskaya et al. / ZooKeys 180: 53–80 (2012) 74 60 m up to 1,080 m above sea level (Fig. 4). Streams and rivers appear from temporary or permanent springs and sink underground through swallow holes called ponors. In general, hydro-systems of all poljes under consideration (Fig. 4), except for the Kona- vosko Polje with direct connection to the Adriatic Sea and the Polje Gradac with no springs or surface flows, belong to the Neretva drainage area and form a complex net of underground flows. Within the Neretva drainage, the poljes belong to catchments of the Buna River (Slato and Nevesinjsko), the Bregava River (Lukavačko and Dabarsko), and the Trebišnijca River (Gatačko, Fatničko and others). At present, no one polje has a direct groundwater (surface) flow connection with Neretva or its tributaries. His- torically, interconnections between variable surface streams and between them and the main Neretva course occurred during different geological epochs. Fish distributions can provide some evidence of this. Conversely, very local distribution of some fishes may indicate the isolation of some surface drainage systems for a long time. y g y g Slato [Zlato] Polje (1,020‒1,080 m above sea level) is situated at the highest el- evation of all Eastern Herzegovina poljes. Ćurčić (1915a) reported finding no fishes at Slato Polje, and no fishes are known from later literature or from museum collec- tions. The Slato Polje is connected with Nevesinjsko Polje through a narrow valley that is now dry. The Nevesinjsko Polje, the largest polje in Eastern Herzegovina, has a surface area of 170 km2 and is located at an elevation from 870 m to 800 m above sea level. Comments on the distribution and conservation of T. dabar, T. miloradi and T. metohiensis The lowest point is Biograd Ponor, which is the terminus of the Zalomka River that starts at Raščelica near the Gatačko Polje. This river has a permanent flow only between Fojnica and Črni Kuk. Along the river bed downstream from Črni Kuk there are a lot of ponors. The most prominent leakage zone is in the Rilja section where the Zalomka is active only 213 days per year, on average (Milanović 1981, 2006). In the warm season almost the entire river bed of the Zalomka within the Nevesinjsko Polje is dry, and fishes are found in its upper section only. Only four species are known from there: Salmo sp., Squalius cf. squalus, Squalius svallize Heckel & Kner, 1858, Phoxinus sp. (PZ personal observations). Ćurčić (1915a) reported that Paraphoxinus metohienis was the most numerous species around Fojnica, but at present only Phoxinus sp. was found there by PZ. Further downstream, T. metohiensis occurs in those very short river sections that are adjacent to permanent springs such as Ljeskovik. This species also oc- curs in upper reaches of the Zavidolka River that temporarily flows to the Zalomka in the east from the Biograd Ponor (our data). Delminichthys ghetaldii is absent from the Nevesinjsko Polje and the Zalomka system. j j Dabarsko Polje, about 20 km long and 1 to 3 km wide, is located close to the Nevesinjsko Polje but isolated from it. The Dabarsko Polje lies more than 400 m of elevation below the Nevesinjsko Polje. At present, the Dabarsko Polje is a closed basin without a possibility for surface runoff. All waters of the Dabarsko Polje catchment flow through underground karst conduits toward the springs of the Bregava River, though historically the Polje drained to the Bregava River canyon that is now dry. The lowest point is the Ponikva Ponor (471 m above sea level), the terminus of a single permanent stream in the polje, the Vrijeka River, which is only 2.5 km long (Milanović 1981, 2006). The Opačica River located in the northwestern part of the polje is longer Two new freshwater fish species of the genus Telestes (Actinopterygii, Cyprinidae)... 75 but intermittent. Only two native species occur in Dabarsko Polje: D. ghetaldii and T. dabar (recorded earlier under the name Phoxinellus metohiensis) in Vrijeka and only T. dabar in Opačica (our data). Comments on the distribution and conservation of T. dabar, T. miloradi and T. metohiensis Delminichthys (as Phoxinellus) ghetaldii was first re- corded in Sušica and Ljelješnica cave springs by Ćurčić (1915a) and then confirmed by PZ’s findings in a stream flowing from the Ljelješnica Cave (Zupančič and Bogutskaya 2002) and in the Vrijeka. It is relatively less abundant in Vrijeka than T. dabar. A trout has been introduced into Vrijeka. j The small Lukavačko Polje (2.5 km2) is located at an elevation of 852–880 m east from the Dabarsko Polje. The two poljes are divided by about 400 m of elevation. Fatničko Polje is also a small closed basin (5.6 km2) located southeast from the Dabar- sko Polje at a much lower elevation, 462–470 m above sea level. The Fatničko Polje is divided from the Dabarsko Polje by an extremely karstified limestone ridge which is about 2 km wide. The most prominent karst features of the Fatničko Polje are the Obod temporary spring and the Pasmica Ponor. About 85% to 90% of the Fatnicko Polje wa- ter flows to the Trebišnjica springs and 10% to 15% to the Bregava springs (Milanović 1981, 2006). Ćurčić (1915a, 1915b), Taler (1953a, 1953b), Sabioncello (1967) and Vuković (1977) reported P. metohiensis from the Lukavačko and Fatničko poljes. How- ever, no extant samples confirm these reports. In 1998–2001, PZ found only D. ghet- aldii in Fatničko Polje and no D. ghetaldii or T. metohiensis in the Lukavačko Polje (Zupančič and Bogutskaya 2002). No other data have been received since then. p g y Gatačko Polje (37.6 km2) consists of two geomorphologically and hydrogeologi- cally interconnected units: Gatačko Polje itself and Small Gatačko Polje. The largest ponor zone is situated in Small Gatačko Polje along the 8 km long tectonic contact between flysch sediments and karstified limestone, from Srdevići to the Šabanov Ponor (936 m above sea level). The entire Gatačko Polje belongs to the catchment area of the Trebisnjica River, except a very small eastern part. The longest underground flow (35 km) in Eastern Herzegovina is between the Srdevići Ponor and the Trebisnjica Springs. The main flow in the Gatačko Polje is the Mušnica River with its tributary Gračanica. The Mušnica is formed by three streams, Vrba, Ulinjski Potok and Jasenički Potok. They flow from the Cemern and Lebršnik mountains. The Mušnica goes from the east- ern to the western border of the polje and along its western border southwards before it completely sinks in the Šabanov Ponor. Comments on the distribution and conservation of T. dabar, T. miloradi and T. metohiensis The flow of the Mušnica River re-appears in the Cerničko Polje where it is named the Ključka River. It originates in a large cave, Vilina Pećina and terminates about 300 m downstream in a ponor that has a recharge capacity of approximately 20 m3 s−l (Milanović 2006). Telestes metohiensis (in Paraph- oxinus or Phoxinellus by earlier authors), Phoxinus sp. and Alburnus neretvae Buj, Sanda et Perea 2010 are known to occur in Mušnica. Phoxinus sp. and, probably, Salmo sp. occur in Gračanica. Many literature sources (see in Zupančič and Bogutskaya 2002) reported Paraphoxinus metohiensis from the Gračanica River at Gacko, but we know of no extant samples. Only T. metohiensis was found (PZC) in short karstic streams of the Cerničko Polje. Telestes metohiensis or close species are absent from other poljes except for the Ko- navosko Polje. This polje is located rather far in the south from poljes inhabited by T. Nina G. Bogutskaya et al. / ZooKeys 180: 53–80 (2012) 76 metohiensis and T. dabar. The Konavosko Polje or Polje Konavli is the lowermost polje in the region (elevation 60 m above sea level) with surface of 48 km2. It is developed along the most important overthrust of the entire dinaric karst region (“High Karst Overthrust”). The largest spring Konavoska Ljuta is located at this contact. In natural conditions it was a temporary flooded polje. Main flows are Ljuta and its tributaries Konavočica and Opačica. At present, a tunnel between the polje and the sea coast drains the polje (Milanović 2006). No recent records exist of a Telestes species in the Ljuta or other springs of the Konavosko Polje. However, we think that efforts are worth trying to find T. miloradi, a new species, described here from the Konavoska Ljuta before it is finally considered extinct. It is known that many species from Phox- inellus, Telestes and Delminichthys genera are able to enter karstic underground waters during droughts and dramatic water table level fluctuation (Milanović 2006; Jelić, 2008; Marčić et al. 2011). However, this phenomenon probably depends considerably on the degree of development, size and depth of caverns, siphonic lakes and pools of estavelles. The ranges of T. metohiensis and T. dabar are extremely small at least during the dry season and in surface water bodies. The recognition of T. dabar as a distinct species will require its Red List evaluation and a re-evaluation of T. Comments on the distribution and conservation of T. dabar, T. miloradi and T. metohiensis metohiensis (now consid- ered as Vulnerable) because of the loss of a part of its former presumed range. Telestes dabar would probably deserve the Critically Endangered status because of its very re- stricted distribution (IUCN 2010). Both species are threatened by habitat destruction, in particular by construction of tunnels for the draining of poljes and controlling their inundations, lining of river beds to obstruct water flow into ponors and reversible com- munication with estavelles. Key to Telestes species of isolated karst river systems of the Adriatic basin in Bos- nia and Herzegovina and Croatia, including Krbavsko Polje 1a Pharyngeal teeth in two rows, usually 2.5–5.2 or 2.5–4.2. Preoperculo-man- dibular canal communicating with infraorbital canal. Postcleithrum present, of moderate size...........................................................................................2 1b Pharyngeal teeth in one row, usually 5–4. Preoperculo-mandibular canal not communicating with infraorbital canal. Postcleithrum minute or absent......3 2a Branched dorsal-fin rays 8½ and branched anal-fin rays 9½. Total lateral-line scales 60–68.................................................................................... T. ukliva 2b Branched dorsal-fin rays 7½ and branched anal-fin rays 8½. Total lateral-line scales 72–79....................................................................................T. turskyi 3a Black stripe on ventral portion of trunk narrow and not reaching caudal pe- duncle. Branched dorsal-fin rays usually 8½................................................4 3b Black stripe on ventral portion of trunk absent or present; if present, stripe wide anteriorly and extending posteriorly to caudal peduncle. Branched dor- sal-fin rays usually 7½..................................................................................5 Key to Telestes species of isolated karst river systems of the Adriatic basin in Bos- nia and Herzegovina and Croatia, including Krbavsko Polje Two new freshwater fish species of the genus Telestes (Actinopterygii, Cyprinidae)... 77 4a 4a Scales overlapping on most of body, scales approximately same size in, above and below lateral line. Lateral line not curving above anal-fin origin. Lower- jaw length 8–10% SL..................................................................T. miloradi 4b Scales non-overlapping on most of body, though densely set. Lateral-line scales larger than scales above and below lateral line. Lateral line making clear curvature above anal-fin origin. Lower-jaw length 10–12% SL........T. dabar 5a Body strongly compressed, abdomen with weakly scaled ventral keel in front of pelvics. Lateral line short, incomplete, terminating between pectoral and anal fins, and usually widely interrupted, with 17–56, usually 23–37, total scales. Branched anal-fin rays 7½............................................... T. fontinalis 5b Body slightly compressed, abdomen rounded. Lateral line long, slightly in- complete or complete, usually terminating on caudal peduncle, and narrowly interrupted, with 24–70, usually 54–68, total scales. Branched anal-fin rays usually 8½...................................................................................................6 6a Black stripe on ventral portion of trunk present......................T. metohiensis 6b Black stripe on vental portion of trunk absent............................ T. croaticus Comparative material Telestes karsticus. PZC 577, 7, 547–750 mm SL, CROATIA: Sušik River at Tomići, Dobra River system (Danube drainage), 24 June 2005, coll. Zupančič. Telestes metohiensis. All from BOSNIA AND HERZEGOVINA. Gatačko Polje: NMW 51176:3, lectotype, mm SL, 87.9 mm SL, ‘Mušica bei Imotski’ [misspelling, should be Mušnica River, Gatačko Polje; Imotski is probably an error because Steindach- ner (1901: 198) clearly indicated that the specimens had been received from karst waters and streams near Gacko, a town in the north of Gatačko Polje (43°09.4'N, 18°31.8'E); see also Zupančič & Bogutskaya 2002], 1899, coll. Redel, Sturani [Sturany]; NMW 51176:1–2, 72.2–82.5 mm SL, same data as holotype; NMW 12972, 1 paralectotype, 92.2 mm SL, same data as holotype; NMW 12973–75 (in one jar with 12972), 3 para- lectotypes, 59.2–95.3 mm SL, same data as holotype; NMW 51172, 2 paralectotypes, 92.0–97.8 mm SL, same data as holotype; NMW 51174, 2 paralectotypes, 76.4–88.5 mm SL, same data as holotype; NMW 51175, 3 paralectotypes, 66.8–75.9 mm SL, same data as holotype; SMF 805, 2, 53.2–65.6 mm SL, Gacko; ZMH 15137, 7, 51.2– 64.4 mm SL, Gacko, Herzegovina; Cerničko Polje: PZC 223, 9, 45.0–90.6 mm SL, source of Ključka River in Vilina Pečina (cave), 43°5.6'N 18°29'E, 16 May 2003, coll. Zupančič; PZC 330, 9, 46.0–88.7 mm SL, same locality and collector, 23 May 2001; PZC 337, 4, 73.7–85.4 mm SL, same locality and collector, 20 Aug. 2000; PZC 368, 4, 49.0–62.2 mm SL, spring at Ključ, 43°5.6'N 18°29.6'E, 23 May 2000, coll. Zupančič; PZC 503, 7, same locality and collector, 3 Aug. 2000. Nevesinjsko Polje: (NMW syn- types [now paralectotypes] of P. metohiensis labeled as Paraphoxinus affinis [handwritten by Steindachner; never published], nomen museale) NMW 9368–9372, 5, Zalomska [Zalomka River], 1896, coll. Hawelka; NMW 51088, 10, 47.6–57.9 mm SL, same Nina G. Bogutskaya et al. / ZooKeys 180: 53–80 (2012) 78 data as above; NMW 51089, 10, 49.6–58.1 mm SL, same data as above; NMW 51092, 5, 51.9–65.5 mm SL, same data as above; NMW 51093, 8, 47.4–58.7 mm SL, same data as above; NMW 51094, 10, same data as above; NMW 51090, 5 (not labeled as syntypes of P. Comparative material metohiensis though exclusion of these specimens from the type series is not evident from the original description), 47.6–57.9 mm SL, same data as above; NMW 51091, 4 (not labeled as syntypes, as above), 63.5–87.1 mm SL, same data as above; PZC 206, 3, 50.4–73.6 mm SL, Batuša River (Zalomka system), 43°19.3'N 18°7.1'E, 9 May 2000, coll. Zupančič; PZC 355, 17, 39.6–63.8 mm SL, same locality and collec- tor, 21 May 2001; PZC 356, 6, 53.2–66.1 mm SL, same locality and collector, 5 May 2000; PZC 312, 19, 50.1–79.8 mm SL, spring Ljeskovik in Zalomka River near Rast and Odžak, 43°12.1'N 18°12.4'E, 21 May 2001, coll. Zupančič; PZC 313, 14, 49.1– 78.7 mm SL, same locality and collector, 1 July 2002; PZC 567, 7, 45.1–86.2 mm SL, same locality and collector, 8 July 2011; PZC 358, 2, 38.2–42.0 mm SL, spring in Zalomka River at Budisavlje, 43°13.3'N 18°13.1'E, 22 May 2011, coll. Zupančič; PZC 523, 15, 39.5–90.9 mm SL, Zovidolka [Zavodoka, Zovidolska] River at Ud- bine, 43°8.6'N 18°14.7'E, 15 Sept. 2006, coll. Zupančič; PZC 293, 12, 49.1–99.2 mm SL, Zovidolka [Zavodoka, Zovidolska] River at Udbine, 43°8.6'N 18°14.7'E, 29 Aug. 2006, coll. Zupančič; PZC 315, 17, 57.8–81.4 mm SL, same locality and collector, 16 July 2002; PZC 523, 15, 39.5–90.9 mm SL, same locality and collector, 15 Sept. 2006; PZC 524, 13, 39.5–90.9 mm SL, same locality and collector, 15 Sept. 2006; PZC 566, 45, same locality and collector, 8 July 2011. y y Telestes polylepis. NMW 18931–41, 11, paralectotypes, 93.6–107.2 mm SL, CRO- ATIA: Josefsthal [Josipdol], 1866; NMW 49713, 3, syntypes, 86.7–90.7 mm SL, CROATIA: Dobra River, 1866; NMW 49714–1, lectotype, 85.9 mm SL, CROATIA: Mresnitza [Mrežnica] River, 1866; NMW 49715, 2, paralectotypes, 80.8–84.1 mm SL, same data as lectotype. Č yp Telestes turskyi. NMW 49629-1, lectotype, 129.5 mm SL, CROATIA: Čicola River [tributary of Krka], Dernis [Drniš] “Heckels Reise, 1840”; 17, paralectotypes, 55.3– 102.3 mm SL. Telestes turskyi. NMW 49629-1, lectotype, 129.5 mm SL, CROATIA: Čicola River [tributary of Krka], Dernis [Drniš] “Heckels Reise, 1840”; 17, paralectotypes, 55.3– 102.3 mm SL. Telestes ukliva. NMW 49639-1, lectotype, 85.0 mm SL, CROATIA: Sign “[aus der Cettina]”, “Heckels Reise, 1840”; NMW 49639-2 and 3, 2, 83.3–84.8 mm SL, same data as lectotype; NMW 49635, 3, paralectotypes, 58.0–75.8 mm SL, same data as lectotype; ZMH 15095, 1, CROATIA: Cetina. Telestes ukliva. Comparative material NMW 49639-1, lectotype, 85.0 mm SL, CROATIA: Sign “[aus der Cettina]”, “Heckels Reise, 1840”; NMW 49639-2 and 3, 2, 83.3–84.8 mm SL, same data as lectotype; NMW 49635, 3, paralectotypes, 58.0–75.8 mm SL, same data as lectotype; ZMH 15095, 1, CROATIA: Cetina. yp Other extensive comparative material is listed in Zupančič and Bogutskaya (2002) and Bogutskaya and Zupančič (2003). yp Other extensive comparative material is listed in Zupančič and Bogutskaya (2002) and Bogutskaya and Zupančič (2003). Acknowledgements We are very thankful to N. Ančić for his assistance during field trips. Sincere appre- ciation goes to B. Herzig, E. Mikschi, C. Prenner and H. Wellendorf (NMW) for providing materials that have been used for comparisons from the collection under their care. We appreciate valuable comments on an earlier version of the manuscript Two new freshwater fish species of the genus Telestes (Actinopterygii, Cyprinidae)... 79 and linguistic revision by two anonymous reviews, the editor Carole Baldwin, and Brian Coad (Canadian Museum of Nature). Contribution by NB and AN to the study was supported by a grant from the Russian Foundation for Basic Research No. 10-04- 01178, All Cypriniformes Species Inventory Project (ACSI-2), and a grant to ZISP from Ministry of Education and Science of Russian Federation. References Bogutskaya NG (1990) Morphological fundamentals in classification of the subfamily Leucis- cinae (Cyprinidae). Communication 1. Journal of Ichthyology 30(3): 63–77. Bogutskaya NG (1991) Morphological fundamentals in classification of the subfamily Leucis- cinae (Cyprinidae). Communication 2. Journal of Ichthyology 31(1): 66–82. Bogutskaya NG, Zupančič P (2003) Phoxinellus pseudalepidotus (Teleostei: Cyprinidae), a new species from the Neretva basin with an overview of the morphology of Phoxinellus species of Ctoatia and Bosnia-Herzegovina. Ichthyological Exploration of Freshwaters 14(4): 369–383. Freyhof J, Lieckfeldt D, Bogutskaya NG, Pitra Ch, Ludwig A (2006) Phylogenetic position of the Dalmatian genus Phoxinellus and description of the newly proposed genus Delminich- thys (Teleostei: Cyprinidae). Molecular Phylogenetics and Evolution 37: 371–384. Ćurčić V (1915a) Narodno ribarstvo u Bosni i Hercegovini II. Hercegovina. Glasnik Zemalj- skog Muzeja Bosne i Hercegovine 27: 37–107. [in Serbian] Ćurčić V (1915b) Narodno ribarstvo u Bosni i Hercegovini II. Hercegovina. Glasnik Zemalj- skog Muzeja Bosne i Hercegovine 27: 313–350. [in Serbian] Heckel JJ (1843) Ichthyologie. In: von Russegger J. Reisen in Europa, Asien und Africa, mit besonderer Rücksicht auf die naturwissenschaftlichen Verhältnisse der betreffenden Länder unternommen in den Jahren 1835 bis 1841. Band 1. Theil 2. Reise in Griechenland, Unte- regypten, im nördlichen Syrien und südöstlichen Kleinasien, mit besonderer Rücksicht auf die naturwissenschaftlichen Verhältnisse der betreffenden Länder; unternommen in dem Jahre 1836. E. Schweizerbart, Stuttgart, 991–1099. IUCN (2010) Guidelines for Using the IUCN Red List Categories and Criteria. Version 8.1. Available at http://www.iucnredlist.org/documents/RedListGuidelines.pdf [accessed on 15 August 2011] Jelić D, Duplić A, Ćaleta M, Žuntić P (2008) Endemske vrste riba jadranskog sliva. Agencija za zaštitu okoliša, Zagreb, 78 pp. [in Croatian] Karaman MS (1972) Süsswasserfische der Türkei. 9 Teil. Revision einiger kleinwüchsiger Cy- prinidengattungen Phoxinellus, Leucaspius, Acanthobrama usw. aus Südeuropa, Kleineasien, Vorder-Asien und Nord Africa. Mitteilungen aus dem Hamburgischen Zoologischen Mu- seum und Institut 69: 115–155. Kottelat M, Freyhof J (2007) Handbook of European freshwater fishes. Kottelat, Cornol, Swit- zerland and Freyhof, Berlin, Germany, 646 pp. Marčić Z, Buj I, Duplić A, Ćaleta M, Mustafić P, Zanella D, Zupančič P, Mrakovčić M (2011) A new endemic cyprinid species from the Danube drainage. Journal of Fish Biology 79: 418–430. Nina G. Bogutskaya et al. / ZooKeys 180: 53–80 (2012) 80 Mrakovčić M, Brigić A, Buj I, Ćaleta M, Mustafić P, Zanella D (2006) Crvena knjiga slatko- vodnih riba. Hrvatske Ministarstvo Kulture, Državni zavod za zaštitu prirode, Zagreb, 253 pp. [In Croatian] Milanović P (1981) Karst Hydrogeology. References Water Resources Publications, Littleton, Colorado, 434 pp. Milanović PT (2006) Karst of Eastern Herzegovina and Dubrovnik Littoral. ZUHRA, Beo- grad, 362 pp. Perea S, Böhme M, Zupančič P, Freyhof J, Šanda R, Özuluğ M, Abdoli A, Doadrio I (2010) Phylogenetic relationships and biogeographical patterns in Circum-Mediterranean sub- family Leuciscinae (Teleostei, Cyprinidae) inferred from both mitochondrial and nuclear data. BMC Evolutionary Biology, 2010: 265. doi: 10.1186/1471-2148-10-265 Sabioncello I (1967) Sistematika slatkovodnih riba. In: Livojevic Z, Bojčić C (Eds) Priručnik za slatkovodno ribarstvo. Agronomski Glasnik, posebna izdanja, 21–90. [in Croatian] Steindachner F (1866) Ichthyologische Notizen (II). Zur Flussfischfauna von Croatien. Sit- zungsberichte der Mathematisch-Naturwissenschaftlichen Classe der Kaiserlichen Akade- mie der Wissenschaften 52(1): 594–599. Steindachner F (1901) Bericht über ein Vorkommen einer bisher noch unbeschriebenen Pa- raphoxinus-Art. Anzeiger der Kaiserlichen Akademie der Wissenschaften. Mathematisch- Naturwissenschaftliche Classe 38(18): 197–198. Taler Z (1953a) Znamenite i osobite ribe u Bosni i Hercegovini. Ribarski List, Sarajevo, 28: 56–57. [in Croatian] Taler Z (1953b) Rasprostranjenje i popis slatkovodnih riba Jugoslavije. Glasnik Prirodnjačkog muzeja Srpske Zemlje, Ser. B (Beograd) 5–6: 423–455. [in Croatian] Vuković T (1977) Ribe Bosne i Hercegovine. Svjetlost, Sarajevo, 205 pp. (in Serbian). Zupančič P, Bogutskaya NG (2000) Description of a new species, Phoxinellus dalmaticus (Cy- prinidae: Leuciscinae), from the Cikola River in the Krka River system, Adriatic basin (Croatia). Natura Croatia 9(2): 67–81. Zupančič P, Bogutskaya NG (2002) Description of two new species, Phoxinellus krbavensis and P. jadovensis, re-description of P. fontinalis Karaman, 1972, and discussion of the distribu- tion of Phoxinellus species (Teleostei: Cyprinidae) in Croatia and in Bosnia and Herzego- vina. Natura Croatica 11(4): 411–437.
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Chemical modification of the 6'‐amino cyclopropyl of abacavir eliminates HLA‐B*57:01‐restricted CD8+ T‐cell activation without loss of antiviral activity
Clinical and translational allergy
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© 2014 Alhaidari et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Authors’ details 1 1University of Liverpool, MRC Centre for Drug Safety Science, UK. 2GlaxoSmithKline, Drug Metabolism & Pharmacokinetics, UK. 1University of Liverpool, MRC Centre for Drug Safety Science, UK. 2GlaxoSmithKline, Drug Metabolism & Pharmacokinetics, UK. Published: 18 July 2014 Published: 18 July 2014 Background Abacavir, a nucleoside reverse transcriptase inhibitor, is used within drug combination therapy to treat the HIV-1 virus. It is a guanosine-analogue pro-drug, and once meta- bolised to carbovir, it acts as a DNA chain terminator. Exposure to abacavir is associated with a high frequency of CD8+ T-cell-mediated hypersensitivity reactions in individuals carrying the HLA risk allele B*57:01. To acti- vate T-cells, abacavir interacts directly with endogenous HLA-B*57:01, altering the repertoire of peptides displayed on the cell surface. Docking studies show that the amino cyclopropyl group at the 6-position of abacavir assists in the arrangement and interaction of peptides with the MHC binding pocket. We hypothesized that chemical modification of abacavir at this position would provide a new series of analogues with potent antiviral activity that do not activate abacavir-specific CD8+ T-cells. Chemical modification of the 6’-amino cyclopropyl of abacavir eliminates HLA-B*57:01-restricted CD8+ T-cell activation without loss of antiviral activity Mohammad Alhaidari1*, Emma Yang1, Neil Berry1, Andrew Owen1, Steve Clarke2, Paul O’Neill1, Dean Naisbitt1, Kevin Park1 From 6th Drug Hypersensitivity Meeting (DHM 6) Bern, Switzerland. 9-12 April 2014 determine which analogues activated the T-cells. All of the abacavir-responsive clones secreted IFN-, in a drug- concentration-dependent fashion (EC50 [50% of maxi- mal IFN- spot forming units] less than 20M for all clones). A minimum of 5 clones were used to compare abacavir specific responses against the analogues. Abacavir and N-propyl abacavir were equally potent at activating clones. In contrast, two very closely related abacavir analogues were devoid of T-cell activity. Conclusion Our findings show that it is possible to block the T-cell activity of abacavir while maintaining anti-viral activity by simple chemical modification. Methods Fifteen analogues were synthesized and their anti-viral activity measured. T cell responses to these analogues was then measured using CD8+ clones generated from HLA-B*57:01+ donors. doi:10.1186/2045-7022-4-S3-P40 Cite this article as: Alhaidari et al.: Chemical modification of the 6’-amino cyclopropyl of abacavir eliminates HLA-B*57:01-restricted CD8+ T-cell activation without loss of antiviral activity. Clinical and Translational Allergy 2014 4(Suppl 3):P40. Alhaidari et al. Clinical and Translational Allergy 2014, 4(Suppl 3):P40 http://www.ctajournal.com/content/4/S3/P40 Alhaidari et al. Clinical and Translational Allergy 2014, 4(Suppl 3):P40 http://www.ctajournal.com/content/4/S3/P40 POSTER PRESENTATION Open Access 1University of Liverpool, MRC Centre for Drug Safety Science, UK Full list of author information is available at the end of the article Results Several analogues, including the key closely related com- pounds, retained antiviral activity, but displayed highly divergent T-cell responses. Three HLA-B*57:01 positive blood donors were selected to generate abacavir-responsive T-cell clones. IFN- release was visualized using ELISpot to measure abacavir-specific T-cell responses and to 1University of Liverpool, MRC Centre for Drug Safety Science, UK Full list of author information is available at the end of the article
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https://www.qeios.com/read/C2E5FZ/pdf
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Linseed Oil
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Qeios · Definition, February 2, 2020 Ope n Pe e r Re v ie w on Qe ios Linseed Oil National Cancer Institute Source National Cancer Institute. Linseed Oil. NCI T hesaurus. Code C107324. T he oil extracted from the seeds of Linum usitatissimum. Linseed oil is used as a nutritional supplement and as a polymerizing resin. Qeios ID: C2E5FZ · https://doi.org/10.32388/C2E5FZ 1/1
https://openalex.org/W2177526619
https://figshare.com/articles/journal_contribution/A_preposterior_analysis_to_predict_identifiability_in_the_experimental_calibration_of_computer_models/1496544/3/files/2193054.pdf
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A preposterior analysis to predict identifiability in the experimental calibration of computer models
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Online Supplement for "Preposterior Analysis to Predict Identifiability in Experimental Calibration of Computer Models," by Paul D. Arendt, Daniel W. Apley, and Wei Chen Online Supplement for "Preposterior Analysis to Predict Identifiability in Experimental Calibration of Computer Models," by Paul D. Arendt, Daniel W. Apley, and Wei Chen Online Supplement for "Preposterior Analysis to Predict Identifiability in Experimental Calibration of Computer Models," by Paul D. Arendt, Daniel W. Apley, and Wei Chen Sensitivity to the Actual Discrepancy Function and to the Choice of σδ 2 In the MC loop of the algorithm of Figure 1, the hypothetical discrepancy functions are generated from a GP model. In reality, however, the actual discrepancy functions can take whatever form the physics of the problem dictate, and we used non-GP discrepancy functions in the examples in Sections 5.1 and 5.2. Here we investigate the sensitivity of the posterior covariance to the actual discrepancy function for the sinusoidal example of Section 5.2. Specifically, we generate different sets of actual physical experimental data via Eq. (5) with the eight different discrepancy functions {δk(x): k = 1, 2, . . ., 8} listed in Table S1, some of which are plotted in Figure S1. The first two discrepancy functions are mathematical functions with different magnitudes but similar shape, and the remaining discrepancy functions are realizations from GP models with the hyperparameters shown in Table S1. Figure S2 shows results analogous to Figure 7 for the eight different discrepancy functions. Most of the scatter plots in Figure S2 exhibit a strong linear trend, which indicates that the preposterior STD provides a reasonable indication of the relative improvement in identifiability that one can expect from running a larger physical experiment. Table S1. Different discrepancy functions used in the sinusoidal example. Table S1. Different discrepancy functions used in the sinusoidal example. Discrepancy function Description δ1(x) 2 0.25(0.1exp{ } 0.05 ) x x − δ2(x) 2 0.1exp{ } 0.05 x x − δ3(x) Each δk(x) (k = 3,4,5) is a realization of a GP model with 2 { , , } {0,1,9.26} δ δ δ β σ ω = δ4(x) δ5(x) δ6(x) Each δk(x) (k = 6,7,8) is a realization of a GP model with 2 { , , } {0,0.028,9.26} δ δ δ β σ ω = δ7(x) δ8(x) Figure S1. Plots of selected discrepancy functions listed in Table S1. Figure S2. Plot of the posterior STD for the 8 different discrepancy functions versus the preposterior STD (both normalized by the prior STD). In Step 3 of the MC loop for calculating the preposterior STD, σδ 2 = 0.028 was used for all 8 cases. The numbers indicate Ne. In the vertical axis, the circles and error bars represent the mean posterior STD and ± 1 standard deviations of the posterior STD, with respect to the prior p(θ). Because the parameter σ 2 specified in Step 0c-i dictates the magnitude of the discrepancy Figure S1. Sensitivity to the Actual Discrepancy Function and to the Choice of σδ 2 Plots of selected discrepancy functions listed in Table S1. Figure S1. Plots of selected discrepancy functions listed in Table S1. Figure S2. Plot of the posterior STD for the 8 different discrepancy functions versus the preposterior STD (both normalized by the prior STD). In Step 3 of the MC loop for calculating the preposterior STD, σδ 2 = 0.028 was used for all 8 cases. The numbers indicate Ne. In the vertical axis, the circles and error bars represent the mean posterior STD and ± 1 standard deviations of the posterior STD, with respect to the prior p(θ). Figure S2. Plot of the posterior STD for the 8 different discrepancy functions versus the preposterior STD (both normalized by the prior STD). In Step 3 of the MC loop for calculating the preposterior STD, σδ 2 = 0.028 was used for all 8 cases. The numbers indicate Ne. In the vertical axis, the circles and error bars represent the mean posterior STD and ± 1 standard deviations of the posterior STD, with respect to the prior p(θ). page 2 Because the parameter σδ 2 specified in Step 0c-i dictates the magnitude of the discrepancy functions that are generated when generating the hypothetical physical experimental data within the MC loop, the sensitivity of the preposterior STD to choice of σδ 2 is of interest. For calculating the preposterior STDs in Figure S2, we used σδ 2 = 0.028 in Step 3 of the MC loop for all 8 cases (regardless of value of σδ 2 listed in Table S1 for generating the actual discrepancy function). Figure S3 shows analogous results but using a much larger σδ 2 = 1.0 in Step 3 of the MC loop. Relative to Figure S2, the primary difference in Figure S3 is that the points are all shifted to the right. This is consistent with what one would expect, since using a larger value of σδ 2 in Step 3 should result in a larger preposterior STD. Notice that there is still a strong linear trend in most of the scatter plots in Figure S3. Figure S3. Plot of the posterior STD for the 8 different discrepancy functions versus the preposterior STD (both normalized by the prior STD). In Step 3 of the MC loop for calculating the preposterior STD, σδ 2 = 1.0 was used for all 8 cases. Figure S3. Sensitivity to the Actual Discrepancy Function and to the Choice of σδ 2 Plot of the posterior STD for the 8 different discrepancy functions versus the preposterior STD (both normalized by the prior STD). In Step 3 of the MC loop for calculating the preposterior STD, σδ 2 = 1.0 was used for all 8 cases. An Additional Example of Using the Preposterior Analysis to Select Ne The beam and sinusoidal examples were used in Section 5.3 to illustrate how to use the preposterior analysis to select the number Ne of experimental runs. These examples involved only a single input variable x and used uniformly spaced grid designs. Here we consider an example that uses Latin hypercube designs with two input variable and a calibration parameter. Suppose the computer model is page 3 page 3 1 2 1 1 2 1 2 ( , , ) sin( ) [0,1] [0,1] [0,4] m y x x x x x x x θ θ θ = + ∈ ∈ ∈ and the physical experimental data are generated from the model and the physical experimental data are generated from the model * 1 2 1 2 1 2 2 ( , ) ( , , ) ( , ) ~ (0,0.01 ) e m y x x y x x x x N θ δ e e = + + * 1 2 1 2 1 2 ( , ) ( , , ) ( , ) e m y x x y x x x x θ δ e = + + * 1 2 1 2 1 2 2 ( , ) ( , , ) ( , ) ~ (0,0.01 ) e m y x x y x x x x N θ δ e e = + + We began with a Latin hypercube design for the computer experiment with 40 points in three dimensions {x1, x2, θ}. An optimal Latin hypercube design based on the maximin criterion was used. A Gaussian process model was fit to the computer experiment data via MLE in Step 0a of Figure 1. For the physical experimental design specified in Step 0b, we used maximin Latin hypercube designs of various size Ne = {10, 20, 30, 40, 50} over two dimensions {x1, x2}. Because of the random nature of the Latin hypercube designs, none of the physical experimental designs had x settings that coincided with the computer experiment x settings. Next, in Step 0c-i, we set the GP hyperparameters of the discrepancy function to be βδ = 0, ωδ = 3, and σδ = 0.75, which corresponds to assigning them point mass priors. The relatively large value of σδ results in a variety of relatively large discrepancy functions being generated within Step 3 of the preposterior algorithm. An Additional Example of Using the Preposterior Analysis to Select Ne In step 0c-ii, we specified λ = 0.012. Lastly, in Step 0c-iii, we specified the prior for θ to be uniform over the entire range [0, 4]. From the preposterior analysis, Figure S4 shows the resulting fixed-θ preposterior standard deviation versus θ t for the designs with different Ne, and Figure S5 shows the (normalized) preposterior standard deviation as a function of Ne. From Figure S5, the preposterior standard deviation does not substantially decrease beyond Ne = 30 roughly. page 4 Figure S4. Fixed-θ preposterior STD (normalized by the prior STD of θ) versus θt for Latin hypercube designs of various size Ne (Ne is specified in the legend). 1 2 3 4 0.4 0.6 0.8 1.0 10 20 30 40 θ t Normalized STD Normalized STD Figure S4. Fixed-θ preposterior STD (normalized by the prior STD of θ) versus θt for Latin hypercube designs of various size Ne (Ne is specified in the legend). Figure S5. Preposterior STD (normalized by the prior STD of θ) verses Ne for the Latin hypercube designs. 10 20 30 40 50 0.4 0.6 0.8 1.0 Ne Preposterior STD (Normalized) 10 20 30 40 50 0.4 0.6 0.8 1.0 Ne Preposterior STD (Normalized) Figure S5. Preposterior STD (normalized by the prior STD of θ) verses Ne for the Latin hypercube designs. page 5
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Detection of Microcystins in South African surface waters by high performance liquid chromatography in the light of Quality by Design statical tool.
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Detection of Microcystins in South African surface waters by high performance liquid chromatography in the light of Quality by Design statical tool. Abstract Cyanobacteria, an algae bloom that is responsible for the creation of deadly toxins. These toxins have the potential to adversely impact human and animal health. Microcystins (MC’s), are harmful toxins that are produced by cyanobacteria species. These deadly toxins are the most investigated toxins worldwide. However, in South Africa their occurrence statistics are deficient due to inaccessibility of reliable, sensitive, and precise analytical methods to investigate them in water surfaces. Thus, a development of analytical methods for the detection and accurate quantification of algal toxins is crucial to consider the health risk of exposure to toxins in wastewater and surface waters. This study recommends an analytical method for the detection and quantification of algal toxins using solid phase extraction (SPE) and high-performance liquid chromatography (HPLC) coupled with photodiode detector (PDA). The developed method was validated based on linearity which ranged from 3–2500 µg L− 1, recoveries at 99.6–113.9%, the limit of detection (LOD) ranging from 0.2–480 µg L− 1, and limit of quantification (LOQ) at 0.7–160 µg L− 1 and the regression coefficients obtained were above 0.9880 in all analytes. The developed method was applied to Darvill Wastewater Treatment Plant, Umgeni water, Durban surface waters, University of KwaZulu-Natal (UKZN) sport centre tap water and UKZN Westville two ponds), South Africa. MC-RR was the most detected analyte at concentration ranging from 0.069–30.521 ng L− 1 compared to other targeted MCs. The developed method is simple and cheap, which can consent developing countries to monitor these deadly MC toxins in water surfaces. Introduction Due to the increase in the population of cyanobacteria in various water resources, recreational activities have been recently forbidden because they are toxic to biotic components [1]. Cyanobacteria, also known as blue-green algae, occur spontaneously in freshwater [2]. There have been several reports on the toxicity of cyanobacterial bloom in surface waters worldwide [3],[4],[5],[6],[7],[8],[9],[10]. These microbes use the energy of sunlight and fix inert atmospheric nitrogen into usable nitrates that can be absorbed by plants [11]. Climate change was discovered to be the most common environmental factor contributing to the rapid increase of cyanobacteria in water bodies [4],[12]. Due to climate change, heavy rain, residential detergents, sunlight, pH, fossil fuels, and industrial pollutants were discovered to be the common factor at increasing of bloom, as water becomes rich in nutrients and trigger a rapid increase in bloom [12],[1]. Many genera of cyanobacteria generate a toxic secondary metabolite called cyanotoxins [13]. These toxins constitute an extensive group of chemical compounds that are different in their molecular structure and toxicological properties [14]. Amongst these toxins, Microcystins (MCs) were the most common toxins produced by cyanobacteria in large quantities during algal blooms [1]. Microcystins are cyanotoxins released by cyanobacteria, which can survive even under extreme conditions such as high or low temperature, sunlight, or in the dark [15]. These MCs were discovered to be the most identified toxin produced by a vast range of cyanobacterial species [16]. Also, MCs are stable and resistant to extreme pH, even in the presence of common hydrolytic enzymes due to their cyclic structure [17]. These toxins are cyclic peptides and are one of the most common contributors in the development of primary liver cancer [18]. MCs’ cyclic structure are noted to be responsible for their stability in temperature and pH [19]. Several variants have been identified in waters worldwide, such as MC-RR, MC-YR, and MC-LR, which were identified as the most toxic and prevalent worldwide [20]. Researchers described multiple ways in which human and animal exposure to toxins has resulted in illnesses or deaths [21],[22]. Pathway exposure to toxins includes contact with contaminated water via swimming, intake of contaminated fish and shells, dietary supplements, or inhalation [23],[22],[24]. Several illnesses or symptoms were reported, such as skin irritation, fever, and respiratory paralysis [21]. Among the three MC variants, MC-LR was declared the most prevalent, exhibiting 46% of the total concentration of MCs in natural blooms [25]. Research Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/20 Introduction Due to its severe and chronic toxicity, the WHO fixed a guideline value of 1 µg L− 1 maximum of MC-LR in drinking water [26]. The structure of MC-LR is shown in Fig. 1. The U.S Environmental Protection Agency (EPA) has recently evaluated the risk to human health from drinking water contaminated with algal toxins [27], [16], [28]. In South Africa, no illnesses or deaths of humans and animals are reported due to cyanotoxins. Maybe we are underestimating these toxins, or the healthcare workers have not been trained to recognise the symptoms. The global occurrence of cyanotoxins in natural water reservoirs promoted the development of methods and approaches for detecting toxins in environmental waters. Most methods currently developed are different in terms of sensitivity and specificity [29]. Although, several methods of MC detection were developed and optimised, there are still challenges with respect to finding a suitable method to detect environmental samples at low concentration. Some of the reported detection methods in the literature are expensive, time consuming and require specialized personnel and other equipment, such as Liquid Chromatogram Mass Spectrometer (LC-MS) [30]. Hence, High Performance Liquid Chromatogram (HPLC) equipped with Photo Diode Array detector (PDA) was selected because of its ability to detect and quantify toxins in environmental waters. The SPE-HPLC/PDA method was developed and optimized to detect and quantify MCs in natural waters and to improve the detection and quantification limits by applying a pre-concentration step in the experiment. Sample Extraction HLB cartridge was conditioned by 3 mL of methanol followed by 3 mL of Milli-Q water. Afterward, the sample was loaded at a flow rate of 15 mL min − 1. Then cartridges were dried under vacuum for a duration of 45 min. The analytes were eluted with 5 mL of methanol and filtered on a 0.45 µm syringe filter. The eluate was then transferred to an amber vial for analysis. Samples were left for 12 hours for evaporation and reconstituted with 1 mL of methanol. Sample Collection Samples were collected from different water surfaces in Durban; UKZN Westville sport centre tap water, UKZN Westville 2 ponds and from Darvill Wastewater Treatment Plant at uMgeni water in Pietermaritzburg (PMB), all were sampled using 1 L amber bottles to prevent the analyte from degradation. Sample bottles were transported in a cooler box under control condition of 5 to the laboratory and kept in the fridge below 4 for further analysis. ℃ ℃ Sample Preparation Samples from the fridge were carried out and left to reach room temperature. Then they were filtered using a gravitational method to remove all sludge and particles. Due to sludge thickness and the large sample volume used, filtration was completed in 2 hours. The amount of sample used was 100 mL and pH were adjusted to pH 6 and pH 9 using 0.1 M HCl and 0.1 M NaOH, respectively. Materials And Instruments Oasis HLB cartridge (3 cc, 60 mg adsorbent, 30 µm particle size, Water, from Sigma-Aldrich, (Steinheim, Germany) was optimized using ultra-pure grade water spiked with MCs standards. Eumax ultrasonic cleaner bath obtained from LABOTEC (Elma, Singen, Germany) was used for degassing the mobile phase. Edward 8 vacuum pump purchase from Busch (Durban, New Germany, and South Africa) was used for drying the SPE cartridges. Vacuum manifold, Holdoph purchased from Holdoph (Germany) was used for sample pre-concentration. Different pH levels were obtained using a pH meter (Eutech) purchased from Labotech (Durban, South Africa) and calibrated with buffers at pH 4, 7, and 10. Analyses were carried out under LC (Shimadzu technology) purchased from (Kyoto, Japan), equipped with gradient pump. The separation of the analyte was accomplished on a Hypersil Gold C18 (Guard column 30 mm x 2.1 mm, 3 µm particle size), thermostated at 35 to 85 . Ultraviolet (UV) spectra were drawn from 238 to 254 nm. ℃ ℃ Apparatus All the following apparatus used were obtained from chemistry stores in University of KwaZulu Natal (UKZN) Westville (Durban, South Africa): such as conical flask (250 mL), measuring cylinder (100 mL – 1 L), universal bottles (250 mL – 1000 L), beakers (500 mL), glass sample tubes, pasteur pipette, SPE tubes, filter paper 125 mm diameter and 0.45 µm syringe filter. All glasswares were washed, rinsed with acetone, and heated to a higher temperature of 150 for 24 hours to prevent emulsion. Small volumes were measured by various sizes of micropipette (5 µL – 1000 µL) plus kit Dragon lab (Shaanxi, China) and larger volumes were measured by volumetric flask. HPLC vials (2 mL) were bought at Chemetrix (Durban, South Africa). ℃ Chemicals and reagents Liquid Chromatogram (LC) grade, methanol (MeOH), acetonitrile (ACN), trifluoroacetic acid (TFA), ≥ 99.9% including MCs standard solution (MC-RR, MC-YR and MC-LR) were obtained from Sigma-Aldrich (Steinheim, Germany). Hydrochloric (HCl) acid 37% and sodium hydroxide (NaOH) pellets were bought from Sigma-Aldrich (Durban, South Africa). Hydrochloric acid, HCl (0.1 M), and sodium hydroxide, NaOH (0.1 M) were prepared using an in-house method from 37% HCl and NaOH pellets. Methanol was used to prepare stock solutions of MCs. Ultra-pure water used for toxins extraction was generated by a gradient Page 2/20 Page 2/20 A10 Milli-Q system bought from Microsep (Durban, South Africa). Solutions prepared were stored in the fridge at -5 oC in an amber bottle to prevent them from degradation. A10 Milli-Q system bought from Microsep (Durban, South Africa). Solutions prepared were stored in the fridge at -5 oC in an amber bottle to prevent them from degradation. Spiking A 100 mL sample was spiked with different concentrations of 20 µL and 50 µL of 5000 µg L− 1 MCs, respectively. The spiked and un-spiked blank samples were prepared in triplicate, extracted using the developed extraction method, and analysed on HPLC for matrix-matched validation. Standard Preparation A working solution of 2500 µg L− 1 of MCs was prepared from 5000 µg L− 1 stock solution of the standard by transferring 500 µL into 1 mL amber volumetric flask and topped up to the mark with methanol. Sequential standards from 3, 5, 10, 36, 60, 120, 250, 375, 750, 1.000, and 2.500 µg L− 1 were prepared in triplicate from the working solution. All standards for calibration curves were prepared in methanol and analysed on the HPLC instrument. The calibration curve was established by plotting peak areas against known concentrations of MC for each analyte. Method development The MCs belong to the family of peptides made from seven amino acids [13]. They have different hydrophobicity but depend on two variant amino acids [35]. Cyanotoxins referred to as molecules that contain an equal number of positively and negatively charged functional groups in a typical pH of 6 relevant to drinking water, making it highly water-soluble [7]. The HPLC/PDA method was developed by optimizing the instrument parameters including, injection volume, flow rate, pH, and mobile phase. Optimisation of solvent The choice of solvent is important since the wavelength of MCs is lower, which might interfere with the detection and quantification. Methanol, water, and acetonitrile were investigated because they are normally used as chromatographic solvents. When methanol was used, there was interference because of the closeness of the methanol wavelength of 220 nm to 238 nm wavelength of MCs. Fig. 4 (a) shows the interference of methanol with the analyte; the red colour is methanol, the blue and green colour are the analytes and the other colours (yellow, blue, pink) observed are interferences because of methanol interaction with the targeted analytes. This interference is well depicted by the purity spectrum in Fig. 4 (b), which shows the baseline of methanol (red line) above the analytes. Fig. 4 (c and d) shows the results of acetonitrile where MCs peaks were clearly visible, and the purity spectrum shows the baseline below the analytes. Acetonitrile and water were chosen as the mobile phase because they did not interfere with the analytes' wavelength. Moreover, this duo yielded better separation [20]. Separation method The separation of MCs was achieved on the HPLC as supported by the retention times of MC-RR, MC-YR, and MC-LR, with chromatographic peaks at 6.969 min, 7.467 min, and 7.622 min, respectively. The chromatogram shown in Fig. 2 was obtained by injecting 10 µL of 2500 µg L-1 MCs standard in a C18 column. This separation is comparable with the MCs achieved by other researchers in the literature [20]. The UV detector was scanned between 200 to 300 nm, and finally, data was recorded at a 238 nm channel as this wavelength corresponds to the maximum absorption of MCs [36]. Chromatogram obtained by injecting 10 µL of 2500 µg L-1 microcystins standard of targeted analytes the first analyte detected, this is supported by previous studies where MC-RR was reported as the first eluted Microcystin-RR was the first analyte detected, this is supported by previous studies where MC-RR was reported as the first eluted analyte [37], [20], [38]. Microcystin-YR and MC-LR analytes were discovered to be very close to each other; therefore, MC-YR was verified by injecting 10 µL-1 of the 2500 µg L-1 MC-YR standard solution. The MC-YR peak, which corresponds to the MCs mixture solution, was found with a retention time of 7.481 min as shown in Fig. 3. analyte [37], [20], [38]. Microcystin-YR and MC-LR analytes were discovered to be very close to each other; therefore, MC-YR was verified by injecting 10 µL-1 of the 2500 µg L-1 MC-YR standard solution. The MC-YR peak, which corresponds to the MCs mixture solution, was found with a retention time of 7.481 min as shown in Fig. 3. Instrumental Procedure Prepared standards of MCs were analysed using HPLC to quantify and separate non-volatile samples in a solution under gradient elution mode. Mobile phase (A) ultra-pure water consisted of 0.025% TFA acid with mobile phase (B) pure ACN consisted of 0.025% TFA acid, were prepared. The initial mobile phase flow rate was 0.7 mL min− 1 and the injection volume of 10 µL was used for the analyte. The optimized gradient for analyte separation as followed: 20% mobile phase A was set at 10 min and kept constant until mobile phase B reached 80%. The linearity gradient was changed from 80–100% mobile phase B until 20 min. At 30 min, the mobile phase was decreased to 30% and kept constant for 15 min to recondition the column. Three chromatographic Page 3/20 Page 3/20 peaks representing each MC were observed at less than 10 min. The retention times at which each analyte eluted were 6.969 min, 7.467 min, and 7.622 min, respectively, for MC-RR, MC-LR, and MC-YR. peaks representing each MC were observed at less than 10 min. The retention times at which each analyte eluted were 6.969 min, 7.467 min, and 7.622 min, respectively, for MC-RR, MC-LR, and MC-YR. Ecological Risk Determination Risk assessment for MCs have been done by previous researchers using different factors such as predicted no effect concentration (PNEC) derived from various species and using a sensitivity distribution model toxicity data.[31–33] In this study, the hazard risk quotient (HQ), was derived from based on a formula which involved the use of the measured environmental concentration (MEC) obtained from this study, and the no observed adverse effect level (NOAEL) obtained from the toxicity studies done by Fawell KJ et al. 1999.[34] This was done for the ecological risk assessment of MCs (LR, RR and YR) studied in surface waters and wastewater. HQ = MEC/NOAEL Where the HQ 1, this indicate that there is a high risk on the measured environmental samples hence, it can cause negative effects on the living organisms on that specific area. In those areas having the HQ greater than 1, it is imperative to take some measures on managing the risk to minimise the risk. If HQ is 0.1–1, then there is a medium risk on the studied area and where HQ 0.1 there is a low potential risk in the area. Optimisation of pH Because toxins can take on different chemical species, such as nickel, mercury, arsenic etc, the pH has a significant impact on their behaviour [19]. Environmental fate is closely tied to both environmental pH and MCs hydrophobicity [19]. It was partially important to optimise the pH as MCs highly depend on it. Due to climate change, unclean wastewater treatment plants effluents, and other factors mentioned earlier; water pH may vary due to cyanobacterial blooms [1]. Milli-Q water samples were adjusted to 3 different pH levels (4, 5, and 6) to obtain the optimum pH and achieve better recoveries. The effect of pH on MCs is shown in Fig. 10, where all three analytes at pH 4 showed higher recoveries above 120%, recovery at pH 4 proved to be significantly higher than those obtained at (pH 5) and (pH 6). At pH 6, the average percentage recoveries of all targeted analyte were found within the acceptable range by IUPAC, which is between 60% and 120% in all MC compounds [44]. Extraction method Solid-phase extraction is usually a reliable technique to extract and concentrate samples from complex matrices and sample preparation is a necessary part of a positive chromatography [43]. Few parameters are crucial for preconcentration, such as sample elution flow rate, sample volume, selected solvent percentage for elution, and drying duration. All these parameters were investigated in this work. The pH and extraction solvent were found to affect the recoveries hence these two parameters were optimized. A 100 mL sample was spiked with 20 µL of 5000 µg L-1 standard containing all the targeted MCs and prepared in triplicate in Milli-Q water. These samples were extracted and analysed as defined in sections 2.8. Optimisation of injection volume The MCs’ peaks depend on the amount of sample volume injected. MCs standard of 2500 µg L-1 was injected with varying volumes between 2 µL and 20 µL. At higher volume, the analyte peaks were not well resolved due to tailing, as observed in Fig. 6 (b). At lower volume, separation of peaks improved, with MC-YR and MC-LR being resolved, as shown in Fig. 6 (a). However, the signal-to-noise ratio at a low injection volume of 2 µL was elevated. The injection volume of 10 µL was chosen because it appeared to have a better resolution and symmetric peaks with a lower signal-to-noise ratio (Fig. 2). The injection volume is normally found to affect the resolution; hence the lower volume than 20 µL is recommended [39]. Instrument stability The instrument stability is performed to ensure the instrument is stable for routine analyse. The stability of the instrument was evaluated on the method developed by injecting 10 µL of 375 µg L-1 MC standard five times over 30 min intervals. Fig. 8 shows the plot of the peak area versus time. The linearity of the graph over 180 min confirmed that the instrument is stable enough for routine analysis. Furthermore, to evaluate the stability of the instrument, the retention times of the analytes over time were compared. The relative standard deviation for peak areas ranged from 1.46 to 2.80, and the obtained results prove the instrument's stability with a confirmation of a stable signal in all analytes. Optimisation of flow rate The flow rate and other parameters, including mobile phase solvent, and injection volume, were investigated to achieve a concise and precise chromatographic method. In this study, the mobile phase flow rate of 0.1 mL min-1, 0.5 mL min-1, and 0.7 mL min-1 were assessed by injecting 10 µL of 2500 µg L-1 into an HPLC/PDA instrument. The analytes were eluted between 21.5 to 24 min; 8 to 9.5 min; and 6.9 to 7.5 min, at 0.1 mL min-1, 0.5 mL min- 1 , and 0.7 mL min-1, respectively, as shown in Fig. 7. Retention times decreased with an increase in flow rate because the MCs interacted more with the mobile phase than the stationary phase. Hence, MCs strongly adsorb with hydrophilic nature [40],[41],[42]. The flow rate of 0.7 mL min-1 was selected because it yielded narrow peaks with a shorter retention time than other flow rates reported in the literature [20]. Calibration study A sensitive method is needed to detect low concentrations of MCs in environmental waters. A calibration curve was obtained by plotting analyte peak area versus analyte concentration. The MC standards ranging from 3 to 2500 µg L-1 were prepared in triplicate and analysed as described in the experimental section above. Fig. 9 shows the calibration curve and regression coefficient analyses for all the analytes above 0.98, with MC-LR having the highest regression value of 0.9895. MC-RR showed more sensitivity at the 3 µg L-1 detection limit, whereas MC-YR showed the least sensitivity for the method. Development of gradient elution When 100% of water was used, there was no elution of the analytes; hence 100% of acetonitrile was introduced, and elution of analyte peaks was identified, but there was no separation, as it is shown in Fig. 5 (b). The gradient program was changed to 50-50% of water and acetonitrile, but there was no separation as shown in Fig. 5 (a). Hence TFA was introduced as an additive in both water and acetonitrile. Elution of peaks was observed, TFA was adjusted to 0.025%, and peaks were separated as shown in Fig. 4 (c). Although the separation was found between 25% (0.025% TFA in water) and 75% (0.025% TFA in acetonitrile), the percentage of 0.025% TFA in acetonitrile was increased up to 100% to remove the excess of toxins [36]. Page 4/20 Page 4/20 Intraday and interday precision Method repeatability and interday and intraday precision were validated by analysing 3 MC standards prepared in triplicate. The lowest, middle, and upper concentrations analysed were 60, 375 and 2500 µg L-1. The obtained results were calculated as the mean percentage of the RSD of the standard solution at the three concentration levels. Results indicated that the method developed has good precision for the analysis of MCs and is accurate for quantitative analysis, as shown in Table 2. Instrumental method The instrument detection limit obtained was 10 µg L-1; results are shown in Table 1. Instrumental linearity and linear regression (R2) were established to be 10 µg L-1 to 2500 µg L-1 and 0.9880 to 0.9895, respectively. Limit of quantification and limit of detection The limit of quantification (LOQ) and limit of detection (LOD) are dependent on the sample matrix [51]. In this work, to study the impact of the matrix, 100 mL of drinking water, river water, Milli-Q water, and wastewater were spiked with 20 µL of 5000 µg L-1 MCs standard solution, and the final concentration after spiking was 1 µg L-1. The results are presented in Table 5. The LOD was found to be lower in drinking water and Milli- Q water compared to river water and wastewater due to that, in drinking and milli-Q water the matrix is purified. Matrix matched studies proved that the method can be applied in natural surface waters but in wastewater it had a higher detection limit. Although they were high, but they were still far below the enzyme-linked immunosorbent assay (ELISA) method. In wastewater, MCs’ recoveries were very low when using a concentration of 1 µg L-1, hence samples were further spiked with 2.5 µg L-1 to obtain LOD and LOQ. In river waters, error margins were large, this was attributed to concentration levels of MCs already present in river water. Though error margins were discovered low in Milli-Q and tap water, nevertheless error margins were within acceptable limits in all matrices. Method robustness The Quality by Design (QbD) process is a strategic method and development process. During this process, the capability and efficiency of the method are ensured. The QbD process involves the systematic design and development of chromatography method. With analytical and risk-management methodologies, Quality by Design (QbD) improves the ability of the method through a systematic approach to their design, development. The proposed method critical method parameters (CMP’s) are identified and studied by utilizing the QbD statical tool. The CMPs are studies and proved the method robustness. Design expert software version 13 is used for the QbD approach. The robustness of an analytical procedure, it is the ability of a method to remain stable even when small and deliberate variations in the method parameters are made, thus providing evidence of its reliability during normal operation. The current proposed analytical procedure was studied by conducting the Design of Experiments (DoE’s) in the approach of QbD with design expert software. The proposed method CMPs are identified flow rate (0.7 mL min-1 ± 10) pH of the mobile phase (5.0 ± 0.2) and column temperature (30   ± 5). The selected CMPs are changed deliberately and constructed the DoE and the tool had given total 19 runs by selecting 3 center points, 2 replicates with zero blocks for 3 factors in the factorial design. The total 19 runs ran on the liquid chromatography and the selected responses Retention time of MC-RR (R1), resolution between MC-RR and MC-YR (R2) and resolution between MC-YR and MC-LR (R3). The total runs and responses were tabulated in Table 3. The responses R1, R2 and R3 were analysed by selecting the major effects. The Analysis of Variance (ANOVA) table was tabulated in Table 4. The ANOVA table clearly shows that the selected design was significant, and the predicted value (P value) found below 0.05 and lack of fit was found not significant. Fig. 11 shows the contour and Pareto charts for all three responses, and it highlights the impact of flow rate on R1, R2 and pH impact on R3. The impacts were clearly shown on Pareto charts. The variation of the responses is in the acceptable range and express the method robustness. The model graphs for three responses Half normal plot, 3D surface, and 3D cube was shown in Fig. 12. Optimisation of solvent Due to the hydrophobicity of MCs, typically, methanol and acetonitrile were used for their extraction from an aqueous matrix [45]. Seeing that methanol is more responsive with MCs in the SPE compared to acetonitrile [20], [46]. Both mentioned solvents were undertaken to study. Each sample was assessed at an optimum pH of 6 and extracted with each solvent, respectively. Methanol demonstrated a good separation and identification of MCs peaks during Page 5/20 Page 5/20 chromatographic analysis. With acetonitrile as a solvent for the extraction of MCs, peaks were not identified. Hence methanol was chosen as the extraction solvent for MCs and showed higher efficiency and was comparable to other studies in literature [47],[48],[49],[50]. chromatographic analysis. With acetonitrile as a solvent for the extraction of MCs, peaks were not identified. Hence methanol was chosen as the extraction solvent for MCs and showed higher efficiency and was comparable to other studies in literature [47],[48],[49],[50]. Application in real water samples Some of the targeted microcystin analytes in these samples were not detected by HPLC. This might be due to that; they were below the LOD of the developed HPLC method. The amount of MCs concentration found in this study were much lower than the WHO recommended level of 1 µg L-1 MC-LR [52]. Application in real water samples Samples were collected from Umgeni River, UKZN Sport Centre tap water, laboratory tap water, and UKZN Westville campus ponds and analysed for MCs using the developed method, the results are shown in Table 6. Microcystins-RR concentration obtained was from ND – 2.160 µg L-1 and MC-YR and MC-LR were not detected in all points. There are three ponds identified within UKZN premises, named pond 1, 2 and 3 respectively in which algae was observed on two ponds, thus were sampled to determine the presence of MCs. Pond 1 which is situated near UKZN building premises. The ponds were sampled due to visibility of algae to investigate the presence of toxins. The results obtained are shown in Table 6 where MC-RR was detected in Pond 2. The higher Page 6/20 Page 6/20 concentration obtained in Pond 2 is a result of algae which was highly visible and moreover there were small fishes observed, which lead to determination of toxins concentration to ensure the safety of living organisms. concentration obtained in Pond 2 is a result of algae which was highly visible and moreover there were small fishes observed, which lead to determination of toxins concentration to ensure the safety of living organisms. Various factors contributed to a higher concentration in Pond 2 such as sunlight, pH, temperature, heavy rain, and floods since the Pond is situated next to human activities and is more exposed to sunlight as a result of higher temperatures which contribute to the increased production of cyanotoxins. To evaluate the concentration of microcystins (µg L-1) from the Darvill wastewater treatment plant, three consecutive campaigns were undertaken for MCs determination. Analysis was carried out on the HPLC/PDA and MC-RR was the most detected toxin with the concentration ranging from ND to 30.521 µg L-1. The digested thickened WAS from the first campaign samples, MC-YR was detected at 2.015 µg L-1 and MC-LR was not detected. On a second campaign, the balancing tank and inlet showed a greater concentration from 4.403 – 6.470 µg L-1 MC-RR and MC-YR was detected in the inlet point at 4.938 µg L-1. On the secondary effluent and Maturation River, MC-RR showed a lower concentration from ND – D compared to other points. On the third campaign, in contrast to MC-RR, which was the most detected across all points at concentrations ranging from ND – 30.521 µg L-1, whereas MC-YR and MC-LR were not detected. Conclusion A sensitive and accurate analytical method of HPLC/PDA with SPE was established and optimized to pre-concentrate, purify, and detect the MC-RR, MC-YR and MC-LR in natural water samples. Good separation and high resolution of targeted MCs analysis were achieved under the C18 Hypersil column by using a suitable solvent (ACN and water) with 0.025% TFA. Most importantly, SPE method was worth it to remove all impurities. During the extraction method, pH was kept constant at 6. The developed method was validated by evaluation of LOD, LOQ, linearity, accuracy, and precision to meet validation criteria for use in drinking and surface waters. The method was then successfully employed to wastewater treatment plant samples and Umgeni River in KZN. Recoveries ranging from 99.6 – 113.9 were obtained at pH 6. Some of the targeted MCs were not detected. Due to reduced algal visibility in winter, MCs are anticipated to be low [55]. Low levels of toxins were caused by a variety of events, including floods. Severe flooding that recently hit KZN cleaned and drained the water surfaces, including algae. The analytical method developed is thus applicable for monitoring MCs in an environmental sample and can replace the ELISA method. Further research is required because these findings are significant for risk assessment. The archival results given in this study will support future risk assessments for surface waters and assist to locate microcystin levels into a broader ecological context. Competing interest/Competing interests The authors declare that they have no competing interests/competing interests. cological Risk assessment of MCs (LR, RR and YR) in surface w Microcystins are cyanobacterial that blooms from algae as a result of various environmental factors such as light, salinity and human activities.[33, 34] Thus, it is imperative to assess the ecological risks of these toxins possess living organisms as a results of water contamination. Moreover, the attention of these studies are performed and stressed to the living species such as the microorganisms.[32, 34] This study uses available data on previous studies done on the organisms, due to that this minimise the use of other organism species, hence, it is recommended. The value of NOAEL 40 µg L-1 for microcystins used for the HQ calculations was obtained from the study done by Fawell KJ et al. 1999[34] using mouse, in realistic water conditions. The NOAEL with the MECs from the study qualified the ecological risk assessment in the points selected on the environment, the results are shown in Table 4. Only the sampling areas were certain microcystins were quantified, the risk assessment was done. The hazard quotients for the studies areas ranged from 0.0503 - 0.764, showing the low risk and medium risk. The was no point having high risk, although there on the Inlet, 3rd  campaign- Inlet (MC-RR) the HQ is close to one, this indicates that there is a potential to risk. Comparison of the current study with literature To compare the analytical methods, factors including the column, the mobile phase, the extraction method, the limit of detection, and the concentration level reported in previous studies were evaluated, as shown in Table 8. Acknowledgements Authors are thankful to the University of KwaZulu-Natal, School of Chemistry and Physics, University Road, Westville 3630, South Africa, for supporting this work and guidance for the complete research. Declarations Competing interest/Competing interests Ethics approval This article does not contain any studies with animals performed by any of the authors. This article does not contain any studies with animals performed by any of the authors. We authorize to publish the article without any conflict. We authorize to publish the article without any conflict. We authorize to publish the article without any conflict. References 1. de la Cruz, A.A., et al., Introduction to cyanobacteria and cyanotoxins. Water treatment for purification from cyanobacteria and cyanotoxins, 2020: p. 1- 35. 1. de la Cruz, A.A., et al., Introduction to cyanobacteria and cyanotoxins. Water treatment for purification from cyanobacteria and cyanotoxins, 2020: p. 1- 35. 2. Chorus, I. and M. 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Tables Factor Response Std Run A: Flow rate B: pH C: Column temp R1 R2 R3 17 1 0.7 5 30 6.538 4.978 1.591 18 2 0.7 5 30 6.531 4.977 1.6 11 3 0.8 4.8 35 5.498 3.986 1.51 15 4 0.8 5.2 35 5.591 4.313 1.791 5 5 0.6 5.2 25 7.598 5.219 1.751 9 6 0.6 4.8 35 7.629 4.886 1.518 3 7 0.8 4.8 25 5.432 5.126 1.66 19 8 0.7 5 30 6.535 4.974 1.595 13 9 0.6 5.2 35 5.591 4.446 1.659 4 10 0.8 4.8 25 5.62 4.198 1.599 2 11 0.6 4.8 25 7.44 5.396 1.558 8 12 0.8 5.2 25 5.595 4.459 1.526 14 13 0.6 5.2 35 7.419 5.146 1.746 12 14 0.8 4.8 35 5.638 4.612 1.541 6 15 0.6 5.2 25 7.549 4.471 1.762 16 16 0.8 5.2 35 5.585 4.448 1.569 10 17 0.6 4.8 35 7.569 5.369 1.592 7 18 0.8 5.2 25 5.578 4.698 1.772 1 19 0.6 4.8 25 7.359 5.12 1.659 R1= Retention time of MC-RR Intraday   MC-RR MC-YR MC-LR Concentration µg L-1 60 380 250 60 380 250 60 380 250 %RSD 3.90 2.80 1.20 1.04 1.92 1.69 0.49 1.46 1.99   Interday   MC-RR MC-YR MC-LR Concentration µg L-1 60 380 250 60 380 250 60 380 250 %RSD 8.85 8.90 8.42 3.69 3.41 0.85 1.87 5.31 5.59 Table 3. DoE’s analysis with responses factors. Table 3. DoE’s analysis with responses factors. Tables Table 1. Analytical standard used to establish MCs LOD and linearity ranged from 3 µg L-1 - 2500 µg L-1 Curve characters MC-RR MC-YR MC-LR R2 0.9890 0.9880 0.9895 LOD µg L-1 60 10 10 Linear range µg L-1 60 – 2500 10 – 2500 10 – 2500 Standard Curve equation y = 26809x + 86.88 y = 23203x + 2872 y = 24966x + 3189.3 Table 1. Analytical standard used to establish MCs LOD and linearity ranged from 3 µg L-1 - 2500 µg L-1 Page 9/20 Table 2. Method repeatability Intraday/Interday precision validation at the lower, middle, and higher concentrations. able 2. Method repeatability Intraday/Interday precision validation at the lower, middle, and higher concentration Table 2. Method repeatability Intraday/Interday precision validation at the lower, middle, and higher concentrations. Intraday   MC-RR MC-YR MC-LR Concentration µg L-1 60 380 250 60 380 250 60 380 250 %RSD 3.90 2.80 1.20 1.04 1.92 1.69 0.49 1.46 1.99   Interday   MC-RR MC-YR MC-LR Concentration µg L-1 60 380 250 60 380 250 60 380 250 %RSD 8.85 8.90 8.42 3.69 3.41 0.85 1.87 5.31 5.59 Table 2. Method repeatability Intraday/Interday precision validation at the lower, middle, and higher concentrations. Intraday   MC-RR MC-YR MC-LR Concentration µg L-1 60 380 250 60 380 250 60 380 250 %RSD 3.90 2.80 1.20 1.04 1.92 1.69 0.49 1.46 1.99   Interday   MC-RR MC-YR MC-LR Concentration µg L-1 60 380 250 60 380 250 60 380 250 %RSD 8.85 8.90 8.42 3.69 3.41 0.85 1.87 5.31 5.59 Table 3. DoE’s analysis with responses factors. Tables Factor Response Std Run A: Flow rate B: pH C: Column temp R1 R2 R3 17 1 0.7 5 30 6.538 4.978 1.591 18 2 0.7 5 30 6.531 4.977 1.6 11 3 0.8 4.8 35 5.498 3.986 1.51 15 4 0.8 5.2 35 5.591 4.313 1.791 5 5 0.6 5.2 25 7.598 5.219 1.751 9 6 0.6 4.8 35 7.629 4.886 1.518 3 7 0.8 4.8 25 5.432 5.126 1.66 19 8 0.7 5 30 6.535 4.974 1.595 13 9 0.6 5.2 35 5.591 4.446 1.659 4 10 0.8 4.8 25 5.62 4.198 1.599 2 11 0.6 4.8 25 7.44 5.396 1.558 8 12 0.8 5.2 25 5.595 4.459 1.526 14 13 0.6 5.2 35 7.419 5.146 1.746 12 14 0.8 4.8 35 5.638 4.612 1.541 6 15 0.6 5.2 25 7.549 4.471 1.762 16 16 0.8 5.2 35 5.585 4.448 1.569 10 17 0.6 4.8 35 7.569 5.369 1.592 7 18 0.8 5.2 25 5.578 4.698 1.772 1 19 0.6 4.8 25 7.359 5.12 1.659 R1= Retention time of MC-RR R1= Retention time of MC-RR R2= Resolution between MC-RR and MC-YR R3= Resolution between MC-YR and MC-LR Table 4. ANOVA statistics analysis R2= Resolution between MC-RR and MC-YR Page 10/20 Page 10/20 Page 10/20 R Source Sum of Squares df Mean Square F-value p-value   R1 Model 11.59 1 11.59 55.94 < 0.0001 significant A-Flow 11.59 1 11.59 55.94 < 0.0001   Curvature 0.0343 1 0.0343 0.1655 0.6896   Residual 3.31 16 0.2072       Lack of Fit 1.61 6 0.2683 1.57 0.2508 not significant Pure Error 1.7 10 0.1705       Cor Total 14.94 18         R2 Model 1.11 1 1.11 9.8 0.0065 significant A-Flow 1.11 1 1.11 9.8 0.0065   Curvature 0.1372 1 0.1372 1.21 0.2873   Residual 1.81 16 0.1132       Lack of Fit 0.4675 6 0.0779 0.5799 0.7393 not significant Pure Error 1.34 10 0.1344       Cor Total 3.06 18         R3 Model 0.0551 1 0.0551 9.28 0.0077 significant B-pH 0.0551 1 0.0551 9.28 0.0077   Curvature 0.0047 1 0.0047 0.7859 0.3885   Residual 0.095 16 0.0059       Lack of Fit 0.026 6 0.0043 0.6293 0.7049 not significant Pure Error 0.069 10 0.0069       Cor Total 0.1548 18 Table 5. Matrix matched analysis at four different surface waters to determine the method LOD and LOQ. Tables Parameters Tap River Wastewater Milli-Q Microcystins RR YR LR RR YR LR RR YR LR RR YR LR Concentration (µg L-1) 1 1 2.5 1 LOD (µg L-1) 0.09 0.01 0.49 0.52 0.24 1.95 0.28 0.36 4.32 0.02 0.03 0.19 LOQ (µg L-1) 0.29 0.03 1.64 1.73 0.79 6.50 0.92 0.25 14.40 0.08 0.09 0.65 Precision (%) 4.72 0.79 7.47 20.36 14.33 14.51 1.24 1.09 3.44 1.56 0.16 0.38 Table 5. Matrix matched analysis at four different surface waters to determine the method LOD and LOQ. hed analysis at four different surface waters to determine the method LOD and LOQ. Table 5. Matrix matched analysis at four different surface waters to determine the method LOD and LOQ. Table 5. Matrix matched analysis at four different surface waters to determine the method LOD Table 6. Application samples from Umgeni River, sport centre tap water, laboratory tap water and UKZN Wetville campus ponds in KZN for MCs determination. Table 6. Application samples from Umgeni River, sport centre tap water, laboratory tap water and UKZN Wetville campus ponds in KZN for MCs determination. Page 11/20 Page 11/20 Sampling Point MC-RR µg L-1 MC-YR µg L-1 MC-LR µg L-1 Blue lagoon dam Blue lagoon outlet Umgeni river inlet Umgeni estuary Sport centre tap Laboratory tap water  Campus pond1 Campus pond2 ND ND ND ND ND ND ND 2.160  ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND Table 7. Application samples from Darvill Wastewater Treatment Plant for MCs determination on three consecutive weeks Sampling Point MC-RR µg L-1 MC-YR µg L-1 MC-LR µg L-1 Return Activated Sludge 1st campaign samples ND ND ND Digested Thickened WAS ND 2.015 ± 0.106 ND Settled sewage D ND ND Raw sludge D ND ND Thickened WAS D ND ND Raw sewage ND ND ND Final effluent ND ND ND                                            2nd campaign samples Sampling points MC-RR µg L-1 MC-YR µg L-1 MC-LR µg L-1 Effluent secondary D ND ND Maturation River D ND ND Balancing tank 6.470  ND ND Inlet 4.403  4.938  ND                                             3rd campaign samples Sampling points MC-RR µg L-1 MC-YR µg L-1 MC-LR µg L-1 Effluent secondary ND ND ND Maturation River ND ND ND Balancing tank 9.384  ND ND Inlet 30.521  ND ND ication samples from Darvill Wastewater Treatment Plant for MCs determination on three consecutive weeks Table 7. Tables Application samples from Darvill Wastewater Treatment Plant for MCs determination on three consec Page 12/20 Page 12/20 Page 12/20 Table 8. Comparison of the analytical method used in literature with the current developed method Page 12/20 Instrument Extraction method Mobile phase Column Analyte Method detection limit Method quantification limit R2 Nature of water Environmental concentration of microcystins Reference HPLC/PDA SPE 0.05% TFA in Acetonitrile and 0.1% TFA in UPW) (Shimadzu, 5 μm, C18, 250x4.6 mm MC-RR, MC-YR, MC-LR 0.02 µg L-1 N/A N/A Reservoir in Brazil MCs total (ND – 1.59 µg L-1) Falconer- Dias et.al[6] HPLC/PDA SPE with empore extraction disks 0.1% TFA in Acetonitrile and 0.1% TFA in UPW Upelcosil ABZþPlus column  (150x 4.6 mm, 5 mm; Supelco,) MC-RR, MC-YR, MC-LR 0.08 µg L-1 0.15 µg L-1 N/A Inland waters in Bulgarian ND Stoyneva- Gartener et.al [53] HPLC/PDA   0.1% TFA in Acetonitrile and 0.1% TFA in UPW Waters C18 Symmetry (3.9 mm internal diameter × 150 mm, 5 μm particle size MC-YR MC-LR N/A N/A 0.9981 Lake District in Turkey 2.9 – 13.5 µg L-1 N/D Gurbuz et.al [54] HPLC- UV/PDA SPE (0.05% TFA in Acetonitrile) and (0.05% TFA in UPW) C18 analytical column (XBridge C18 – 3.5 µm – 4.6 x100mm, Waters) MC-RR, MC-YR, MC-LR 0.10 µg L-1 N/A 0.990 Pigeard pond in France MCs total (ND – 34.4 µg L-1 Thurest- Benoist et.al [20] HPLC/PDA SPE (0.025% TFA in Acetonitrile) and (0.025% TFA in UPW)   Hypersil Gold C18 (Guard column 2.1 x 10 mm, 3 µm particle size) MC- RR,  MC- YR,  MC-LR 0.0003 µg mL-1 0.48 0.16 0.0007 µg mL-1 1.50 0.58 0.9890 0.9880 0.9895 Darvill wastewater in KZN   MCs total (ND – 30.521 ng L- 1) Current method Table 9:  MCs ecological risk assessment in real water samples Sampling point Campus pond1 Digested thickened WAS 2nd campaign (balancing tank) 2nd campaign- inlet 3rd campaign (balancing tank) 3rd     campaign (Inlet) Analyte MC-RR µg/L MC- YR µg/L MC-RR µg/L MC-YR µg/L MC-RR µg/L MC- YR µg/L MC-RR µg/L MC-YR µg/L MC-RR µg/L MC- YR µg/L MC-RR µg/L MC- YR µg/L MEC 2.160 ± 0.003 - - 2.015 ± 0.106 6.470 ± 0.149 - 4.403 ± 0.261 4.938 ± 0.181 9.384 ± 0.087 - 30.521± 0.357 - RCR 0.0540 - - 0.0503 0.161 - 0.110 0.123 0.234 - 0.764 - Ecological risk assessment Low risk - - Low risk Medium risk - Medium risk Medium risk Medium risk - Medium risk - Table 9:  MCs ecological risk assessment in real water samples Page 13/20 Page 13/20 Figure 1 Microcystins – LR general structure Figure 2 Microcystins chromatogram obtained by injecting 10 µL of 2500 µg L-1 microcystins standard of targeted analytes Figure 1 Microcystins – LR general structure Figure 1 Figure 1 Microcystins – LR general structure Figure 2 Microcystins chromatogram obtained by injecting 10 µL of 2500 µg L-1 microcystins standard of targeted analytes Figure 2 Microcystins chromatogram obtained by injecting 10 µL of 2500 µg L-1 microcystins standard of targeted analytes Page 14/20 Page 14/20 Figure 3 MC-YR chromatogram obtained when injecting 10 µL of 2500 µg L-1 standard solution in HPLC. Page 12/20 Figure 4 MCs separation spectrum (a) when methanol was used there was no separation, (b) purity curve above the analytes, (c) when acetonitrile was used, 3 Figure 3 MC-YR chromatogram obtained when injecting 10 µL of 2500 µg L-1 standard solution in HPLC. MC-YR chromatogram obtained when injecting 10 µL of 2500 µg L-1 standard solution in HPLC. Figure 4 MCs separation spectrum (a) when methanol was used there was no separation, (b) purity curve above the analytes, (c) when acetonitrile was used, 3 analytes were identified, (d) purity curve below analytes Figure 4 MCs separation spectrum (a) when methanol was used there was no separation, (b) purity curve above the analytes, (c) when acetonitrile was used, 3 analytes were identified, (d) purity curve below analytes Figure 4 Figure 4 MCs separation spectrum (a) when methanol was used there was no separation, (b) purity curve above the analytes, (c) when acetonitrile was used, 3 analytes were identified, (d) purity curve below analytes MCs separation spectrum (a) when methanol was used there was no separation, (b) purity curve above the analytes, (c) when acetonitrile was used, 3 analytes were identified, (d) purity curve below analytes Page 15/20 Page 15/20 Figure 5 Chromatograms of microcystins (a) separation chromatogram (b) where there was no separation. Figure 6 Chromatograms of microcystins at different injection volume (a) 20 µL and (b) 2 µL obtained by analysing 2500 µg L-1 MCs standard. Figure 5 Chromatograms of microcystins (a) separation chromatogram (b) where there was no separation. Figure 5 Chromatograms of microcystins (a) separation chromatogram (b) where there was no separation. Figure 6 Chromatograms of microcystins at different injection volume (a) 20 µL and (b) 2 µL obtained by analysing 2500 µg L-1 MCs standard. Figure 6 Chromatograms of microcystins at different injection volume (a) 20 µL and (b) 2 µL obtained by analysing 2500 µg L-1 MCs standard. hromatograms of microcystins at different injection volume (a) 20 µL and (b) 2 µL obtained by analysing 2500 µ Page 16/20 Page 16/20 Figure 7 Chromatograms of microcystins obtained by injecting 10 µL of 2500 µg L-1 for separation at different flow rate (a) MCs at 0.1 mL min-1, (b) MCs at 0.5 mL min-1 and (c) MCs at 0.7 mL min-1 gure 7 Figure 7 Chromatograms of microcystins obtained by injecting 10 µL of 2500 µg L-1 for separation at different flow rate (a) MCs at 0.1 mL min-1, (b) MCs at 0.5 mL min-1 and (c) MCs at 0.7 mL min-1 Chromatograms of microcystins obtained by injecting 10 µL of 2500 µg L-1 for separation at different flow rate (a) MCs at 0.1 mL min-1, (b) MCs at 0.5 mL min-1 and (c) MCs at 0 7 mL min-1 Page 17/20 Figure 8 Microcystins instrument stability line graph obtained by injecting 375 µg L-1 MCs standard. Page 17/20 Figure 8 Microcystins instrument stability line graph obtained by injecting 375 µg L-1 MCs standard. Figure 8 Figure 9 Calibration curves obtained for (a) Microcystins (MC-RR); (b) MC-YR; (c) MC-LR on HPLC/PDA. Figure 10 Figure 9 Calibration curves obtained for (a) Microcystins (MC-RR); (b) MC-YR; (c) MC-LR on HPLC/PDA. Figure 9 Calibration curves obtained for (a) Microcystins (MC-RR); (b) MC-YR; (c) MC-LR on HPLC/PDA. Figure 9 Calibration curves obtained for (a) Microcystins (MC-RR); (b) MC-YR; (c) MC-LR on HPLC/PDA. Calibration curves obtained for (a) Microcystins (MC-RR); (b) MC-YR; (c) MC-LR on HPLC/PDA. Figure 10 Figure 10 Page 18/20 pH optimisation of microcystins at pH 4, 5, and 6 to obtain MCs recoveries. pH optimisation of microcystins at pH 4, 5, and 6 to obtain MCs recoveries. pH optimisation of microcystins at pH 4, 5, and 6 to obtain MCs recoveries. Figure 11 The contour and Pareto charts for all three responses. Page 19/20 Figure 11 The contour and Pareto charts for all three responses. Figure 11 The contour and Pareto charts for all three responses. The contour and Pareto charts for all three responses. Page 19/20 Page 19/20 Figure 12 Three responses Half normal plot, 3D surfaces, and 3D cube. Figure 12 Three responses Half normal plot, 3D surfaces, and 3D cube. Three responses Half normal plot, 3D surfaces, and 3D cube. GraphicalAbstract.png Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Page 20/20 Page 20/20
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Ultra-small microorganisms in the polyextreme conditions of the Dallol volcano, Northern Afar, Ethiopia
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Ultra-small microorganisms in the polyextreme conditions of the Dallol volcano, Northern Afar, Ethiopia Received: 10 April 2018 Accepted: 15 May 2019 Published: xx xx xxxx Received: 10 April 2018 Accepted: 15 May 2019 Published: xx xx xxxx The Dallol geothermal area in the northern part of the Danakil Depression (up to 124–155 meter below sea level) is deemed one of the most extreme environments on Earth. The area is notable for being part of the Afar Depression, an incipient seafloor-spreading center located at the triple junction, between Nubian, Somali and Arabian plates, and for hosting environments at the very edge of natural physical- chemical extremities. The northern part of the Danakil Depression is dominated by the Assale salt plain (an accumulation of marine evaporite deposits) and hosts the Dallol volcano. Here, the interaction between the evaporitic deposit and the volcanisms have created the unique Dallol hot springs, which are highly acidic (pH ~ 0) and saline (saturation) with maximum temperatures ranging between 90 and 109 °C. Here we report for the first time evidence of life existing with these hot springs using a combination of morphological and molecular analyses. Ultra-small structures are shown to be entombed within mineral deposits, which are identified as members of the Order Nanohaloarchaea. The results from this study suggest the microorganisms can survive, and potential live, within this extreme environment, which has implications for understanding the limits of habitability on Earth and on (early) Mars. The study of environmental limits of life provides useful information for assessing the limits of habitability both on Earth and elsewhere in the Solar System1,2. Understanding, and defining these limits, using extreme terrestrial environments and Earth analogues sites3,4 is therefore a crucial step in selecting sites for future life detection missions. The Dallol geothermal area (14°14′21″N; 40°17′55″E) is located in the northern part of the Danakil Depression5. This is a narrow lowland salt plain (up to 124 m below sea level) running inland, quasi-parallel to the coast of the Red Sea (Fig. 1a), which formed when part of the Red Sea was isolated during the Pleistocene.h g g The area is located at the northern segment of the Afar triple junction and is characterized by an attenuated continental crust, which is less than 15 km thick with shallow (3–5 km deep) magma chambers beneath its axial zones6,7. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 10 April 2018 Accepted: 15 May 2019 Published: xx xx xxxx Scientific Reports | (2019) 9:7907 | https://doi.org/10.1038/s41598-019-44440-8 Ultra-small microorganisms in the polyextreme conditions of the Dallol volcano, Northern Afar, Ethiopia The Dallol dome-shaped deposits formed as a result of the interaction between the evaporitic deposits and the shallow magma intrusions7,8.h g The hydrothermalism of the Dallol area was possibly generated by a phreatic explosion in 1926. Since then volcanic episodes, seismicity and hydrothermal activity have been reported in the area5. As a consequence of geo- thermal activity7,9 several minerals, for example, pyrolusite, chlorargyrite, wurtzite and halite, precipitate forming colourful chimneys (Fig. 1b). It has been postulated that microbial activity does not play a major role in mineral precipitation and geochemical cycling within Dallol10.h At the surface, the water temperature at the source is above 100 °C and highly acidic (pH ~ 0). The resultant hot pools vary in color depending on the high metal concentration- (e.g., iron 35.6 g/L, copper 93 mg/l) (Table 1). A 1Centro de Astrobiología (INTA-CSIC) Crtera. Ajalvir km 4 Torrejón de Ardoz, Madrid, 28850, Spain. 2Dipartimento di Scienze Biologiche, Geologiche e Ambientali (BiGeA), Università di Bologna, Bologna, Italy. 3Department of Geology, University of Johannesburg, Johannesburg, South Africa. 4Centro de Biología Molecular “Severo Ochoa” Cantoblanco, Madrid, Spain. 5IRSPS, Universitá d’Annunzio, Pescara, Italy. 6Ibn Battuta Centre, Université Cadi Ayyad, Marrakech, Morocco. 7School of Environment, Earth and Ecosystems Sciences, The Open University, Milton Keynes, UK. 8Department of Earth Sciences, Mekelle University, Mekelle, Tigre, Ethiopia. Correspondence and requests for materials should be addressed to F.G. (email: gomezgf@cab.inta-csic.es) Scientific Reports | (2019) 9:7907 | https://doi.org/10.1038/s41598-019-44440-8 1 www.nature.com/scientificreports/ Figure 1. Study area. (a) Location map of the Dallol geothermal area (red dot) in the Danakil Depression (map data © 2018 Google). (b) Panoramic view of the sampling sites (D9: central small chimney and D10: water from the blue pool at the bottom of the chimney). Figure 1. Study area. (a) Location map of the Dallol geothermal area (red dot) in the Danakil Depression (m data © 2018 Google). (b) Panoramic view of the sampling sites (D9: central small chimney and D10: water fr the blue pool at the bottom of the chimney). Figure 1. Study area. (a) Location map of the Dallol geothermal area (red dot) in the Danakil Depression (map data © 2018 Google). (b) Panoramic view of the sampling sites (D9: central small chimney and D10: water from the blue pool at the bottom of the chimney). Ultra-small microorganisms in the polyextreme conditions of the Dallol volcano, Northern Afar, Ethiopia Sample D9 D10 pH 0.25 2.42 T 86 47 Cond 188 262 Eh 411 412 H2% 0.02 0.13 CO2% 0 0.15 Na 119333 89323 Mg 3408 5998 Al 383 533 P 10.1 14.9 K 8248 16594 Ca 3810 5927 Mn 538 950 Fe 19159 35652 Cu 40.6 93 Zn 39.6 72 Rb 11.9 20.9 Sr 71.1 116.7 Pb 1.4 0.2 Table 1. Physico-chemical parameters for two sampling sites, D9 and D10 (concentrations in mg/l, average of three measurements). The units are stated as followed T: °C, Eh: mV; conductivity: mS/cm2. Sample D9 D10 pH 0.25 2.42 T 86 47 Cond 188 262 Eh 411 412 H2% 0.02 0.13 CO2% 0 0.15 Na 119333 89323 Mg 3408 5998 Al 383 533 P 10.1 14.9 K 8248 16594 Ca 3810 5927 Mn 538 950 Fe 19159 35652 Cu 40.6 93 Zn 39.6 72 Rb 11.9 20.9 Sr 71.1 116.7 Pb 1.4 0.2 Table 1. Physico-chemical parameters for two sampling sites, D9 and D10 (concentrations in mg/l, average of three measurements). The units are stated as followed T: °C, Eh: mV; conductivity: mS/cm2. Table 1. Physico-chemical parameters for two sampling sites, D9 and D10 (concentrations in mg/l, average of hree measurements). The units are stated as followed T: °C, Eh: mV; conductivity: mS/cm2. Table 1. Physico-chemical parameters for two sampling sites, D9 and D10 (concentrations in mg/l, average three measurements). The units are stated as followed T: °C, Eh: mV; conductivity: mS/cm2. combination of these extreme chemical and physical parameters (e.g. temperature, pH, salinity and heavy metals) has resulted in a unique multi-extreme environment.i combination of these extreme chemical and physical parameters (e.g. temperature, pH, salinity and heavy metals) has resulted in a unique multi-extreme environment.i First reported thermoacidophilic prokaryote was isolated from coal refuse piles11 and the first eukaryote from upper oxygenic zones of acidic geothermally heated waters in solfataras12. In 1995 two species of a moderately thermophilic archaea living around pH 0 were reported13.i p g p p Here, we describe for the first time morphological and molecular evidence of thermohaloacidophilic nanomi- croorganism existing in this novel multi-extreme environment. Ultra-small microorganisms in the polyextreme conditions of the Dallol volcano, Northern Afar, Ethiopia h h d l f ll l6 7 d l l f d Due to the unique geochemistry and volcanic activity of Dallol6,7 it is an ideal analogue site for studying Martian hydrothermal environments, such as Nili Patera caldera9, where hydrothermal sinter deposits are found in direct association with volcanic activity14, and Gusev crater15, the landing site of the Spirit MER rover. y g p Despite the Dallol hot springs being deemed one of the most extreme environments on Earth the possibility of microbial life existing within the springs has not been studied. www.nature.com/scientificreports/ 4d–f, members of the Nanohaloarchaea group were detected with the Narc1214 FISH probe in the salt precipitates at the hydrothermal fluid source from D9, which corroborates the phylogenetic data. l The presence of ultra-small microorganisms was further investigated using Transmission Electron Microscopy (TEM) (Fig. 5), Scanning Electron Microscopy (SEM) and Scanning Transmission Electron Microscopy (STEM) (Fig. 6). Using these techniques, it was possible to identify ultra-small cellular morphologies (between 50 nm and 500 nm), which supports the molecular data (Figs 5 and 6). The Energy-dispersive X-ray (EDX) elemental micro- analysis suggested that the cellular morphologies were unambiguously biological given their high carbon content (Fig. 5A–F). Whilst, SEM-EDX showed the presence of silica precipitates (Fig. 6G–M) surrounding and encrusting the ultra-small cellular morphologies (Fig. 5A–D), suggesting a hydrothermal origin with a high Fe content (Fig. 5E–F).h Figure 6 shows ultra-small microorganisms entombed within precipitated silica minerals (Fig. 6D–L). The cellular morphologies were associated with the needle-shaped crystals (Fig. 6M), which suggests a relationship between the microorganisms and the biomineralization process as has been previously reported in other acidic environments17.hh The ultra-small microorganisms were entombed within the precipitated mineral structures (Fig. 6(D–F)). The mineral precipitation process gave rise to thin mineral layers (Fig. 6(D)). Figure 6(E,F) show an enlarged view of the thin mineral layers. Figure 6(G) shows ultra-small microorganisms, which were not covered by minerals, whilst Fig. 6(H,I) show ultra-small microorganisms partially covered with mineral precipitates. More biominer- alized ultra-small microorganisms are visible in Fig. 6(J,K). Figure 6(M) shows STEM images with close views of ultra-small microorganisms surrounded by mineral needles and Fig. 6(F) shows mineral spheres resulting from the mineralization process. Needles with spike forms are observed in Fig. 6(J–L). Finally, Fig. 6(N,O) show ultra-small microorganisms surrounded by minerals in a close view using STEM. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 2. X-Ray Diffraction analyses showing sample D9 mineral composition. (a) chlorargyrite (C) and wurtzite (W); (b) halite (H), and (c) pyrolusite (P) and halite (H). Figure 2. X-Ray Diffraction analyses showing sample D9 mineral composition. (a) chlorargyrite (C) and wurtzite (W); (b) halite (H), and (c) pyrolusite (P) and halite (H). DNA was extracted from the salt precipitates at the hydrothermal fluid source (D9), 16S rDNA was amplified using Archaea primers (Fig. 4c) and phylogenetic analysis was performed. The OTU named Dallol 9 showed close similarity (95.8%) with the Nanohaloarchaea environmental clone ARDARCSS13 (GenBank accession number EU869371) (Fig. 3) which closely matched to uncultured sequences from Nanohaloarchaeales Order.h DNA was extracted from the salt precipitates at the hydrothermal fluid source (D9), 16S rDNA was amplified using Archaea primers (Fig. 4c) and phylogenetic analysis was performed. The OTU named Dallol 9 showed close similarity (95.8%) with the Nanohaloarchaea environmental clone ARDARCSS13 (GenBank accession number EU869371) (Fig. 3) which closely matched to uncultured sequences from Nanohaloarchaeales Order.h g y q The clone ARDARCSS13 and Dallol 9 sequences are close to Candidatus Holaredivivus sp. G17 (Fig. 3) that has been described16 in a Bras of Port saltern pond (Alicante, Spain) as a photoheterotrophic microbe that present rhodopsin and photolyase. Candidatus Holaredivivus sp. G17 is capable to degrade polysaccharides, likewise presents typical genes of archaea. In relation to survival strategies in hypersaline environments, Haloredivivus sp. G17 is a salt-in strategist as the isoelectric point of his proteins shows16.i To confirm the presence of members of the Nanohaloarchaea group in the samples Fluorescence In Situ Hybridization (FISH) was carried out. Samples fixed in situ for FISH were sequentially filtered through 0.45, 0.22 and 0.1 µm filters to select for small (<0.1 µm) microorganisms. Inspection of the DNA stained material retained in the 0.2 µm filters with SybrGold suggested the presence of ultra-small microorganisms forming compact dense colonies, including mucilaginous films (Fig. 4a). Ultrasonication was further used to disintegrate the colonies prior to sequentially filtration. The ultra-small microorganisms passed through the 0.22 µm filter, but were again retained on the 0.1 µm filters (Fig. 4b). Figure 4c shows 16S rDNA amplification using universal archaea primers. i g g pi g p As it is shown in Fig. Results and Discussion In this system many protuberances can be observed (Figs 1b and 6A–C), which are generated by the precipi- tation of minerals from the superheated underground solutions. The chimney selected for this study was Site D9 (Figs 1b and 6A) and composed of chlorargyrite (Fig. 2a), wurtzite (Fig. 2a), halite (Fig. 2b) and pyrolusite (Fig. 2c). Table 1 reports the physico-chemical parameters of Site D9 and the surrounding pool (Site D10) as shown in Fig. 6A. The pH and temperature between the two sites varied, for example at Site D9, the source of the hydrothermal fluid, the fluid was pH 0.25 and 86 °C compared to 2.42 and 47 °C at site D10. Scientific Reports | (2019) 9:7907 | https://doi.org/10.1038/s41598-019-44440-8 2 Concluding Remarks g We demonstrated the presence of living ultra-small microorganisms in a multi-extreme environment with adverse conditions for life: extreme low pH (0.25), temperature (90 °C), redox potential, salinity and heavy metals content. Molecular studies, electron microscopies observation and phylogenetic analyses of amplified rDNA sequences showed the presence of ultra-small microorganisms related to the Order Nanohaloarchaea. Scientific Reports | (2019) 9:7907 | https://doi.org/10.1038/s41598-019-44440-8 3 www.nature.com/scientificreports/ Figure 3. Neighbor-joining 16S rRNA phylogenetic tree of DPANN archaea. Open circles indicate bootstrap values between 70–89. Solid circles indicate bootstrap values bootstrap values higher than 90. Figure 3. Neighbor-joining 16S rRNA phylogenetic tree of DPANN archaea. Open circles indicate bootstrap values between 70–89. Solid circles indicate bootstrap values bootstrap values higher than 90. Figure 3. Neighbor-joining 16S rRNA phylogenetic tree of DPANN archaea. Open circles indicate bootstrap values between 70–89. Solid circles indicate bootstrap values bootstrap values higher than 90. The ultra-small bacteria observed in the samples were morphologically spherical and entombed in the mineral layers that form the small chimneys in Dallol under high acidity and high temperature conditions. The saturation of salts and minerals in the superheated water, which results in precipitation and the formation of the chimneys may be influenced by entombed microorganisms. We described the presence of ultra-small microorganisms in a natural environment which is an Earth analogue of some regions of Mars such as Nili Patera Caldera9,10. The pres- ence of life in the Dallol hot springs expands our understanding of the limits of habitability on Earth and beyond. However, future work is needed to understand how these nanobacteria survive in such an extreme environment and whether they play a role in geochemical cycling. Materials and Methodsh Dehydration was completed with a mixture of ethanol/propylene oxide (1:1) for 10 min and pure propylene oxide 3 × 10 min. Infiltration of the resin was accomplished with propylene oxide/ Epon (1:1) for 45 min and pure LR White resin (London Resin Company limited, England), overnight at room temperature. Polymerization of infiltrated samples was done at 60 °C for 2 days. Ultrathin sections of the samples were stained with uranyl acetate and lead citrate by standard procedures. y y p Four types of electronic microscopy techniques were used for this study: Scanning Electron Microscope (SEM) (JEOL-5600 coupled to an EDX, INCA) with an Energy Dispersive X-Ray Analyzer (EDX) and SEM-FI (Philips XL30-FEG); Scanning Electron Microscopy-Field Emission Gun (SEM-FEG) (Philips XL30-FEG); Transmission Electronic Microscope (TEM) (JEM-1010); TEM/STEM electronic microscope (JEOL 2100 K) with FEG. Samples were mounted on conductive graphite stubs and sputter and gold-coated in a Quorum, Q150T-S apparatus to ensure electrical conductivity and prevent charging under electron beams. pp y p g g For SEM analysis, samples were also analyzed with EDX to obtain semiquantitative chemical data. A STEM unit was coupled to the microscope with acquisition of contrast images z (HAADF). The qualitative element composition of samples was determined using an INCA-X-SIGHT with a Si-Li detector (Oxford, England) with a detection limit of 10% for the main element. The operating energy was 200 kv. For TEM analyses, the instrument was operated at 200 kv (Catalysis Institute and PETROQUIMICA-CSIC), equipped with an energy dispersive X-RAY microanalysis instrument INCA (Oxford, England). The TEM/STEM was operated at 200 kv with an EDX, which was coupled to a STEM unit, with acquisition of contrast images z (HAADF). The same grids were used for both TEM and TEM-STEM. Fluorescence microscopy. Samples were fixed in 4% formaldehyde in phosphate-buffered saline (PBS) for 2 h and stored at 4 °C until further processing. Fixed samples were dispersed by 3 cycles of sonication of 30 sec, with a 30 sec break in between with 1 pulse per second (intensity of 20%). The samples were filtered sequentially through a 0.4 μm pore size polycarbonate filters, a 0.2 μm pore size filter and a 0.1 μm pore size filter (Millipore, Germany). Filters were pretreatment as previously described18. FISH was performed as described by Glöckner, et al.19 using Cy3 single-labeled Narc1214 probe20 (Biomers, Ulm, Germany). Materials and Methodsh Sampling. The sampling campaign was carried out in January 2017 and samples were collected from a fuma- role’s wall (14°14′21″N; 40°17′55″E) located at the main Dallol hydrothermal outcrop in the Danakil depression and a blue pool surrounding the small fumarole. Samples were collected aseptically, using sterilized spatulas and plastic aseptic materials in 12 mL sterile glass vials. The vials were completely filled to prevent head space. To ensure that the samples did not oxidize during transportation and storage, the vials were sealed with a septum tap, covered with parafilm tape and kept under anaerobic conditions. Samples were transported at room temperature. “In Situ” physico-chemical parameters measurement. Physico-chemical parameters (T: °C, Eh: mV; conductivity: mS/cm2) were measured “in situ” using a multi-parametric probe, YSI 556 MPS. Gas composition by gas chromatography and elemental composition using ICP-MS. Electron microscopy. Samples were fixed in 4% paraformaldehyde and 2% glutaraldehyde, in 0.1 M phos- phate buffer (pH 7.2), for 2 h at room temperature. Fixed samples were washed three times in the same buffer and post-fixed with 1% OsO4 in water for 60 min at room temperature in the dark. After three washes in distillate water, samples were incubated with 2% aqueous uranil acetate for 1 h at room temperature, washed again, and Scientific Reports | (2019) 9:7907 | https://doi.org/10.1038/s41598-019-44440-8 4 www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 4. Living microorganisms at the Dallol hydrothermal outcrop. (a) SybrGold DNA staining of microorganisms retained in 0.2 µm filter before ultrasonication. (b) SybrGold DNA stain of microorganisms retained in 0.1 µm filter after ultrasonication. (c) Electrophoresis of archaeal amplified DNA from sample D9. (d–f) FISH images of Nanohaloarchaea microorganisms. In green, SybrGold DNA staining (d). In red, Narc1214 FISH probe signal (e). (f) d and e merge. Scale bars, 5 μm. Figure 4. Living microorganisms at the Dallol hydrothermal outcrop. (a) SybrGold DNA staining of microorganisms retained in 0.2 µm filter before ultrasonication. (b) SybrGold DNA stain of microorganisms retained in 0.1 µm filter after ultrasonication. (c) Electrophoresis of archaeal amplified DNA from sample D9. (d–f) FISH images of Nanohaloarchaea microorganisms. In green, SybrGold DNA staining (d). In red, Narc1214 FISH probe signal (e). (f) d and e merge. Scale bars, 5 μm. dehydrated in increasing concentrations of ethanol 30, 50, and 70% 20 min each, 90% 2 × 20 min, and 100% 2 × 30 min at room temperature. Materials and Methodsh Stringencies were regulated adjust- ing formamide and NaCl concentration in hybridization and washing buffer at 30% (vol/vol) and 0.112 M respec- tively. Filters were counterstaining by incubation with SYBR® Gold (Molecular Probes, Eugene, OR, USA) 1X diluted in milliQ water for 15 min, before being mounted on glass a slide using Vectashield (Vector Laboratories, Burlingame, CA, USA): Citifluor (Citifluor, London, United Kingdom) (1:4). Samples were imaged using a con- focal laser scanning microscope LSM710 (Carl Zeiss, Jena, Germany) equipped with diodo (405 nm), argon (458/488/514 nm) and helium and neon (543 and 633 nm) lasers. Fiji software21 was used to process images. Scientific Reports | (2019) 9:7907 | https://doi.org/10.1038/s41598-019-44440-8 5 www.nature.com/scientificreports/ Figure 5. Transmission Electron Microscopy (TEM) images of sample D9 showing ultra-small biological morphologies. Arrows indicate where the EDX analyses were performed. Spectra show the high carbon content associated with high Si and Fe content in these spots. Figure 5. Transmission Electron Microscopy (TEM) images of sample D9 showing ultra-small biological morphologies. Arrows indicate where the EDX analyses were performed. Spectra show the high carbon content associated with high Si and Fe content in these spots. DNA extraction and amplification of ribosomal genes. Samples were filtered through polycarbonate membranes (0.45, 0.2 and 0.1 µm diameter; Millipore, USA). Total genomic DNA was extracted from those mem- branes using the DNeasy PowerSoil Kit (Qiagen, Germany) as described in the manufacture’s instructions, and con- centrated using a SpeedVac Concentrator. The quantity of the extracted DNA was analysed by fluorimetry using a Qubit 2.0 fluorometer (Thermo Fhiser Scientific, USA). Total genomic DNA were stored at −20 °C for sequencing. Ribosomal genes were amplified using a primers set specific to amplify V2-V3 region of 16S rRNA in Archaea domain (Arch1F 5′-CGGRAAACTGGGGATAAT-3′ and Arch1R 5′-TRTTACCGCGGCGGCTGBCA-3′). PCR reactions were performed as described22,23. Gel electrophoreses (1% agarose Conda, Spain in 0.5X TBE buffer, 90 mV during 30 min and staining with GreenSafe Premium NZYTech) were carried out to check the size and quality of PCR products. Ribosomal genes library preparation and sequencing. Library preparation and 2 × 300 pair-end sequencing by Illumina MiSeq were made by Genomic Unit in Parque Científico de Madrid Foundation/FPCM (Madrid Spain) Total genomic DNAs was quantified by Picogreen. Materials and Methodsh Then, an input of 16 pg of DNA and 27 cycles were used in a first PCR with Q5® Hot Start High-Fidelity DNA Polymerase (New England Biolabs, USA) in the presence of 100 nM primers for 16S amplification (5′-ACACTGACGACATGGTTCTACACCTACGGGNGGCWGCAG-3′ and 5′- TACGGTAGCAGAGACTTGGTCTGACTACHVGGGTATCTAATCC-3′, these primers amplify the V3-V4 region of 16S), 200 nM primers for Archaea amplification (5′-ACACTGACGACATGGTTCTACACGGRAAACTGGGGATAAT -3′ and 5′-TACGGTAGCAGAGACTTGGTCTTRTTACCGCGGCGGCTGBCA-3′),ti After the first PCR, a second PCR of 15 cycles was perfomed with Q5® Hot Start High-Fidelity DNA Polymerase (New England Biolabs, USA) in the presence of 400 nM of primers (5′-AATGATACGGCGACCA CCGAGATCTACACTGACGACATGGTTCTACA-3′ and 5′-CAAGCAGAAGACGGCATACGAGAT-[10 nucleotides barcode]-TACGGTAGCAGAGACTTGGTCT-3′) of the Access Array Barcode Library for Illumina Sequencers (Fluidigm, USA).hii q g The amplicons were validated and quantified by a Bioanalyzer. An equimolecular pool was purified by gen extraction and titrated by quantitative PCR using the “Kapa-SYBR FAST qPCR kit for LightCycler480” with a reference standard for quantification. The pooled amplicons were denatured and added to the flowcell at a density of 9 pM. Clusters formed, which were sequenced using a “MiSeq Reagent Kit v3”, in a 2 × 300 pair-end sequencing run on an Illumina MiSeq sequencer. Scientific Reports | (2019) 9:7907 | https://doi.org/10.1038/s41598-019-44440-8 6 www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports tificreports/ Figure 6. (A) General view of the sampling site, (B) the small chimneys (temperature of water 90 °C. (C) D9 sample from a small chimney in (A). (D–L) SEM and (M–O) Scanning TEM images of sample D9 showing the morphologies of ultra-small microorganisms entombed in the mineral layers. Figure 6. (A) General view of the sampling site, (B) the small chimneys (temperature of water 90 °C. (C) D9 sample from a small chimney in (A). (D–L) SEM and (M–O) Scanning TEM images of sample D9 showing the morphologies of ultra-small microorganisms entombed in the mineral layers. Detection of DPANN OTUs by microbiome analyse. Quality of reads was evaluated by means of FastQC software. PANDAseq Assembler was using for assembling forward and reverse reads and convert in a fasta file24. Sequencing data were processed using Qiime software package version 1.9.025. High qual- ity contigs were clustered into OTUs based on 94% sequence similarity with UCLUST. The first sequence for each OTU as the representative OTU, which were aligned using PYNAST26. The taxonomic identity of each phylotype was determined using the SILVA_132_QIIME database (https://www.arb-silva.de/down- load/archive/qiime/)27. g Europlanet 2020 RI received funding from the European Union’s Horizon 2020 research and innovation program under Grant Agreement No 654208. g Europlanet 2020 RI received funding from the European Union’s Horizon 2020 research and innovation program under Grant Agreement No 654208. References Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing- based diversity studies. Nucleic acids research. 41(1), 1–11 (2013). y 24. Bartram, A. K., Lynch, M. D., Stearns, J. C., Moreno-Hagelsieb, G. & Neufeld, J. D. Generation of multimillion-sequence 16S rRNA gene libraries from complex microbial communities by assembling paired-end Illumina reads. Applied and environmental microbiology. 77(11), 3846–3852 (2011). gy 25. Caporaso, J. G. et al. QIIME allows analysis of high-throughput community sequencing data. Nature methods. 7(5), 335 (2010).l ( ) 26. Caporaso, J. G. et al. PyNAST: a flexible tool for aligning sequences to a template alignment. Bioinformatics. 26(2), 266 (2010).h 27. Quast, C. et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic acids research. 41(D1), D590–D596 (2012).t 28. Ludwig, W. et al. ARB: a software environment for sequence data. Nucleic acids research 32(4), 1363–1371 (2004). 28. Ludwig, W. et al. ARB: a software environment for sequence Author Contributions Felipe Gómez led the work, did the sampling and the experimentation and wrote the article, including Figures 1–3. Barbara Cavalazzi contributed to design the field campaign, to the description of the geological setting and to the corrections of the article. Nuria Rodríguez contributed in the sampling, experimental and electromicroscopy of the work. She did the Figures 4 and 5 and Table 1. Ricardo Amils advised on the microbiological part of the work and contributed to the correction of the manuscript. Gian Gabriele Ori contributed to design the field campaign and advised on the geological part of the work. Karen Olsson-Francis contributed to the microbiological work and to the correction of the article. Cristina Escudero contributed to the hybridization and staining experiments. Jose M. Martínez contributed with the metagenomic study of the work. Hagos Miruts contributed to the sampling campaign and advised on the contributed to the geological interpretation of the geological Dallol model. References F., Ehlmann, B. L., Milliken, R. E. & Murchie, S. L. Silica deposits in the Nili Pa volcanic complex on Mars. Nature Geoscience. 3, 838–841, https://doi.org/10.1038/ngeo990 (2010). 4. Skok, J. R., Mustard, J. F., Ehlmann, B. L., Milliken, R. E. & Murchie, S. L. Silica deposits in the Nili Patera caldera on the Syrtis Majo volcanic complex on Mars. Nature Geoscience. 3, 838–841, https://doi.org/10.1038/ngeo990 (2010).f p , , p g g ( ) 15. Ruff, S. W. & Farmer, J. D. Silica deposits on Mars with features resembling hot spring biosignatures at El Tatio in Chile. Nature communications. 7, 13554, https://doi.org/10.1038/ncomms13554 (2016).i 15. Ruff, S. W. & Farmer, J. D. Silica deposits on Mars with features resembling hot spring biosignatures at El Tatio in Chi i ti 7 13554 htt //d i /10 1038/ 13554 (2016) 15. Ruff, S. W. & Farmer, J. D. Silica deposits on Mars with features resembling hot spring biosignatu communications. 7, 13554, https://doi.org/10.1038/ncomms13554 (2016).i 15. Ruff, S. W. & Farmer, J. D. Silica deposits on Mars with features resembling hot spring biosignatures at El Tatio in Chile. Nature communications. 7, 13554, https://doi.org/10.1038/ncomms13554 (2016).i p g 16. Ghai, R. et al. New abundant microbial groups in aquatic hypersaline environments. Scientific reports 1, 135 (2011). g y fi p 17. Oggerin, M., Tornos, F., Rodriguez, N., Pascual, L. & Amils, R. Fungal Iron biomineralization in Río Tinto. Minerals 6, 37, https:// doi.org/10.3390/min6020037 (2016).li g 8. Ishii, K., Mussmann, M., Macbregor, B. J. & Amann, R. An improved fluorescence in situ hybridization protocol for the identification of bacteria and archaea in marine sediments. FEMS Microbiol Ecol. 50, 203–13 (2004).i 19. Glöckner, F. O. et al. An In Situ Hybridization Protocol for Detection and Identification of Planktonic Bacteria. Systematic and Applied Microbiology 19, 403–406, https://doi.org/10.1016/S0723-2020(96)80069-5 (1996). 20. Narasingarao, P. et al. De novo metagenomic assembly reveals abundant novel major lineage of Archaea in hypersaline microbial communities. The ISME journal 6(1), 81 (2012). h j 1. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 9(7), 676–82, https://doi.org/10.1038 nmeth.2019 (2012).l 22. Cruaud, P. et al. Influence of DNA extraction method, 16S rRNA targeted hypervariable regions, and sample origin on microbial diversity detected by 454 pyrosequencing in marine chemosynthetic ecosystems. Applied and Environmental Microbiology. 80(15), 4626–4639 (2014). 23. Klindworth, A. et al. Materials and Methodsh Since Ultra small microorganisms are the target of this study, their representative sequences were filtered from biom table in order to analysis only these taxa using filter_taxa_from_otu_ table.py and filter_fasta.py scripts in Qiime. Finally, the ARB software package28 was used to reconstruct the phylogenetic tree. The neighbour-joining with Felsenstein correction included in the ARB package was used for phylogenetic inference. The robustness of the reconstructed trees was evaluated by bootstrap analysis of 1000 resampled datasets. Scientific Reports | (2019) 9:7907 | https://doi.org/10.1038/s41598-019-44440-8 7 www.nature.com/scientificreports/ References 1. Horneck, G. et al. AstRoMap European Astrobiology Roadmap. Astrobiology. 16(3), 201–243, https://doi.org/10.1089/ast.2015.1441 (2016). 1. Horneck, G. et al. AstRoMap European Astrobiology Roadmap. Astrobiology. 16(3), 201–243, https://doi.org/10.1089/ast.2015.1441 (2016). 2. Stetter, K. O. Extremophiles and their adaptation to hot environments. FEBS Letters. 452, 1–2, https://doi.org/10.1016/S0014 5793(99)00663-8 (2003).h 3. Fairén, A. G. et al. Astrobiology through the Ages of Mars: The Study of Terrestrial Analogues to Understand the Habitability o Mars. Astrobiology. 10(8), 821–843, https://doi.org/10.1089/ast.2009.0440 (2010). gy p g 4. Rothschild, L. J. Earth analogs for Martian life. Microbes in evaporites, a new model system for life on Mars. Icarus. 88-1, 246–260 https://doi.org/10.1016/0019-1035(90)90188-F (1990). p g ( ) ( ) 5. Miruts, H., Bheemalingeswara, K. & Jemal, A. A preliminary geological and generalized stratigraphy of western margin of northerm Afar depression, Dallol area, northern Ethiopia. Momona Ethiopian Journal of Science. 8(1), 1–22 (2016). p p p f 6. Pagli, C. et al. Shallow axial magmachamber at the slow spreading Erta Ale Ridge. Nat Geosci 5, 284–288 (2012). 7. Nobile, A. et al. Dyke-fault interaction during the 2004 Dallol intrusion at the northern edge of the Erta AleRidge (Afar, Ethiopia) Geophys Res Lett 39, L19305 (2012).h p y 8. Cavalazzi, B. et al. The Dallol Geothermal Area, Northern Afar (Ethiopia)—An Exceptional Planetary Field Analog on Earth Astrobiology Published Online: 17 Jan, https://doi.org/10.1089/ast.2018.1926 (2019).h gy p g ( ) 9. Fawdon, P. et al. The geological history of Nili Patera, Mars. JGR. 120(5), 951–977, https://doi.org/10.1002/2015JE004795 (201 gy p g 9. Fawdon, P. et al. The geological history of Nili Patera, Mars. JGR. 120(5), 951–977, http h 10. Kotopoulou, E. et al. A Polyextreme hydrothermal system controlled by iron: the case of Dallol at the Afar triangle. ACS Earth Space Chem. 3(1), 90–99, https://doi.org/10.1021/acsearthspacechem.8b00141 (2019).f p g p 11. Darland, G., Brock, T. D., Samsonoff, W. & Conti, S. F. A thermophilic acidophilic Mycoplasma isolated from a coal refuse Science 170, 1416–1418 (1970).h ( ) 12. Brock, T. D. Thermophilic microorganisms and life at high temperatures. Springer-Verlag, New York (1978). h 13. Schleper, C., Pühler, G., Kühlmorgen, B. & Zillig, W. Life at extremely low pH. Nature 375, 741–742 (1995). 14. Skok, J. R., Mustard, J. F., Ehlmann, B. L., Milliken, R. E. & Murchie, S. L. Silica deposits in the Nili Patera caldera on the Syrtis Major volcanic complex on Mars. Nature Geoscience. 3, 838–841, https://doi.org/10.1038/ngeo990 (2010). 14. Skok, J. R., Mustard, J. Additional Informationh Competing Interests: The authors declare no competing interests. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 8 Scientific Reports | (2019) 9:7907 | https://doi.org/10.1038/s41598-019-44440-8 www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. 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Salivary diagnostic markers in males and females during rest and exercise
Journal of the International Society of Sports Nutrition
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7,334
Abstract Background: Saliva is a useful diagnostic tool for analysis in sports, exercise and nutrition research, as collection is easy and non-invasive and it contains a large number of analytes affected by a range of physiological and pathological stressors and conditions. This study examined key salivary electrolytes and stress and immune markers in males and females at rest and during exercise. Methods: Unstimulated whole saliva from 20 healthy, recreationally active participants (8 males and 12 females) was analysed for flow rate, osmolality, sodium (Na+), potassium (K+), chloride (Cl−), secretory immunoglobulin A (SIgA), α-amylase activity and cortisol during both rest and moderate intensity (70% peak power) cycling exercise in a randomised crossover design. Each trial lasted 60 min and sampling was carried out at 15 and 45 min after the start of the trial. Saliva was collected using the gold-standard drool method; participants were required to provide at least 1 mL sample over 2 or 3-min period. Results: Females showed a greater response to steady-state exercise stress than males, with significant increases in osmolality (P < 0.001), α-amylase activity (P = 0.001) and secretion rate (P = 0.023) and SIgA secretion rate (P = 0.023), with trends for an increase in K+ (P = 0.053) and decrease in Cl−(P = 0.067). There were no differences between rest and exercise for any salivary analytes in males. In addition, females showed a trend for higher levels of cortisol than males at both rest (P = 0.099) and exercise (P = 0.070), as well as a higher heart rate (P < 0.001) and greater ratings of perceived exertion (P < 0.001) during the exercise trial. The coordination of the two stress response pathways (α-amylase vs cortisol) was positive in males (r = 0.799; P = 0.017) yet negative in females (r = −0.475; P = 0.036). Conclusions: Males and females show a markedly different response to steady-state exercise stress as measured in Conclusions: Males and females show a markedly different response to steady-state exercise stress as measured in unstimulated whole saliva. Keywords: Hydration, Stress response, Immune markers, Electrolytes, Sex Keywords: Hydration, Stress response, Immune markers, Electrolytes, Sex Hydration, electrolyte status, stress and immune re- sponses are key markers for exercise performance and health status [2, 3, 10]. © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: A.Ali@massey.ac.nz 2Centre for Metabolic Health Research, Massey University, Palmerston North, New Zealand 5School of Sport, Exercise and Nutrition Massey University, Auckland, New Zealand Full list of author information is available at the end of the article Rutherfurd-Markwick et al. Journal of the International Society of Sports Nutrition (2017) 14:27 Rutherfurd-Markwick et al. Journal of the International Society of Sports Nutrition (2017) 14:27 DOI 10.1186/s12970-017-0185-8 Open Access Open Access Kay Rutherfurd-Markwick1,2, Carlene Starck2,3, Deborah K. Dulson4 and Ajmol Ali2,5* Kay Rutherfurd-Markwick1,2, Carlene Starck2,3, Deborah K. Dulson4 and Ajmol Ali2,5* Abstract A 2–3% dehydration-associated body mass loss has been linked with a reduction in heat regulation, cardiovascular function and exercise per- formance [11] and results in significant changes in salivary composition [1]. Water is the predominant fluid constituent of saliva, thus hypo-hydration is expected to decrease salivary flow-rate, increase osmolality, and may alter the concentrations of key electrolytes, hormones and proteins [11–13]. Background Saliva is gaining momentum as a relevant fluid for clinical and forensic diagnosis, as well as for analysis in sports, exercise and nutrition research, as collection is easy and non-invasive and it contains a large number of analytes affected by a range of physiological and patho- logical stressors and conditions [1–5]. In addition, saliva is less complex than serum, including lower protein content, thus requiring substantially less preparation for analysis [2, 3]. Furthermore, saliva may be used to exam- ine the role of sex hormones in stress and disease [6–9]. Exercise modulates both the innate and acquired arms of the immune system [7] and activates the two major neuroendocrine stress response arms, the hypothalamic- pituitary-adrenal (HPA) axis and the sympathetic- Rutherfurd-Markwick et al. Journal of the International Society of Sports Nutrition (2017) 14:27 Page 2 of 8 participants were required to be free of injury, chronic disease and infection in the 4 weeks prior to the study. adreno-medullary (SAM) axis (sympathetic nervous sys- tem). While SAM activation is an immediate response to exercise, the HPA axis shows a delayed response [6, 14]. Saliva carries two primary markers of HPA and SAM ac- tivation, cortisol and α-amylase, respectively [15]. Alpha- amylase is a reliable indicator of the established blood markers for SAM, epinephrine and norepinephrine, as well as playing a role in mucosal immunity [16]. How- ever, salivary secretory immunoglobulin A (SIgA) is the most widely recognised marker of mucosal immunity [17, 18] and, there appears to be a relationship between decreases in SIgA and increased risk of upper respiratory tract infection (URTI) [10, 19]. Immune and stress re- sponses work together to combat exercise stress [6], with both the HPA and SAM axes modulating the function of the immune system. Preliminary procedures A preliminary session was undertaken to familiarise par- ticipants with the experimental protocol. Upon arrival to the laboratory, participants were shown the correct tech- nique for saliva specimen collection by the passive drool method for the collection of unstimulated whole saliva (UWS). Each participant then performed an incremental exercise test on a cycle ergometer (Ergomedic 874E, Monark Exercise AB, Vansbro Sweden) starting at 60 W, with intensity increasing by 30 W·min−1 until volitional fatigue. Following a brief rest of 5–10 min, participants cycled at a resistance corresponding to 70% of their previously determined peak power for 10 min, then re- ported their perceived exertion and level of confidence regarding completing 60 min of continuous exercise at this intensity. y While blood sampling has historically been used to measure hydration, electrolyte status and markers of stress and immunity, blood sampling procedures may not be practical for the setting, they can be expensive and the invasive approach may not be appealing for all participants [4]. The analysis of other bodily fluids such as saliva holds promise in these situations; however, our understanding of the actions and interactions of the key salivary diagnostic markers in response to stress is in- complete. Most studies have presented information about a selection of markers only and differences in methodology between studies have led to equivocal in- formation [4]. Information in the literature is limited mostly to reviews, in which correlations have been made between studies using different types of participants and varying protocols [2, 3, 10]. Moreover, while it appears that there are sex-related differences in the response of salivary markers to exercise stress [16, 20] most of the research focuses on men or a mixed cohort [3, 10]; hence data pertaining to women in isolation, or compar- ing the male and female response, is scarce [3, 15]. Main trials In a randomised cross-over design, participants per- formed either an exercising or resting protocol; the alternative protocol was performed on their subsequent visit (3–7 days later). The exercising protocol involved 60 min of steady-state cycling at 70% peak power, whereas for the resting trial participants sat quietly for 60 min. Participants were asked to refrain from consuming caf- feine and alcohol and avoid exercise in the 24-h period prior to the trial. They were also asked to replicate the same food and beverage intake prior to each trial and report to the laboratory 3 h post-prandial. Four hours prior to their arrival to the laboratory, participants were reminded (via text message) to consume the 7 mL·kg−1 BM quantity of water provided by the researcher in the preliminary session. Upon arrival to the laboratory, a midstream urine sample was obtained for immediate de- termination of hydration status by urine specific gravity (USG) using a handheld refractometer (Sur-Ne, Atago Co Ltd., Japan); all participants’ USG levels were below 1.020 and therefore were well hydrated prior to exercise. Body mass was measured before and immediately after each trial period. Our aim was to conduct a thorough analysis of a wide range of salivary analytes in males and females both at rest and in response to exercise, in order to provide a valuable reference dataset for future studies. Methods Participants In total, 20 recreationally active participants completed the study (males n = 8; females n = 12: mean age 27.4 ± 5.9 years). Males (height 1.77 ± 0.04 m; weight 81.1 ± 6.5 kg) were significantly taller and heavier than females (height 1.66 ± 0.06 m; weight 62.8 ± 8.4 kg; P < 0.001). All procedures had prior approval by the local institutional ethics committee. Following comple- tion of a health screening questionnaire, written in- formed consent was obtained from all participants. In order to be considered for inclusion in this study Saliva was collected via the UWS drool method at two time points (15 min and 45 min) during each protocol. Both trials were conducted at the same time of day (15:00–18:00 h) to overcome any circadian influences. Heart rate (HR; T31 Polar heart rate monitor, Kempele, Finland) was measured continuously and ratings of perceived exertion (RPE) were monitored at 10-min intervals during exercise. Rutherfurd-Markwick et al. Journal of the International Society of Sports Nutrition (2017) 14:27 Page 3 of 8 Page 3 of 8 Page 3 of 8 Hydration parameters h h There was no change in UWS flow rate between rest and exercise for males (P = 0.248) or females (P = 0.801; Fig. 1a). However, males produced a higher flow rate than females during exercise (P = 0.007) and there was a trend for increase in flow rate at rest (P = 0.056). Within the exercise trial itself, females showed an increase in flow rate from 15 to 45 min (P = 0.031; Table 1); this was not observed in males (P = 0.730). However, females (P = 0.010) also produced a significant increase in flow rate within the rest trial (Table 1). Saliva osmolality was measured using a freezing point depression osmometer according to the manufacturer’s instructions (Osmomat 030, Gonotec, Berlin, Germany). Salivary electrolyte levels were measured using an Easy- Lyte analyser according to the manufacturer’s instruc- tions (Medica Corporation, Bedford, MA, USA). Salivary secretory IgA concentration was determined by ELISA as described elsewhere [22]. Salivary cortisol concentra- tion was determined by radioimmunoassay according to the manufacturer’s instructions (IBL International GMBH, Tecan, Hamburg, Germany, IBMG1206). Saliv- ary α-amylase activity was determined using the Infinity Amylase Liquid stable reagent (Thermoscientific, Wor- thing, UK) according to the manufacturer’s instructions. 1 There was a rise in saliva osmolality for females (P = 0.01) during exercise compared to rest, but no change for males (P = 0.838) and no difference between Fig. 1 Mean data for (a). Flow rate (g·min−1) and (b). osmolality (mOsmol·kg−1) and associated errors between rest and exercise for males and females. A significant difference between rest and exercise is indicated with an asterisk (**, P < 0.001; *, P < 0.05) and between males and females with letters (a, P < 0.001; b, P < 0.05) g g The secretion rates of SIgA (μg·min−1) and α-amylase (U·min−1) were calculated by multiplying the saliva flow rate (mL·min−1) by the IgA concentration (mg·L−1) and α-amylase activity (U·mL−1), respectively. Statistical analysis Independent t-tests were used to compare data between males and females. Paired t-tests were used to compare 15 min vs. 45 min at rest and 15 min vs. 45 min during exercise. Paired t-tests were used to compare rest vs. exercise data for males and females, separately (mean of rest vs mean of exercise). Pearson’s correlation was used to examine the relationships between independent variables. The results are presented as mean values ± standard deviation. Statistical significance was accepted at P < 0.05. Saliva collection and analysis (P < 0.001) but there were no sex differences for HR at rest (P = 0.136). RPE increased with duration of exercise (P = 0.006) and was higher at 60 min compared to 10 min (P < 0.05). Females reported higher average RPE than males during exercise (P < 0.001). There was no difference in body mass loss between males and females during exercise although females lost more fluid than males in both absolute mass (P = 0.014) and as a percentage of body mass (P = 0.001). (P < 0.001) but there were no sex differences for HR at rest (P = 0.136). RPE increased with duration of exercise (P = 0.006) and was higher at 60 min compared to 10 min (P < 0.05). Females reported higher average RPE than males during exercise (P < 0.001). There was no difference in body mass loss between males and females during exercise although females lost more fluid than males in both absolute mass (P = 0.014) and as a percentage of body mass (P = 0.001). Saliva was collected into a disposable pre-weighed 60 mL plastic container. Participants were instructed to sit leaning forwards with their head tilted downwards and swallow before any sampling took place. During sampling participants were asked to perform minimal orofacial movement and to allow the saliva to dribble into the tube. At least 1 mL was collected over a 2-min period per participant. If insufficient sample was ob- tained after this time, a further minute of collection was performed. Saliva was weighed (Sartorius LE3235, Germany) and flow rate was calculated on the assump- tion that saliva density was 1 g·mL−1 [21]. Saliva speci- mens were then stored at −80 °C until analysis. Exercise trial Although there were no differences in USG between rest and exercise trials in males (P = 0.178) or females (P = 0.972), pre-exercise, females exhibited lower USG than males (P = 0.018). HR was higher during exercise than rest (P < 0.001), increased during exercise for both sexes (P < 0.001), but remained constant at rest. During exercise, females had a higher average HR than males Fig. 1 Mean data for (a). Flow rate (g·min−1) and (b). osmolality (mOsmol·kg−1) and associated errors between rest and exercise for males and females. A significant difference between rest and exercise is indicated with an asterisk (**, P < 0.001; *, P < 0.05) and between males and females with letters (a, P < 0.001; b, P < 0.05) Fig. 1 Mean data for (a). Flow rate (g·min−1) and (b). osmolality (mOsmol·kg−1) and associated errors between rest and exercise for males and females. A significant difference between rest and exercise is indicated with an asterisk (**, P < 0.001; *, P < 0.05) and between males and females with letters (a, P < 0.001; b, P < 0.05) Rutherfurd-Markwick et al. Exercise trial Journal of the International Society of Sports Nutrition (2017) 14:27 Page 4 of 8 Page 4 of 8 Table 1 Levels of salivary analytes at two time points during rest and exercise trials in males and females, degree of change between the time points by percentage (Δ) and the significance of this change (P) Rest Exercise T1 T2 Δ (%) P T1 T2 Δ (%) P Flow rate (g · min-1) Males 0.64 ± 0.47 0.72 ± 0.57 8.9 ± 26.6 0.176 0.81 ± 0.38 0.77 ± 0.52 -1.7 ± 39.5 0.730 Females 0.34 0.13 0.40 ± 0.12 21.3 ± 31.0 0.010 ↑ 0.32 ± 0.19 0.38 ± 0.24 16.7 ± 24.4 0.031 ↑ Osmolality (mOsmol · kg-1) Males 65.4 ± 15.8 65.9 ± 18.7 0.31 ± 9.1 0.850 62.1 ± 18.5 71.3 ± 34.8 11.9 ± 27.1 0.287 Females 57.3 ± 15.1 56.0 ± 11.8 -0.20 ± 15.1 0.650 66.3 ± 16.6 68.8 ± 11.2 6.1 ± 14.0 0.434 Na (mmol · L-1) Males 4.5 ± 2.2 5.1 ± 1.8 28.3 ± 48.9 0.451 5.1 ± 2.5 9.3 ± 12.0 37.7 ± 69.8 0.285 Females 6.4 ± 3.2 5.0 ± 1.3 -11.0 ± 31.0 0.136 5.3 ± 2.4 6.6 ± 1.8 35.9 ± 61.6 0.199 K (mmol · L-1) Males 19.7 ± 4.1 21.3 ± 6.2 10.6 ± 40.5 0.528 22.3 ± 4.6 22.8 ± 5.1 2.2 ± 11.0 0.626 Females 21.9 ± 6.0 19.5 ± 4.8 -9.3 ± 15.1 0.060 ↓ 23.5 ± 5.5 24.3 ± 5.5 3.9 ± 7.5 0.150 Cl (mmol · L-1) Males 32.6 ± 13.2 35.9 ± 20.4 13.8 ± 64.7 0.634 32.2 ± 13.5 36.6 ± 22.6 10.6 ± 26.2 0.343 Females 49.6 ± 27.2 43.1 ± 23.3 -9.7 ± 26.7 0.193 37.6 ± 19.1 38.7 ± 16.6 5.9 ± 14.3 0.543 SIgA concentration (mg · L-1) Males 65.7 ± 42.2 66.8 ± 52.0 -3.5 ± 31.1 0.895 56.1 ± 41.2 61.2 ± 49.2 12.0 ± 53.4 0.526 Females 86.4 ± 49.0 60.2 ± 35.8 8.3 ± 130.1 0.120 95.2 ± 103.9 112.9 ± 91.3 46.1 ± 63.4 0.083 ↑ SIgA secretion rate (μg · min-1) Males 33.6 ± 17.5 39.1 ± 37.2 12.8 ± 59.9 0.559 39.6 ± 35.1 31.1 ± 12.9 7.5 ± 62.4 0.486 Females 25.2 ± 17.1 24.1 ± 18.8 34.6 ± 137.1 0.275 29.7 ± 27.8 45.5 ± 34.4 78.8 ± 110.3 0.032 ↑ α-amylase activity (U∙mL-1) Males 34.6 ± 20.4 38.2 ± 22.1 12.3 ± 12.2 0.050 ↑ 34.8 ± 19.5 44.0 ± 24.6 26.5 ± 19.0 0.034 ↑ Females 25.7 ± 23.5 29.5 ± 24.9 24.9 ± 27.4 0.030 ↑ 41.7 ± 21.9 56.3 ± 22.3 35.2 ± 32.9 <0.001 ↑ α-amylase secretion rate (U · min-1) Males 20.8 ± 18.9 27.6 ± 26.1 26.6 ± 34.4 0.559 28.1 ± 22.2 30.5 ± 22.6 22.9 ± 53.5 0.672 Females 7.3 ± 5.9 11.3 ± 9.6 68.4 ± 51.5 0.275 13.1 ± 9.5 20.6 ± 12.1 68.4 ± 51.5 0.004 ↑ Cortisol (nmol · L-1) Males 4.21 ± 0.73 4.02 ± 0.48 -3.7 ± 8.2 0.210 4.63 ± 0.71 4.55 ± 0.60 -1.0 ± 8.5 0.617 Females 5.95 ± 2.26 5.26 ± 2.16 -11.4 ± 8.3 <0.001 ↓ 6.00 ± 1.88 5.96 ± 2.08 -0.3 ± 10.8 0.894 UWS was sampled at 15 min (T1) and 45 min (T2) during each protocol. Exercise trial Arrows indicate whether there was an increase (↑) or decrease (↓) in the levels of the corresponding analyte over time Table 1 Levels of salivary analytes at two time points during rest and exercise trials in males and females, degree of change between the time points by percentage (Δ) and the significance of this change (P) sexes at rest (P = 0.191) or during exercise (P = 0.926; Fig. 1b). There was no difference in saliva osmolality over time within the rest or exercise trials for either sex (P > 0.05; Table 1). Concentrations of salivary K+ showed a trend for an in- crease between rest and exercise in females (P = 0.053); however, there was no change in males (P = 0.107; Fig. 2b). While there was no change in salivary K+ levels within the rest or exercise trials for either sex (P > 0.05), there was a trend for a decrease in salivary K+ during rest in females (P = 0.060; Table 1). Salivary Cl−levels showed a trend for a decrease in females between rest and exercise (P = 0.067); however, there was no change for males (P = 0.971; Fig. 2c). There was no change in salivary Cl−levels during the rest or exercise trials for ei- ther sex (P > 0.05; Table 1). Electrolytes mucosal immunity, SIgA (mg·L−1); (b) the sympathetic stress response, α-amylase activity (U·mL−1) and c. the adrenal stress response, cortisol (μg·L−1), and associated errors between rest and exercise for males and females. A significant difference between rest and exercise is indicated with an asterisk (**, P < 0.001; *, P < 0.05) and between males and females with letters (b, P < 0.001; b, P < 0.05) Fig. 2 Mean electrolyte data and associated errors between rest and exercise for males and females. a. Na (mmol·L−1); b. K (mmol·L−1) and c. Cl (mmol·L−1) Fig. 2 Mean electrolyte data and associated errors between rest and exercise for males and females. a. Na (mmol·L−1); b. K (mmol·L−1) and c. Cl (mmol·L−1) Electrolytes There was no change in salivary Na+ levels between rest and exercise in males or females; nor was there any change in Na+ levels between males and females in the rest or exercise trials (P > 0.05; Fig. 2a). There was no change in saliva Na+ levels during the rest or exercise trials for males or females (P > 0.05; Table 1). Rutherfurd-Markwick et al. Journal of the International Society of Sports Nutrition (2017) 14:27 Page 5 of 8 Mucosal immune and stress markers Fig. 2 Mean electrolyte data and associated errors between rest and exercise for males and females. a. Na (mmol·L−1); b. K (mmol·L−1) and c. Cl (mmol·L−1) Fig. 3 Mean data for salivary markers of (a). mucosal immunity, SIgA (mg·L−1); (b) the sympathetic stress response, α-amylase activity (U·mL−1) and c. the adrenal stress response, cortisol (μg·L−1), and associated errors between rest and exercise for males and females. A significant difference between rest and exercise is indicated with an asterisk (**, P < 0.001; *, P < 0.05) and between males and females with letters (b, P < 0.001; b, P < 0.05) Fig. 2 Mean electrolyte data and associated errors between rest and exercise for males and females. a. Na (mmol·L−1); b. K (mmol·L−1) and c. Cl (mmol·L−1) Fig. 3 Mean data for salivary markers of (a). mucosal immunity, SIgA (mg·L−1); (b) the sympathetic stress response, α-amylase activity (U·mL−1) and c. the adrenal stress response, cortisol (μg·L−1), and associated errors between rest and exercise for males and females. A significant difference between rest and exercise is indicated with an asterisk (**, P < 0.001; *, P < 0.05) and between males and females with letters (b, P < 0.001; b, P < 0.05) Fig. 2 Mean electrolyte data and associated errors between rest and exercise for males and females. a. Na (mmol·L−1); b. K (mmol·L−1) and c. Cl (mmol·L−1) Fig. 3 Mean data for salivary markers of (a). mucosal immunity, SIgA (mg·L−1); (b) the sympathetic stress response, α-amylase activity (U·mL−1) and c. the adrenal stress response, cortisol (μg·L−1), and associated errors between rest and exercise for males and females. A significant difference between rest and exercise is indicated with an asterisk (**, P < 0.001; *, P < 0.05) and between males and females with letters (b, P < 0.001; b, P < 0.05) Fig. 3 Mean data for salivary markers of (a). Mucosal immune and stress markers There was no change in SIgA concentration in response to exercise in males or females (P > 0.05); nor was there a change between the sexes at rest (P = 0.728) or exercise (P = 0.235; Fig. 3a). There was no change in SIgA concen- tration during the rest trial for either sex (P > 0.05; Table 1). There was a trend for an increase in SIgA concentra- tion during exercise in females (P = 0.083). Alpha-amylase activity increased in the exercise trial compared to rest for females (P = 0.001; Fig. 3b) but not males (P = 0.501). There was no change in salivary α-amylase activity be- tween males and females at rest (P = 0.429) or exercise (P = 0.345). However, there were significant increases in salivary α-amylase activity during both the rest and exer- cise trials for both sexes (P < 0.05; Table 1). increased from 15 min to 45 min during exercise in fe- males (P < 0.05 for both) with α-amylase secretion rates increasing in the exercise trial compared to rest (P = 0.023). There were no differences between sexes at rest (P > 0.05) or during exercise (P > 0.05) for SIgA or α-amylase secretion rate. There was a trend for higher salivary cortisol levels in females compared to males at both rest (P = 0.099) and during exercise (P = 0.070). There was no difference in salivary cortisol levels between rest and exercise for either sex (P > 0.05). Within the rest trial, a significant decrease in salivary cortisol was observed for females (P = 0.003; Table 1) but not males (P = 0.206). Salivary cortisol levels remained unchanged during the exercise trial. When expressed as a secretion rate (which takes flow rate into account) SIgA and α-amylase secretion rates Rutherfurd-Markwick et al. Journal of the International Society of Sports Nutrition (2017) 14:27 Page 6 of 8 Fig. 4 Correlations between α-amylase activity and cortisol, representing the sympathetic and adrenal stress responses, respectively, for (a). males; (b). females for both rest (filled markers) and exercise (empty markers). c shows a direct comparison of the exercise response at 15 min between males (squares) and females (circles) Discussion The aim of this study was to examine salivary analytes in males and females both at rest and in response to exer- cise. The main finding was that males and females show a markedly different response to steady-state exercise stress as measured in unstimulated whole saliva. This study provides separate novel datasets of salivary re- sponses to exercise stress in males and females that can be used as a reference point for future research. A recognised limitation of the gold standard drool collection method [23] for UWS is the low flow rate rela- tive to stimulated methods, and females have a lower UWS flow rate than males due to smaller salivary glands [24]. Our data showed females had lower flow rates during both exercise (P = 0.007) and rest (P = 0.056) compared to males. This difference in salivary flow rate may become limiting for data analysis, as flow rate has been suggested to influence the concentrations of some salivary analytes [1, 10, 24]. Flow rate has been suggested to be affected by exercise [10], however no difference in salivary flow rate was observed between rest and exercise for either males or females in this study. Exercise increased salivary osmolality in females (Fig 1b); in conjunction with an increase in both α- amylase activity (Fig 3b) and secretion rate (Table 1), this supports the consistently reported exercise-driven ac- tivation of the SAM axis [16, 25]. These results were not seen in males, indicative of sex-specific differences in the salivary response to exercise stress. The reason for the in- crease in α-amylase activity, which was observed during exercise and rest for both sexes, is unclear but it is possible that in the absence of exercise stimulation, the antimicrobial and/or digestive roles of α-amylase may affect exercise-independent measurements. In addition, anticipation of sampling, causing premature activation of the autonomic stress response, may also affect α-amylase activity levels [6]. Fig. 4 Correlations between α-amylase activity and cortisol, representing the sympathetic and adrenal stress responses, respectively, for (a). males; (b). females for both rest (filled markers) and exercise (empty markers). c shows a direct comparison of the exercise response at 15 min between males (squares) and females (circles) Fig. 4 Correlations between α-amylase activity and cortisol, representing the sympathetic and adrenal stress responses, respectively, for (a). males; (b). females for both rest (filled markers) and exercise (empty markers). Discussion c shows a direct comparison of the exercise response at 15 min between males (squares) and females (circles) Unstimulated resting salivary electrolyte levels are typically in the range of 3–29 mmol·L−1 (Na+), 6.4– 36.6 mmol·L−1 (K+) and 0–27 mmol·L−1 (Cl−) [2]. The broad ranges reflect the various factors which affect salivary electrolyte levels such as hydration status and salivary flow rate; the Na+ and K+ values from this study fall within normal ranges while the Cl−levels are higher than the quoted ranges. Although salivary K+ levels in- creased in females in response to exercise, there were no accompanying increases in Na+ or Cl−. In fact, there was a trend for a decrease in Cl−in females. While SAM ac- tivation may induce electrolyte release, research indi- cates substantial variation in the effects of exercise on salivary electrolyte levels which are impacted by exercise intensity and saliva collection methods [10]. reported a decrease in SIgA in response to exercise [10]. However, SIgA may be affected by training status and the type of training carried out [10]; although we recruited recreationally active participants the inter- individual variation in SIgA measurements was large and therefore this result must be interpreted with caution. Taken together our results suggest a notable difference in the physiological response to exercise stress between males and females, particularly with respect to activation of the SAM and HPA axes, represented by α-amylase and cortisol, respectively. While the coordination of the SAM and HPA pathways indicates a functional stress response, the activation of one without the other may represent a dysfunctional response due to the physio- logical consequences of chronic exposure to fluctuating or heightened neuroendocrine responses resulting from In this study females showed an increase in SIgA during the exercise trial, whereas other studies have Rutherfurd-Markwick et al. Journal of the International Society of Sports Nutrition (2017) 14:27 Page 7 of 8 Page 7 of 8 Page 7 of 8 however, in males the cortisol-SIgA relationship was positive (r = 0.255; P = 0.592) while in females it was negative (r = −0.177; P = 0.440). Such weak non- significant associations limit the conclusions that can be drawn, but do lend some support to both the SAM ac- tivity and HPA axis potentially regulating SIgA response. repeated or chronic stress (‘allostatic load’) [26]. Abbreviations Cl Chl id HPA Abbreviations Cl: Chloride; HPA: Hypothalamic-pituitary-adrenal; HR: Heart rate; K +: Potassium; Na+: Sodium; RPE: Ratings of perceived exertion; SAM: Sympathetic-adreno-medullary; SIgA: Secretory immunoglobulin A; URTI: Upper respiratory tract infection; USG: Urine specific gravity; UWS: Unstimulated whole saliva The stress pathways also modulate immune responses [6] with increases in the circulating stress hormones cat- echolamines and cortisol mediating SIgA responses to exercise. Whilst there is scarce human research it has been suggested that cortisol inhibits transepithelial transport of SIgA, while adrenaline appears to enhance IgA transcytosis [29, 30]. In the current study α-amylase was positively associated with SIgA in both males (r = 0.457; P = 0.255) and females (r = 0.214; P = 0.505), Discussion Interest- ingly, the relationship between α-amylase activity and cortisol levels produced during exercise gave opposing trends for males and females (Fig. 4). While both sexes showed a weak but negative α-amylase versus cortisol relationship at rest, during exercise this relationship be- came strongly positive for males (r = 0.799; P = 0.017; Fig. 4a) yet remained negative for females (r = −0.475; P = 0.036; Fig. 4b). Considering the dysfunctional stress response to allostatic load it is unlikely that all females exhibit dysfunctional stress systems. One explanation is that the difference between males and females is compli- cated by the delayed response of cortisol to stress, in comparison to that of α-amylase [14]. To address this, we performed correlational analysis at 15 min and 45 min as well as a lagged comparison with α-amylase at 15 min and cortisol at 45 min. The strongest correlation for both males (r = 0.890; P = 0.003) and females (r = −0.652; P = 0.021) was observed at 15 min (Fig. 4c), indicating a result consistent with the overall exercise stress relationship. Males showed a coordinated increase in both cortisol and α-amylase, whereas females exhib- ited a negative relationship, indicating that the stress axes function independently. This study has some limitations. It is apparent from this study that there are sex-specific responses to exercise, likely due to the steroid hormones and thus, to fully understand these differences, the measurement of these hormone levels is necessary. In addition, although the drool method is considered to be the gold standard approach for diagnosis it has inherent issues, particularly a low flow rate that differs between males and females. Although menstrual cycle phase has been shown not to influence neuroendocrine responses [27, 28] future studies may wish to control for menstruation when using female participants. Our data show several differences between males and females but we also report a few non- significant trends, which may indicate that a larger sample size may have been required, and so universal conclusions cannot be made. Conclusions h While exercise is considered to stimulate both HPA and SAM stress response systems, the response of each is dependent on a number of factors including exercise intensity, duration, training status and the sex hormones [6]. The spread of the female α-amylase versus cortisol plot in Fig. 4c indicates three different scenarios: points in the middle of the plot indicate the two systems work- ing together at a moderate level; points in the lower right region show sympathetic activity but no HPA re- sponse and points in the top left quartile represent HPA activity without SAM activation. Since exercise intensity and duration was consistent between the sexes, and training status is unlikely to be sex-dependent, the most likely explanation for the contrasting neuroendocrine re- sponses observed in females involves menstrual status or menstrual cycle phase, although the latter has been repeatedly shown not to influence these pathways [27, 28]. We did not collect hormonal data, and so a detailed study into the effects of the female hor- mones on exercise-associated stress pathway coordin- ation is warranted. Monitoring hydration status, exploring immune re- sponses to exercise and examining exercise stress are important considerations for sports and exercise nutri- tion scientists and practitioners. Saliva sampling is be- coming increasingly important with regards non-invasive monitoring of athletes as well as non-athletes. Our data provides an overview of the electrolyte, immune and stress response to steady-state submaximal exercise in both males and females using the current gold standard drool method to collect unstimulated whole saliva. This data has revealed some important differences in the re- sponse of males and females to steady-state exercise stress, particularly, opposing associations between the two major neuroendocrine stress axes. While we are un- able to make specific conclusions about the mechanisms involved, future studies directly comparing exercise stress in males and females is warranted. Acknowledgements The authors would like to thank Olivier Morin and John Taulu for help with data collection, Simon Bennett for technical support and the participants for providing their time and effort to complete this research. Competing interests Competing interests 20. Li C-Y, Hsu G-S, Suzuki K, Ko M-H, Fang S-H. Salivary Immuno factors, Cortisol and testosterone responses in athletes of a competitive 5,000 m race. Chinese J Physiol. 2015;58(4):263–9. 20. Li C-Y, Hsu G-S, Suzuki K, Ko M-H, Fang S-H. Salivary Immuno factors, Cortisol and testosterone responses in athletes of a competitive 5,000 m race. Chinese J Physiol. 2015;58(4):263–9. References A review of sex differences in immune function after aerobic exercise. Exerc Immunol Rev. 2011;17:104–21. 7. Gillum T, Kuennen M, Schneider S, Moseley P. A review of sex differences in immune function after aerobic exercise. Exerc Immunol Rev. 2011;17:104–21 8. Mastorakos G, Pavlatou M, Diamanti-Kandarakis E, Chrousos GP. 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Kaufman E, Lamster IB. The diagnostic applications of saliva- a review. Crit Rev Oral Biol Med. 2002;13(2):197–212. 26. Ali N, Pruessner JC. The salivary alpha amylase over cortisol ratio as a marker to assess dysregulations of the stress systems. Physiol Behav. 2012;106(1): 65–72. 2. Nunes LAS, Mussavira S, Bindhu OS. Clinical and diagnostic utility of saliva as a non-invasive diagnostic fluid:a systematic review. Biochem Med (Zagreb). 2015;25(2):177–92. 2. Nunes LAS, Mussavira S, Bindhu OS. Clinical and diagnostic utility of saliva as a non-invasive diagnostic fluid:a systematic review. Biochem Med (Zagreb). 2015;25(2):177–92. 27. Boisseau N, Enea C, Diaz V, Dugué B, Corcuff JB, Duclos M. Oral contraception but not menstrual cycle phase is associated with increased free cortisol levels and low hypothalamo-pituitary-adrenal axis reactivity. J Endocrinol Investig. 2013;36(11):955–64. 3. Papacosta E, Nassis GP. 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Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. Availability of data and materials 14. de Vries WR, Bernards NT, de Rooij MH, Koppeschaar HP. Dynamic exercise discloses different time-related responses in stress hormones. Psychosom Med. 2000;62(6):866–72. Submit your next manuscript to BioMed Central and we will help you at every step: 9. Tiidus P. Estrogen and gender effects on muscle damage, inflammation, and oxidative stress. Can J Appl Physiol. 2000;25(4):274–87. • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit p y y p 10. Chicharro J, Lucía A, Pérez M, Vaquero A, Ureña R. 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Pupil Dilations Reflect Why Rembrandt Biased Female Portraits Leftward and Males Rightward
Frontiers in human neuroscience
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INTRODUCTION women’s portraits, but only 56% of male portraits have a leftward bias (McManus and Humphrey, 1973). Portraitures have been shown to exhibit a leftward bias, where the left check is exposed more often than the right. This occurs more often in female than male portraits which may be due to a desire to portray female’s more emotive left-side. Hemispheric lateral- ization may also play a role by projecting negative emotions to the left-side of the face and positive emotions to the right-side of the face. This distinction will be addressed. Given that conscious judgments of pleasingness are beset with problems, it is impor- tant to also use an unconscious measure such as pupil size. Hess (1965, 1972) claimed that pupils enlarge when viewing pleasant images and constrict when viewing unpleasant images. Yet since pupil size also covaries with luminance, a novel technique was employed to measure pupil size in response to portraits and their mirror image. I found that Hess was incorrect to focus on valence and should have emphasized arousal instead. This finding was evi- dent in male but not female portraits, which may be due to the dominance exhibited in male portraitures. p y This gender difference has had several explanations (Lindell, 2013). One promising interpretation suggests that the leftward bias results from the poser’s preference to portray the left-side of the face’s emotional qualities (Nicholls et al., 1999). Nicholls et al., 1999, p. 665) instructed participants to pose for a portrait to either “put as much real emotion and passion into a portrait as you can” or “to avoid depicting any emotion at all.” In the first case, participants were more likely to turn their left-cheek toward a camera during a picture-taking session, whereas in the second case participants were more likely to turn their right-cheek toward the camera. Likewise, multiple studies report that the left-side of the face is more intense in exhibiting voluntary emotional expres- sion, especially for women (Sackeim and Gur, 1978; Sackeim et al., 1978; Borod and Caron, 1980; Borod et al., 1988; Nicholls et al., 2000). For example,Nicholls et al. (2002a) demonstrated that peo- ple who are more emotionally expressive are more likely to pose for a portrait offering the left-cheek; the paper argues that as females score higher on measures of emotional expressivity, they are more likely to pose offering the left-cheek. Nicholls et al. HUMAN NEUROSCIENCE HUMAN NEUROSCIENCE REVIEW ARTICLE published: 13 January 2014 doi: 10.3389/fnhum.2013.00938 REVIEW ARTICLE REVIEW ARTICLE published: 13 January 2014 doi: 10.3389/fnhum.2013.00938 REVIEW ARTICLE published: 13 January 2014 doi: 10.3389/fnhum.2013.00938 INTRODUCTION (2002b) then demonstrated that viewers perceive images of models offering the left-cheek as more emotionally expressive. Lindell (2013) offers a review of this literature. These findings are consistent with the idea that facial expressions are related to cerebral hemispheric lateral- ity, and that the right brain hemisphere is dominant in processing and also displaying emotional expressions (to the left-side of the face) (Bryden and Ley, 1983). Edited by: Edited by: Sarah Rose Cavanagh, Assumption College, USA Reviewed by: Sarah Rose Cavanagh, Assumption College, USA Kristin Prehn, Charité Universitätsmedizin Berlin, Germany *Correspondence: James A. Schirillo, Department of Psychology, Wake Forest University, 428 Greene Hall, Winston-Salem, NC 27109, USA e-mail: schirija@wfu.edu *Correspondence: James A. Schirillo, Department of Psychology, Wake Forest University, 428 Greene Hall, Winston-Salem, NC 27109, USA e-mail: schirija@wfu.edu Keywords: hemispheric laterality, pupil size, face perception, emotion, esthetic judgments James A. Schirillo* Department of Psychology, Wake Forest University, Winston-Salem, NC, USA James A. Schirillo* James A. Schirillo* Department of Psychology, Wake Forest University, Winston-Salem, NC, USA Department of Psychology, Wake Forest University, Winston-Salem, NC, USA Portrait painters are experts at examining faces and since emotional content may be expressed differently on each side of the face, consider that Rembrandt biased his male portraits to show their right-cheek more often and female portraits to show their left-cheek more often. This raises questions regarding the emotional significance of such biased positions. I presented rightward and leftward facing male and female portraits. I mea- sured observers’ pupil size while asking observers to report how (dis)pleasing they found each image. This was a methodological improvement over the type of research initially done by Eckhard Hess who claimed that pupils dilate to pleasant images and constrict to unpleasant images. His work was confounded since his images’ luminances and contrasts across conditions were inconsistent potentially affecting pupil size. To overcome this limi- tation I presented rightward or leftward facing male and female portraits by Rembrandt to observers in either their original or mirror-reversed position. I found that in viewing male portraits pupil diameter was a function of arousal. That is, larger pupil diameter occurred for images rated both low and high in pleasantness. This was not the case with female portraits. I discuss these findings in regard to the perceived dominance of males and how emotional expressions may be driven by hemispheric laterality. Pupil dilations reflect why Rembrandt biased female portraits leftward and males rightward James A. Schirillo* Department of Psychology, Wake Forest University, Winston-Salem, NC, USA PORTRAITURE’S LEFTWARD BIAS So, pos- itive emotions should be more prevalent on the right-side of the face (since it is governed by the left cerebral hemisphere) and negative emotional expressions should be more prevalent on the left-side of the face (since it is governed by the right cerebral hemi- sphere) (Rossi and Rosadini, 1967; Gainotti, 1969, 1972; Ahern and Schwartz, 1979; Schwartz et al., 1979; Sackeim et al., 1982; Fridlund and Izard, 1983; Natale et al., 1983; Sackeim and Gur, 1983; Davidson, 1984; Silberman and Weingartner, 1986; Schiff and Lamon, 1989; Mandal et al., 1991; Borod et al., 1997; Jasari et al., 2000; for a literature review see Powell and Schirillo, 2009). PORTRAITURE’S LEFTWARD BIAS Portraits often expose more of one side of their face than the other side (McManus and Humphrey, 1973; Grüsser et al., 1988). For example, in a study of 1,474 Western European portraits created from the fourteenth to the twentieth century, 891 posers (~60%) exposed more of their left-cheek, whereas 583 (~40%) exposed more of their right-check (McManus and Humphrey, 1973). This left-cheek asymmetry is stronger for women portraits (Gordon, 1974; Grüsser et al., 1988; Conesa et al., 1995). That is, ~68% of January 2014 | Volume 7 | Article 938 | 1 Frontiers in Human Neuroscience www.frontiersin.org www.frontiersin.org Laterality, pupil size, and esthetics Schirillo Given these findings, males may not want to portray their emo- tive left-side as much as females (or by the behest of the artist). Likewise, artists may prefer to portray women as being more emo- tive than men, thereby exposing their left-cheek more often. These notions are supported by Grüsser et al. (1988), who examined fifteenth to twentieth century portraits and found a left-cheeked bias, which was always stronger for female than for male portraits. Thus, the current study examines the bias a portrait conveys, not the viewer’s preference for a right or left-side of an image. This tests the sitter’s hemispheric asymmetry (contralateral control of facial musculature) rather than the perceiver will produce differences in valence or arousal. This makes it peculiar to portray the left-cheek more often. Log- ically it may follow that the valence hypothesis is less compelling than the lateralized one. Thus, the field of lateralized portraiture has sought to deter- mine whether one side of the face is more pleasant than the other. Schirillo and Fox (2006) (Figure 1) showed observers all 373 of Rembrandt’s portraits and found that left-cheeked females were assessed as more approachable than right-cheeked females portraits while males portraits (for both sides of the face) were assessed as preferably avoided. Thus, observers were more likely overall to want to approach female rather than male Rembrandt portraits. Unfortunately, this study did not use mirror-reversed images so it could not determine overall effect of cheek. Alternatively,a valence hypothesis suggests that since each cere- bral hemisphere controls predominantly the musculature on the lower two-thirds of the contralateral side of the face (Brodal,1965) each side of the face portrays different emotive qualities. CONSCIOUS EMOTIONAL JUDGMENTS MEASURED USING AN UNCONSCIOUS PUPIL SIZE MEASURE Research investigating the interaction of lateralized portraiture and pleasantness ratings is beset with problems (Rinn, 1984). For example, the most popular methodology is to obtain an observer’s subjective impression of the stimuli such as degree of liking (Rus- sell and George, 1990). This method may cause observers to use separate, immeasurable, criteria in making judgments (e.g., one observer may use an image’s contrast, whereas another may use facial features, such as eyebrows). This obfuscates linking esthetic judgments to cerebral laterality. Thus, it makes sense to 1 3 5 -90 0 d io v A h c a o r p p A Orientation (Deg.) Left-Cheeked Females 1 3 5 -90 0 d io v A h c a o r p p A Orientation (Deg.) Left-Cheeked Males 1 3 5 0 90 Approach Avoid Orientation (Deg.) Right-Cheeked Females 1 3 5 0 90 Approach Avoid Orientation (Deg.) Right-Cheeked Males FIGURE 1 | Scatterplots of the distribution of each of 373 Rembrandt portrait angles as a function of ratings of approach/avoidance ratings (collapsed over 73 subjects’ ratings). Upper left (closed squares) – shows left-cheeked males (−90° to −1° orientation), upper right (open squares) – shows right-cheeked males (+1° to +90° orientation), lower left (closed diamonds) – shows left-cheeked females (−90° to −1° orientation), lower right (open diamonds) – shows right-cheeked females (+1° to +90° orientation). Linear regressions are plotted for each graph. Here 2 indicates a rating of “mildly approach,” 3 indicates “neutral,” and 4 indicates “mildly avoid.” Frontiers in Human Neuroscience www.frontiersin.org January 2014 | Volume 7 | Article 938 | 2 1 3 5 0 90 Approach Avoid Orientation (Deg.) Right-Cheeked Females 1 3 5 -90 0 d io v A h c a o r p p A Orientation (Deg.) Left-Cheeked Females Left-Cheeked Females Right-Cheeked Females Approach Avoi Approach 1 3 5 0 90 Approach Avoid Orientation (Deg.) Right-Cheeked Males 1 3 5 -90 0 d io v A h c a o r p p A Orientation (Deg.) Left-Cheeked Males Left-Cheeked Males Right-Cheeked Males Approach FIGURE 1 | Scatterplots of the distribution of each of 373 Rembrandt portrait angles as a function of ratings of approach/avoidance ratings (collapsed over 73 subjects’ ratings). CONSCIOUS EMOTIONAL JUDGMENTS MEASURED USING AN UNCONSCIOUS PUPIL SIZE MEASURE Upper left (closed squares) – shows left-cheeked males (−90° to −1° orientation), upper right (open squares) – shows right-cheeked males (+1° to +90° orientation), lower left (closed diamonds) – shows left-cheeked females (−90° to −1° orientation), lower right (open diamonds) – shows right-cheeked females (+1° to +90° orientation). Linear regressions are plotted for each graph. Here 2 indicates a rating of “mildly approach,” 3 indicates “neutral,” and 4 indicates “mildly avoid.” Frontiers in Human Neuroscience January 2014 | Volume 7 | Article 938 | 2 www.frontiersin.org www.frontiersin.org Laterality, pupil size, and esthetics Schirillo try to understand portrait laterality to what may be a correlate to automatic affective reactions. study of “social appealingness” of male and female right and left- cheeked portraits differs from Schirillo and Fox’s (2006) study of approach/avoidance of the same? Seemingly, “social appeal” dif- fers from the desire to approach or avoid. Further work on this discrepancy is needed. Pupil size is one previously measured unconscious indicator of affective processing (Hess and Polt,1960; Hess,1965,1972; Janisse, 1974; Loewenfeld, 1999). Hess (1965, 1972), attempted to trans- form the field of esthetics by claiming that pupils enlarge when viewing pleasant images and constrict when viewing unpleasant images (see also Hess and Polt,1960;Hess et al.,1965;Simms,1967; Fitzgerald, 1968; Goldwater, 1972). However, pupil size also varies with luminance (Loewenfeld, 1966, 1999; Woodmansee, 1966; Miller, 1967; Kohn and Clynes, 1969; Goldwater, 1972; Janisse, 1973, 1974; Loftus, 1985; Mannan et al., 1995; Locher, 1996), and since Hess compared different images with different intensities and contrast levels (e.g., a snake versus a naked women), his work was confounded. Given that verbal ratings of pleasingness are a self-report mea- sure,it is of interest to determine their relationship with an uncon- scious indicator of pleasingness (i.e., pupil diameter) as suggested by Hess and others, and how this in turn might be related to the emotional content of facial musculature. If self-report interpreta- tions drive assumptions regarding dominance, they may show up in the portraits’ emotional qualities which are reflected in pupil size relationships. However, it may be that pupil diameter is more related to arousal rather than pleasingness. The current study will help clarify this dependent variable. In an earlier study of Rembrandt portraits (Schirillo, 2000) a factor analysis revealed that females with their left-cheek exposed were judged to be much less socially appealing than less commonly painted right-cheeked females. CONSCIOUS EMOTIONAL JUDGMENTS MEASURED USING AN UNCONSCIOUS PUPIL SIZE MEASURE Conversely, the more commonly painted right-cheeked males were judged to be more socially appealing than either left-cheeked males or females facing either direction. It was hypothesized that hemispheric asymmetries reg- ulating emotional facial displays of approach and avoidance influ- enced the side of the face Rembrandt’s models exposed due to prevailing social norms. Thus, females would be considered more appealing than males and left facing males would be considered the least appealing. A second experiment had different subjects judge a different collection of 40 portraits by Rembrandt and their mirror images. Portraits were matched for valence, arousal and dominance by a second set of 20 subjects. I hypothesized that mirror-reversed images would produce the same pattern of results as their original orientation counterparts. I also hypothesized that hemispheric asymmetries that specify the emotional expression on each side of the face will account for the obtained results, that is, original left-cheeked males will be preferred,due to their perceived dominance while there will be no difference in female portraits. A NOVEL METHODOLOGY Since I used original and mirror-reversed portraits, I was also able to explore the hemispheric laterality of emotional expression. Rembrandt may have turned his subject’s faces sideways to display specific emotional content of their facial musculature. If this is the case, self-reports regarding dominance especially of male subjects should show up as emotional responses which might be quantified by pupil size relationships. It was found that Hess was incorrect to focus on valence. Instead, he should have focused on arousal, since it was found that arousal, not valence, drives pupil diameter for male portraits. That is pupils dilated for males that were rated both most pleasant and most unpleasant (Powell and Schirillo, 2009). Thus, verbal ratings of pleasantness are a self-report mea- sure which relate to an unconscious indicator of pleasantness (i.e., pupil diameter) as suggested by Hess and others (Hess and Polt, 1960; Hess, 1965, 1972; Hess et al., 1965). DOMINANCE, VALENCE, AND AROUSAL Dominance (typically associated with positive, e.g., self-assurance, arrogance, and feeling bold or triumphant) or negative affective states (e.g., hostility, irritability, and anger) (Demaree et al., 2005) can lead to larger pupil size (Darwin, 1872). These arousal dif- ferences should only be present in viewing male portraits since they accentuate dominance (Dunbar and Burgoon, 2005). This is emphasized in images where each side of the face reflects different emotional expressions a concept first posited by Darwin (1872), especially as it relates to dominance. Frontiers in Human Neuroscience MATERIALS AND METHODS SUBJECTS Forty right-handed observers (20 males; ages 18–23) with nor- mal or corrected-to-normal vision (but no eyeglasses) from the introductory psychology research pool at Wake Forest Univer- sity participated in the study. Handedness was determined using Annett’s Peg-Moving task as right-minus-left latency (peg-moving speed) (Annett and Kilshaw, 1983). The study was performed in accordance with the ethical standards of the Declaration of Helsinki. The use of Rembrandt portraits allowed for the exploration of esthetic judgments of a famous artist while investigations of Rembrandt’s work in the context of hemispheric laterality provide also examining potential differences in facial emotion expression. First, using artwork should elicit stronger esthetic reactions than photographs of faces. Second,prior preliminary evidence that per- ceived dominance is greater when viewing his right-cheeked male portraits (Schirillo, 2000). It is possible that this is the reason for their prevalence; however due to the correlational nature of the data it may also be that Rembrandt may have chosen to selec- tively portray the right-cheeks of more dominant males. Thus, I attempt to show how hemispheric asymmetries may regulate dis- plays of facial emotion which are reflected by an observer’s esthetic judgment of a portrait. It is interesting that Schirillo’s (2000) STIMULI Forty black and white images taken from oil paintings were cho- sen from a collection of 373 portraits painted by Rembrandt. The specific portraits chosen represent his most rightward and leftward facing portraits (Schirillo and Fox, 2006). Grayscale images were used instead of colored images because color can lead to changes in pupil diameter (Miller, 1967; Kohn and Clynes, 1969). Ten were right-cheeked males (none were self-portraits of Rembrandt), 10 were left-cheeked males, 10 were right-cheeked January 2014 | Volume 7 | Article 938 | 3 www.frontiersin.org Laterality, pupil size, and esthetics Schirillo Schirillo females, and 10 were left-cheeked females (Lists Painting Names in Appendix). Next, these images were used to produce 40 mirror- reversed images using PhotoShop IBM. The portraits were only of busts. Each portrait was scanned into PhotoShop and was pro- jected to each observer individually using an IBM CRT computer monitor using Microsoft PowerPoint. Viewing distance was 24′′ making the image size range from 11.7°(height) × 8.5°(width) to 11.8° × 12.4°. The observers close distance to the screen lim- ited their ability to spend considerable time viewing off-screen. To verify this notion, since the head-mounted Applied Science Laboratories (ASL; series 6000) eye-tracker could also measure eye position, I determined that observers were only off-screen ~3% of their total viewing time. My data also showed that time with no record (due to eye closure) was minimal. In addition, 80 blurred images were created (40 from original and 40 from mirror- reversed images) in PhotoShop using a Gaussian blur function (See Figure 2). There was no ambient lightning in the experimen- tal chamber, in that it was a room without windows. Since the door was closed, the only light available came directly from the computer screen that showed the images. females, and 10 were left-cheeked females (Lists Painting Names in Appendix). Next, these images were used to produce 40 mirror- reversed images using PhotoShop IBM. The portraits were only of busts. Each portrait was scanned into PhotoShop and was pro- jected to each observer individually using an IBM CRT computer monitor using Microsoft PowerPoint. Viewing distance was 24′′ making the image size range from 11.7°(height) × 8.5°(width) to 11.8° × 12.4°. The observers close distance to the screen lim- ited their ability to spend considerable time viewing off-screen. STIMULI FIGURE 2 | (A) Blurred original orientation, (B) original orientation of a left-cheeked female, (C) blurred mirror-reverse, and (D) mirror-reverse orientation (A woman in fanciful costume). Baltimore, The Walters Art Gallery; Br. 386). Copyright 1969 Phaidon Press Ltd. Rembrandt, The Complete Edition of Paintings. A. Bredius, revised by H. Gerson. The eye tracking device was used to determine the pupil size of the left-eye. The right-eye pupil size was not measured because pupil size is believed to be conjugate across the two eyes (Loewen- feld,1999). Pupil diameter was recorded automatically every 17 ms foreachentire15 strial.Theaveragesizeacrosstheentire15 sview- ing period was computed minus any time the pupil computation was off-line (due to blinks, etc.). Given that the ASL eye-tracker stops recording when the eye closes more than 50% (assumedly due to blinks or partial eye closure) I have no record of this data. FIGURE 2 | (A) Blurred original orientation, (B) original orientation of a left-cheeked female, (C) blurred mirror-reverse, and (D) mirror-reverse orientation (A woman in fanciful costume). Baltimore, The Walters Art Gallery; Br. 386). Copyright 1969 Phaidon Press Ltd. Rembrandt, The Complete Edition of Paintings. A. Bredius, revised by H. Gerson. Instead of using linear interpolation to estimate the pupil size during this off-line period (Steinhauer et al., 2004) I felt it best to simply eliminate these segments from my dataset since blinks can alter pupil size (Nakayama, 2006). Given that the ASL eye-tracker is fixed to the head, head-movements did not alter pupil diameter recordings or result in loss of tracking. Thus, other than eye-blink time I did not remove any artifacts from the data. The remaining data was imported into Excel to do the data cleaning which was then converted to SPSS to do the statistical analysis. Average pupil size was calculated for each image across the observation period which excluded instances where the observer blinked or had par- tial eye lid closures (since when the eye closes the ASL machine cannot record any pupil size). Observers used a chinrest to ensure a fixed 24′′ distance between themselves and the screen to retain a constant depth of field across the images (Simms, 1967). Figure 2 shows images in their original and mirror-reverse orien- tation (Figures 2B,D) along with their corresponding preceding blurred images (Figures 2A,C). These images are significantly blurred, so that facial pleasantness cannot be extracted from the blurred images (Bachman, 2007). Frontiers in Human Neuroscience STIMULI Observers viewed 40 images in their original posed orienta- tion, and in their mirror-reversed orientation (resulting in 80 images in total). Since I only compared pupil size across original versus mirror-reversed images, my within-subject’s design elimi- nates potential confounding factors such as age and medication. Right-cheeked mirror-reversed images are portraits that originally faced rightward but due to reversal appeared to be of original left-cheeked images. Likewise, left-cheek mirror-reversed images appeared to be of original right-cheek portraits. Images were ran- domized, and presented to each observer in random order. Each of the 80 images was viewed for 15 s and was preceded by a blurred version of the image for 15 s. Fifteen seconds was decided upon because of three previous findings. First, Smith and Smith (2001) found that art viewers examined The Metropolitan Museum of Art paintings for a median of 17 s. Second, Aboyoun and Dabbs (1998) showed that pupil size rapidly decreases upon image pre- sentation, which then recovers to either baseline or above baseline levels. Consequently, pupil size must be measured for at least sev- eral seconds to overcome this initial depression. Lastly,Richer et al. (1983) found that pupil size increases begin about 1.5 s before To circumvent Hess’ luminance and contrast confounds, I had observers view left- and right-cheeked portraits, and their mir- ror images (e.g., see Figures 2B,D), while they determined the esthetic pleasantness of each face. Simultaneously, I monitored their pupil size allowing for a correlate between portrait pleasant- ness and pupil size. Since original and mirror images have the same luminance profiles, and I only compared pupil size and pleasant- ness ratings across matched pairs of faces, Hess’ confounds were eliminated. Since Woodmansee (1966, p. 133) found “significant pupil- lary constriction with shifts in gaze from darker to brighter areas of the picture,” I, like him, presented a blurred image prior to its clear image to minimize changes in pupil size. For example, January 2014 | Volume 7 | Article 938 | 4 www.frontiersin.org www.frontiersin.org Laterality, pupil size, and esthetics Schirillo stimulus presentation and peak around a second after presen- tation. As a result, I gave observers more time than needed to generate an entire response to an image. Observers’ pupil size was measured during each non-blurred image presentation while they contemplated how pleasant they found the non-blurred images. PLEASANTNESS RATINGS Before examining individual pleasantness ratings, I obtained each observer’s average pleasantness ratings for each of the eight por- trait types (e.g., original right and left, mirror-reverse right and left,males and females). This resulted in each observer having eight data points. Then, pleasantness ratings for male and female por- traits were submitted to a 2 (Portrait Gender: male vs. female) × 2 (Orientation: original vs. mirror-reversed) × 2 (Side of Face: left vs. right) repeated measures ANOVA. Males and female observers were included as a between-subjects factor, but showed no effect. j , Figure 4 shows the means for each portrait group. There was a main effect for Side of Face with left-cheeks rated higher than right-cheeked individuals F(1, 39) = 11.55, p = 0.002, d = 1.07. Additionally, there were three significant interactions. First, there was a Side of Face by Orientation interaction F(1, 39) = 12.54, p = 0.001, d = 1.12. While left-side portraits (origi- nal and reversed) were rated higher than right portraits (see Side of Face main effect), right mirror reversals were rated higher than right originals (M = 4.23 vs. M = 4.13), while left originals were rated higher than left mirror reversals (M = 4.75 vs. M = 4.57, respectively) [t(1, 78) = 2.56, p = 0.01, d = 0.59; t(1, 78) = 2.91, p = 0.005,d = 0.65].Thatis,leftwardappearingportraits,leftorig- inals and right reversals whose appearance to the observer seems to be left faced, are rated higher than right faced originals and left mirror-reversed (portraits viewed as seemingly right faced). Second, a Side of Face by Portrait Gender interaction was found F(1, 39) = 15.12, p = 0.001, d = 1.23 with left-side females por- traits rated higher than right-side females portraits (M = 5.07 vs. M = 4.38), whereas the opposite is true for males portraits though to a lesser degree (M = 4.23 for left male portraits and M = 4.41 for right male portraits) [t(1, 78) = 3.22, p = 0.002, d = 0.72; t(1, 78) = 2.96, p = 0.004, d = 0.66]. Third, there was an Portrait Gen- der by Orientation F(1, 39) = 7.42 p = 0.097, d = 0.86, such that female mirror reversals were rated lower than original female por- traits (M = 4.38 vs. M = 4.58), whereas the opposite relationship was found for males (M = 4.35 vs. RESULTS The rationale for presenting a blurred image of a given por- trait prior to presenting that portrait was to avoid the following confound prevalent in the pupillometry literature. That is, if a constant blank gray screen was used as a baseline the subsequent test-image would produce the following effect (See Figure 2 taken from Bradley et al., 2008). That is, the most important natural function of the pupil is to dynamically respond to changes in envi- ronmental illumination with an initial constriction (i.e., the light reflex) that is related to stimulus luminosity (Beatty and Lucero- Wagoner, 2000). Thus, if a constant blank gray screen were used as a baseline the brighter images would produce a larger constriction than the dimmer images. This effect takes up to 6 s before reach- ing a plateau. To circumvent this effect I choose to first present for 15 s a blurred image of the subsequent test-portrait. This does two things. First, it makes the large constriction (i.e., light reflex) occur during the blurred image rather than during the test-image. By the end of the 15 s of viewing the blurred image the pupil has adjusted to the light level of the image that will subsequently be presented. Given this very long duration the pupil will no longer carry-over any information from the previous clear portrait. This is because the pupil is reflexive and does not contain a memory loop so by the end of presenting a blurred image there are no resid- ual effects from the proceeding clear image. However, the blurred images differ in luminance thus setting a different baseline for the subsequent portrait. This is actually desired,so that the magnitude of the effect is not the result of a shift in overall luminance level (as occurs in Figure 2). Instead, each original and mirror-reversed blurred image sets the same baseline for their subsequent clear image. This is important since it is only the results of these two (original and mirror-reversed test-image) pupil diameters that will be compared against each other. PLEASANTNESS RATINGS M = 4.29) [t(1, 78) = 2.72, p = 0.008, d = 0.61; t(1, 78) = 2.51, p = 0.015, d = 0.015]. I manually recorded pleasantness scores for each face by taking verbal esthetic judgments using a 1–9 numerical scale, with one meaning most displeasing, five meaning neutral, and nine mean- ing most pleasing. Pleasingness is just one dimension of esthetics, but seemed to be appropriate based on a study that used five evaluative scales (e.g., pleasingness, likeability, preferability, inter- estingness, and complexity) (Russell and George, 1990). In Russell and George’s (1990) study,pleasingness was highly correlated with likeability and preferability, and was the highest in inter-subject agreement. The difference in verbal rating between the original and mirror-reverse images was then correlated with the difference between the average pupil diameters. Frontiers in Human Neuroscience STIMULI Observers were instructed to think about the esthetic pleasingness of each image for the entire 15 s it was shown and then report their judgment after the image was removed. This occurred during the presentation of the subsequent blurry image. session progressed. To determine if observers noticed these mir- ror duplications, I asked whether they noticed anything unusual about the images at the end of the session. Sixteen of 40 observers reported noticing that a number of images were mirror-reversed. Only three observers had formal art training and nine reported that they had seen less than 25% of the portraits before. Thus, while many of these images are famous, most observers were not familiar with them. PUPIL SIZE This variation is termed the light reflex. (Bottom) correlation of the magnitude of the light reflex as a function of image luminance (Taken from Bradley et al., 2008). FIGURE 4 | Verbal ratings (striped bars) as a function of portrait type on a 1–9 scale, with 1 indicating most displeasing, 5 indicating neutral, and 9 indicating most pleasant. Pupil size in mm (solid bars) as a function of portrait type (R = Right-cheeked, L = Left-cheeked, M = Males, F = Females, Rev = Mirror-reversed images). Error bars = SEM. FIGURE 5 | Correlation between pupil diameter given the average luminance for each portrait (n = 40 observers). The figure includes the portraits with the highest and lowest average luminance. FIGURE 5 | Correlation between pupil diameter given the average luminance for each portrait (n = 40 observers). The figure includes the portraits with the highest and lowest average luminance. FIGURE 3 | (Top) Average pupil diameter over time as a function of luminance of the image viewed. This variation is termed the light reflex. (Bottom) correlation of the magnitude of the light reflex as a function of image luminance (Taken from Bradley et al., 2008). FIGURE 5 | Correlation between pupil diameter given the average luminance for each portrait (n = 40 observers). The figure includes the portraits with the highest and lowest average luminance. females portraits (M = 5.3) [t(1, 78) = 3.07, p = 0.003, d = 0.69]. This analysis also yielded a significant Side of Face × Orientation interaction F(1, 39) = 14.72, p = 0.001, d = 1.21. Original right- cheeked portraits (M = 5.42) elicited greater average pupil size than right-cheeked reversed portraits (M = 5.15) [t(1, 78) = 3.55, p = 0.001, d = 0.76]. Conversely, average pupil size was largest for left-cheeked reversed portraits (M = 5.44) than for origi- nal left-cheeked portraits (M = 5.23) [t(1, 78) = 3.13, p = 0.002, d = 0.72]. When these findings are examined alongside the Side of Face × Orientation interaction for verbal ratings, I find that images with the appearance of being right-cheeked yielded larger average pupil size and lower verbal ratings. image luminance reinforces my decision to use mirror-reversed images. PUPIL SIZE Before examining individual pupil size, I obtained each observer’s average pupil size for each of the eight types of portraits. Mean pupil sizes for these groups are shown in Figure 3 along- side the means of the previous three-way ANOVA conducted for verbal pleasingness. There was a main effect of portrait gender across the eight types of portraits F(1, 39) = 39.14, p < 0.0001, d = 1.98 (Figure 4). Average pupil diameter when viewing male portraits was larger (M = 5.5) than when viewing Following the stimulus presentation, observers were admin- istered a questionnaire that pertained to their art training and their familiarity with the portraits. They were not told prior to the experimental session that they would see original and mirror- reversed images, but they may have become aware of this as the January 2014 | Volume 7 | Article 938 | 5 Frontiers in Human Neuroscience www.frontiersin.org Laterality, pupil size, and esthetics Schirillo FIGURE 4 | Verbal ratings (striped bars) as a function of portrait type on a 1–9 scale, with 1 indicating most displeasing, 5 indicating neutral, and 9 indicating most pleasant. Pupil size in mm (solid bars) as a function of portrait type (R = Right-cheeked, L = Left-cheeked, M = Males, F = Females, Rev = Mirror-reversed images). Error bars = SEM. FIGURE 5 | Correlation between pupil diameter given the average luminance for each portrait (n = 40 observers). The figure includes the portraits with the highest and lowest average luminance. FIGURE 3 | (Top) Average pupil diameter over time as a function of luminance of the image viewed. This variation is termed the light reflex. (Bottom) correlation of the magnitude of the light reflex as a function of image luminance (Taken from Bradley et al., 2008). FIGURE 4 | Verbal ratings (striped bars) as a function of portrait type on a 1–9 scale, with 1 indicating most displeasing, 5 indicating neutral, and 9 indicating most pleasant. Pupil size in mm (solid bars) as a function of portrait type (R = Right-cheeked, L = Left-cheeked, M = Males, F = Females, Rev = Mirror-reversed images). Error bars = SEM. FIGURE 5 | Correlation between pupil diameter given the average luminance for each portrait (n = 40 observers). The figure includes the FIGURE 3 | (Top) Average pupil diameter over time as a function of luminance of the image viewed. Frontiers in Human Neuroscience LINEAR AND QUADRATIC RELATIONSHIPS BETWEEN PLEASANTNESS RATINGS AND PUPIL DIAMETER Yet, when the mirror-reversed por- trait was preferred (whether right- or left-cheeked) the pupil was smaller while viewing the mirror-reversed portrait compared to the original. Figure 6A shows that there was a significant quadratic effect for male portraits b = 0.40, t(797) = 3.22, p = 0.002, d = 0.22 when the linear relationship was held constant. Pupil diameter was largest when there were the greatest differences in verbal ratings between an original and mirror-reversed male image. That is, when images were extremely liked or disliked, pupil size increased. This quadratic relationship accounted for significantly moreof thevariancethanthelinearrelationship,R2 change = 0.32, p < 0.004, d = 0.22. However, for female portraits, the regression model failed to find a significant linear or quadratic relation- ship between pupil diameter and pleasantness F(2, 797) = 0.59, p = 0.32, d = 0.07 (Figure 6B). Pupil size could be confounded by the luminance of an image, so difference scores were calculated to compare across images. If difference scores were not used, and instead, pupil size for original and mirror-reversed images were used in the regression, then my findings would be seriously con- founded because these values do not account for the changes in luminance/contrast across images and comparisons across images would be misleading. In sum, there were 800 total cases [i.e., 40 observers × 40 portraits (original-minus mirror-reversed)] used in the analysis. Next, linear and quadratic functions were entered into a regres- sion model to evaluate if the relationship is better categorized by a quadratic function. The following quadratic regression model was computed separately for male and female portraits: Pupil = b0 + b1 pleasantness  + b2 pleasantness2 Pupil = b0 + b1 pleasantness  + b2 pleasantness2 FIGURE 6 | Pupil size difference score (original-minus mirror-reversed) as a function of the difference in 1–9 verbal rating scores (between an original-minus mirror-reversed image) for all (A) male portraits, (B) female portraits. It is important to realize that my images were not necessar- ily less pleasing in their non-original orientation. Instead, what I found was that only if there were a large difference in the rating between original and mirror-reversed images (where either orien- tation could have been the more pleasing image) there would also be a large difference in pupil size between those images. LINEAR AND QUADRATIC RELATIONSHIPS BETWEEN PLEASANTNESS RATINGS AND PUPIL DIAMETER I examined linear and quadratic relationships between pleasant- ness and pupil size. First, a regression was computed to examine whether there was a linear relationship between pleasantness and pupil diameter. This was done by taking each portrait (original or mirror-reversed) for each observer as an individual case. Then difference scores between verbal pleasingness were regressed using the predictor variables of pupil size difference and quadric pupil size difference. Figure 6A shows that, for males, as the original verbal ratings became more positive (represented on the x-axis Overall, pupil diameters were well within the normal range, where, as expected, the luminance of the portrait viewed dramatically affected pupil size (Figure 5; range = 4.71–5.82 mm). The fact that pupil contraction increased with an increase in January 2014 | Volume 7 | Article 938 | 6 www.frontiersin.org Schirillo Schirillo Laterality, pupil size, and esthetics by verbal rating differences that were greater than zero), the orig- inal pupil size got smaller. Likewise, as original ratings became more negative (represented on the x-axis by verbal rating dif- ferences that were less than zero), the original pupil size got larger. This negative slope was statistically significant for male portraits r(39) = −0.41, p < 0.009, d = −0.90 (Figure 6A) but I failed to find a relationship for female portraits r(39) = −0.22, p = 0.178, d = −0.90 (Figure 6B). This means that when an orig- inal male portrait was preferred (whether right- or left-cheeked) the pupil was smaller while viewing the original portrait compared to the mirror-reversed image. Yet, when the mirror-reversed por- trait was preferred (whether right- or left-cheeked) the pupil was smaller while viewing the mirror-reversed portrait compared to the original. by verbal rating differences that were greater than zero), the orig- inal pupil size got smaller. Likewise, as original ratings became more negative (represented on the x-axis by verbal rating dif- ferences that were less than zero), the original pupil size got larger. This negative slope was statistically significant for male portraits r(39) = −0.41, p < 0.009, d = −0.90 (Figure 6A) but I failed to find a relationship for female portraits r(39) = −0.22, p = 0.178, d = −0.90 (Figure 6B). This means that when an orig- inal male portrait was preferred (whether right- or left-cheeked) the pupil was smaller while viewing the original portrait compared to the mirror-reversed image. DISCUSSION They also found an increase in pupil diameter when observers expected to hear a gunshot. Like- wise, Polt (1970) found larger pupil size during mental arithmetic tasks when observers believed they would be shocked for incorrect answers. This may also account for why females failed to show an effect of arousal. I suggest that females are considered pleasing (not aversive) compared to males, thus when viewing female portraits one’s pupil size is already approaching the floor, and thereby has less room to show arousal effects. Given that my verbal ratings of pleasingness are a self-report measure of what may be an emotive expression, it was of interest to determine their relationship with an unconscious indicator of pleasingness (i.e., pupil diameter) as suggested by Hess and others, and how this in turn might be related to the emotional content of the facial musculature displayed in the images. For example, self-report assumptions regarding dominance can be present in the emotional qualities of the portraits, which may be captured by pupil size relationships. I have explored these variables while simultaneously eliminating Stern and Strock’s (1987) concern that one drawback to pupillometry is that changes associated with such variables are considerably smaller than those associated with illumination effects. The esthetic verbal pleasantness judgments suggest that the observers were attending to the actual facial physiognomy of the posers and that pleasantness was determined to a lesser degree by the orientation the portrait faced. In agreement with prior research (Schirillo and Fox, 2006), the left-side of women’s faces were rated as more esthetically pleasant than their right-side. This suggests that for women it is important to express more emo- tive facial qualities than males, in agreement with Nicholls et al. (1999) and the right-hemisphere model of emotion lateralization. Yet how emotion may be lateralized in the cerebral hemispheres is still under debate (Davidson, 1995; Demaree et al., 2005; Kill- gore and Yurgelun-Todd, 2007). One recent argument is that an approach/withdrawal model may provide a more appropriate fit to the data as opposed to the positive/negative hemispheric difference model (Demaree et al., 2005). Davidson and others interpretation of theleft/rightdifferencesinemotionvalenceisthattherightcere- bral hemisphere regulates withdrawal behaviors whereas the left cerebral hemisphere regulates approach behaviors (Kinsbourne, 1982;Davidson,1984,1992,1995;Davidsonetal.,1990;Fox,1991). If this is the case, as stated in Schirillo (2000), I speculate that Rembrandt preferred to paint females left-side because it captured the attractive quality of being demure. DISCUSSION Male portraits showed both a linear and quadratic relationship between pupil diameter and esthetic judgments of pleasantness. Male portraits showed both a linear and quadratic relationship between pupil diameter and esthetic judgments of pleasantness. The linear model indicates that pupil diameter increased when viewing negative male portraits and decreased when viewing pos- itive male portraits, whereas a quadratic model shows that pupil diameter increases to both highly pleasant and unpleasant male portraits. Thus, it is plausible that researchers who used only a linear function found that unpleasant images were associated with larger pupil sizes compared to pleasant images (Woodmansee, 1967; Tinio and Robertson, 1969; Libby et al., 1973). These find- ings do not support Hess’ (1965) prediction, which suggested that unpleasant images would have been associated with a smaller pupil diameter. While it has been shown that pupils get larger to intense (arousing) stimuli, I only replicated this for male portraits. Schirillo’s (2000) analysis of Rembrandt’s portraits suggests this may be because the perceived dominance of male portraits was rated higher than for female portraits. Dominance may be associated with positive (e.g., self-assurance, arrogance, and feeling bold or triumphant) or negative affective states (e.g., hostility, irritability, and anger) (Demaree et al., 2005). It is possible that while Rembrandt painted males to exhibit these positive dominant traits (see Humphrey and McManus, 1973), negative dominance traits may have also been captured. Thus, the negative linear relationship between pleas- antness ratings and pupil diameter for males is consistent with Tinio and Robertson (1969), who found that aggressive Thematic Apperception Test cards elicited larger pupil size than control cards. This implies that Rembrandt’s male portraits may actu- ally be perceived as domineering which is consistent with Libby et al. (1973) and Woodmansee (1967) who found that unpleas- ant images were associated with larger pupil sizes compared to pleasant images. However, a quadratic relationship accounts for significantly more variance than a linear relationship. In this case, pupil size increased with large differences among pleasantness ratings for male faces. This is consistent with previous findings that pupil size is related to stimulus intensity rather than their specific positive or negative content (Janisse, 1973, 1974, 1977; Aboyoun and Dabbs, 1998). For example, arousing situations have been shown to pro- duce larger pupil diameter in non-visual stimuli studies. Nunnally et al. (1967) found that painfully loud sounds increased muscle tension causing larger pupil size. LINEAR AND QUADRATIC RELATIONSHIPS BETWEEN PLEASANTNESS RATINGS AND PUPIL DIAMETER As expected, there were almost as many zero differences between original and reversed image verbal ratings as there were difference scores (Figure 7). This inevitable outcome can potentially affect any of my linear and quadratic relationships. FIGURE 7 | Number of cases for all observers in which there was either a zero difference in verbal rating or there was a difference of any magnitude (with the original score being either more or less than the mirror-reversed image). (R = Right-cheeked, L = Left-cheeked, M = Males, F = Females). FIGURE 7 | Number of cases for all observers in which there was either a zero difference in verbal rating or there was a difference of any magnitude (with the original score being either more or less than the mirror-reversed image). (R = Right-cheeked, L = Left-cheeked, M = Males, F = Females). FIGURE 6 | Pupil size difference score (original-minus mirror-reversed) as a function of the difference in 1–9 verbal rating scores (between an original-minus mirror-reversed image) for all (A) male portraits, (B) female portraits. FIGURE 6 | Pupil size difference score (original-minus mirror-reversed) as a function of the difference in 1–9 verbal rating scores (between an original-minus mirror-reversed image) for all (A) male portraits, (B) female portraits. January 2014 | Volume 7 | Article 938 | 7 Frontiers in Human Neuroscience www.frontiersin.org Schirillo Laterality, pupil size, and esthetics DISCUSSION This new methodology eliminates Hess’ (1972) fluctuations in luminance and contrast across images allowing us to observe how pupil size varies as a function of the differences in verbal reports of pleasant and less pleasant original and mirror-reversed portraits. This was done by only comparing measurements between origi- nal and mirror-reversed images which makes image contrast and luminance irrelevant. This also means I do not require a baseline pupil size from the blurred images; since pupil size should not vary between original and mirror-reversed images because such images do not vary in image contrast and luminance. If they do differ, this must be because the emotional content of the images vary, not their image contrast or luminance. Given that it is impossible to equate apparent contrast or mean luminance across images, research testing Hess’ hypothesis had ceased. However, my improved methodology does not need to equate apparent contrast or mean luminance since this automati- cally occurs by comparing pupil size only across an original and its mirror-reversed image. In essence, I created a methodology that reexamined the relationship between a self-report and emotive measure (i.e., pleasingness) and an unconscious physiological measure (i.e., pupil size). I show that Hess (1965, 1972) was incorrect by focusing on valence. Instead he should have focused on arousal, since arousal drives the effect for male portraits not valence. Prehn et al. (2013) demonstrated that oxytocin increased saliency of all social stimuli regardless whether faces were male or female. In the placebo group (without oxytocin treatment), however, they found a strong gender effect (which decreased January 2014 | Volume 7 | Article 938 | 8 Frontiers in Human Neuroscience www.frontiersin.org www.frontiersin.org Laterality, pupil size, and esthetics Schirillo after oxytocin treatment). However, they used Winmorph 3.01 (www.debugmode.com/winmorph) to transform neutral expres- sions into all of the emotional ones in 5% steps. My study had no such control. Interestingly, their placebo“happy”male and female faces did not significantly differ in recorded intensity levels, nor recorded pupil size. This would have been the condition that I most like replicated. Like me, they found increased pupil dila- tions during the processing of male compared with female faces, which they attribute to reflecting men’s (they only used male sub- jects) lower interest in male faces. However, I used both sexes as subjects and found no significant differences between groups. DISCUSSION Yet their oxytocin manipulation is evidence that increased pupil- lary responses have been observed when stimuli are emotionally salient. But I controlled for saliency as much as possible by match- ing for valence and dominance, so I do not believe this was a factor in my experiment. Brodal, A. (1965). The Cranial Nerves. Oxford: Blackwell. Brodal, A. (1965). The Cranial Nerves. Oxford: Blackwell. Bryden, M. P., and Ley, R. G. (1983). “Right-hemispheric involvement in the per- ception and expression of emotion in normal humans,” in Neuropsychology of Human Emotion, eds K. M. Heilman, and P. Satz (New York: Guilford Press), 6–44. Conesa, J., Brunold-Conesa, C., and Miron, M. (1995). Incidence of the half- left profile pose in single-subject portraits. Percept. Mot. Skills 81, 920–922. doi:10.2466/pms.1995.81.3.920 Darwin, C. (1872). The Expression of the Emotions in Man and Animals. New York: D. Appleton and Co. Davidson, R. J. (1984). “Affect, cognition, and hemispheric specialization,” in Emo- tions, Cognition, and Behavior, eds C. E. I. Izard, J. Kagan, and R. B. Zajonc (Cambridge, MA: Cambridge University Press), 320–365. (Cambridge, MA: Cambridge University Press), 320–365. Davidson, R. J. (1992). Emotion and affective style: hemispheric substrates. Psychol. Sci. 3, 39–43. doi:10.1111/j.1467-9280.1992.tb00254.x Davidson, R. J. (1995). “Cerebral asymmetry, emotion, and affective style,” in Brain Asymmetry, eds R. J. Davidson, and K. Hughdahl (Cambridge, MA: MIT Press), 361–388. Davidson, R. J., Ekman, P., Saron, C. D., Senuluis, J. A., and Friesen, W. V. (1990). Approach-withdrawal and cerebral asymmetry: emotional expression and brain physiology I. 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Asymmetrical facial expressions in por- traits and hemispheric laterality: a literature review. Laterality 14, 545–572. doi:10.1080/13576500802680336 This article was submitted to the journal Frontiers in Human Neuroscience. j Copyright © 2014 Schirillo. 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 licen- sor 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. j Copyright © 2014 Schirillo. 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 licen- sor 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. Prehn, K., Kazzer, P., Lischke, A., Heinrichs, M., Herpertz, S. C., and Domes, G. (2013). Effects of intranasal oxytocin on pupil dilation indicate increased salience of socioaffective stimuli. Psychophysiology 50, 528–537. doi:10.1111/psyp.12042 Richer, F., Silverman, C., and Beatty, J. (1983). Response selection and initiation in speeded reactions: a pupillometric analysis. J. Exp. Psychol. Hum. Percept. Perform. 3, 360–370. January 2014 | Volume 7 | Article 938 | 10 Frontiers in Human Neuroscience www.frontiersin.org Laterality, pupil size, and esthetics Schirillo 5. A Young Girl Seated. 1660. London, E. S. Borthwick Norton Sale, May 15. 1953 (Br. 393). Left facing males 1. Rembrandt’s Sister, 1632. Stockholm, National Museum (Br. 85) 1. Head of an Old Man. Copenhagen, Statens Museum (Br. 136). 2. Saskia. 1643. Cassel, Gemaldegalerie (Br. 101). 2. A Man in Oriental Costume. 1633. Munich, Alte Pinakothek (Br. 178). 3. Saskia. 1636. Hartford, Conn., Wadsworth Atheneum (Br. 105). 3. A Bearded Man. 1646. Cassel, Germaldegalerie (Br. 230). 4. Hendrickje Stoffels as Flora. 1654. New York, Metropolitain of Art (Br. 114). 4. Study of an Old Man. 1640. Detroit, Mrs. Standish Backus (Br. 244). 5. Amalia van Solms. 1632. Paris, Musee Jacquemart-Andre (Br. 99). 5. Portrait of an Old Man in a Pearl-Trimmed Hat. 1662. Dresden, Gemaldegalirie (Br. 324). Frontiers in Human Neuroscience January 2014 | Volume 7 | Article 938 | 11 Right facing females 1. Rembrandt’s Mother as a Biblical Prophetess (Hannah?). 1631. Amsterdam, Rijksmuseum (Br. 69). 1. Self-Portrait. 1629. Munich, Alte Pinakothek (Br. 2). 1. Self-Portrait. 1629. Munich, Alte Pinakothek (Br. 2). 2. Self-Portrait. Not dated. The Hague, Mauritshuis (Br. 24). 2. Saskia. 1633. Washington, National Gallery of Art (Widener Collection) (Br. 96). 3. An Old Man with a Gold Chain. 1630. Los Angeles, Hans Cohn (Br. 149). 3. Rembrandt’s Sister. 1634. Indianapolis, John Herron Art Museum (Br. 100). 4. Portrait of a Man Reading. 1645. Williamstown, Mass., The Sterling and Francine Clark Art Museum (Br. 238). 4. A Woman in Fanciful Costume. 1648. Baltimore, The Walters Art Gallery (Br. 386). 5. Study of the Head of an Old Man. 1661. New York, John Hay Whitney (Br. 261). January 2014 | Volume 7 | Article 938 | 11 www.frontiersin.org Frontiers in Human Neuroscience
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Implementation and Validation of a Self-Consumption Maximization Energy Management Strategy in a Vanadium Redox Flow BIPV Demonstrator
Energies
2,016
cc-by
6,636
Academic Editor: Xiaoliang Wei Academic Editor: Xiaoliang Wei g Received: 12 May 2016; Accepted: 20 June 2016; Published: 29 June 2016 g Received: 12 May 2016; Accepted: 20 June 2016; Published: 29 June 2016 Abstract: This paper presents the results of the implementation of a self-consumption maximization strategy tested in a real-scale Vanadium Redox Flow Battery (VRFB) (5 kW, 60 kWh) and Building Integrated Photovoltaics (BIPV) demonstrator (6.74 kWp). The tested energy management strategy aims to maximize the consumption of energy generated by a BIPV system through the usage of a battery. Whenever possible, the residual load is either stored in the battery to be used later or is supplied by the energy stored previously. The strategy was tested over seven days in a real-scale VRF battery to assess the validity of this battery to implement BIPV-focused energy management strategies. The results show that it was possible to obtain a self-consumption ratio of 100.0%, and that 75.6% of the energy consumed was provided by PV power. The VRFB was able to perform the strategy, although it was noticed that the available power (either to charge or discharge) varied with the state of charge. Keywords: vanadium redox flow battery; building integrated photovoltaics (BIPV); energy management strategy; self-consumption maximization; real-scale battery www.mdpi.com/journal/energies Implementation and Validation of a Self-Consumption Maximization Energy Management Strategy in a Vanadium Redox Flow BIPV Demonstrator Luis Fialho 1,2,*, Tomás Fartaria 1,2, Luis Narvarte 3 and Manuel Collares Pereira 1,2 1 Renewable Energies Chair, Universidade de Évora, 7002-554 Évora, Portugal; tomasfartaria@uevora.pt (T.F.); collarespereira@uevora.pt (M.C.P.) 2 Instituto de Investigação e Formação Avançada (IIFA), Universidade de Évora, Palácio do Vimioso, Largo Marquês de Marialva, Apart. 94, 7002-554 Évora, Portugal 3 Instituto de Energía Solar, Universidad Politécnica de Madrid, 28040 Madrid, Spain; navarte@ies-def.upm.es 1 Renewable Energies Chair, Universidade de Évora, 7002-554 Évora, Portugal; tomasfartaria@uevora.pt (T.F.); collarespereira@uevora.pt (M.C.P.) 2 Instituto de Investigação e Formação Avançada (IIFA), Universidade de Évora, Palácio do Vimioso, Largo Marquês de Marialva, Apart. 94, 7002-554 Évora, Portugal 3 Instituto de Energía Solar, Universidad Politécnica de Madrid, 28040 Madrid, Spain; navarte@ies-def.upm.es * Correspondence: lafialho@uevora.pt; Tel.: +351-967-886-820 * Correspondence: lafialho@uevora.pt; Tel.: +351-967-886-820 Academic Editor: Xiaoliang Wei energies energies 2. Real-Scale VRFB Demonstrator The methodology used in this paper is based on the implementation of a real-scale demonstrator, installed at the University of Évora (Portugal). It consists of a rooftop mounted PV system (BIPV) and an electrical storage system (Vanadium Redox Flow battery), both connected to a building, which in turn is connected to the public electric grid (see Figure 1). Figure 1. Demonstrator (photovoltaic (PV) plant and Vanadium Redox Flow battery (VRFB) with respective converters) simplified schematic. Inverters convert power from PV and battery (DC) to the grid (AC). Figure 1. Demonstrator (photovoltaic (PV) plant and Vanadium Redox Flow battery (VRFB) with respective converters) simplified schematic. Inverters convert power from PV and battery (DC) to the grid (AC). 1. Introduction With the current increase of distributed renewable energies generators (such as rooftop photovoltaic power) in electrical grids, and more affordable energy storage systems (ESS), it is becoming much more viable to match individual demand for electricity with on-site photovoltaic (PV) production [1–3]. In the case of residential installations, the use of ESS for short-term management of supply—i.e., allowing the shift of PV energy to higher consumption periods (such as the evening peak or at night)—could increase self-consumed electricity from 30% to 70% [4–6]. To allow this increase, a battery must be integrated in the grid-connected PV system and an ad-hoc energy management strategy (EMS) must be implemented in a control unit that correctly manages the electricity flow between the PV generator, the battery, and the grid. In recent years, several EMSs focused on the maximization of the self-consumption of on-site-produced PV electricity have been presented [7–10], in which they assert that this strategy is useful and economically viable, even without feed-in-tariffs. This EMS requires a reliable and efficient battery technology with a low power/energy ratio, such as the Vanadium Redox Flow Batteries (VRFB). This promising technology has as Energies 2016, 9, 496; doi:10.3390/en9070496 www.mdpi.com/journal/energies 2 of 13 Energies 2016, 9, 496 main advantages the decoupling of power and capacity, a very long lifetime (reported as above 10000 cycles, or 25 years of operation), very low maintenance, and deep discharge capability (almost 100.0%) [11]. Very few batteries of this kind exist in laboratories, especially at such a scale that allows real-life test conditions, so the investigation of these batteries is mostly done with simulations (and sometimes in small lab battery cells) [12,13]. In order to test the performance of this kind of EMS, a real-scale VRB demonstrator was built in the University of Évora, part of the FP7 PVCROPS European project [14]. This demonstrator consists of a PV generator of 6.74 kWp, a PV inverter of 10 kW, a battery inverter of 7.4 kW, and a VRFB of 60 kWh. The main objective of this paper is to present the results of the validation tests of the self-consumption energy management strategy mentioned above and to show its suitability for building integrated PV applications. Useful conclusions for the future operation of these kinds of systems are also discussed. 2.1. Equipment The BIPV system is composed of two strings, a 3.24 kWp of monocrystalline PV panels and 3.5 kWp of polycrystalline cells, connected to a three-phase power inverter [15] with two independent maximum power point tracking (MPPT), connected to the building AC power grid. The energy storage is provided by a Vanadium Redox Flow Battery with 5 kW nominal power and 12 h storage [11], providing a total capacity of 60 kWh. The battery is also connected to the triphasic AC grid by three synchronized inverters (one per phase) [15]. Figure 2 shows the rooftop mounted PV system while Figure 3 shows the battery and four inverters (PV inverter in gray and the three battery inverters in green). To operate the demonstrator, a control system was implemented using a PC with Labview software to send and receive commands, and for overall system communication. This PC also acts as data logger to receive and store data. This PC receives data with parameter values from all the equipment (inverters, battery, and other sensors), runs the Labview software with the EMS, and continuously ensures the operation safety. In addition, each equipment has its own safety measures and surveillance implemented. 3 of 13 Energies 2016, 9, 496 Energies 2016, 9, 496 3 of 13 Figure 2. Roof mounted PV system. Figure 3. VRFB (right) and the inverters (left). Figure 2. Roof mounted PV system. Figure 2. Roof mounted PV system. Figure 3. VRFB (right) and the inverters (left). Figure 3. VRFB (right) and the inverters (left). The main innovative feature of this demonstrator is the usage of a real-scale Vanadium Redox Flow Battery. Table 1 shows the main technical specifications of the VRFB installed in the demonstrator. Table 2 shows the three battery inverters specifications, and Table 3 shows the photovoltaic inverter manufacturer specifications. In this battery, the electrolyte is stored in two tanks (for positive and negative species). The electrolyte is then pumped through the piping system to a stack of cells where the reduction–oxidation reaction occurs. The electron exchange takes place in cells (connected in series to constitute the stack) where a special ion membrane separates the two electrolytes and only permits H+ and V+ to pass. 2.1. Equipment The use of two tanks and a stack allows the decoupling of the battery power density (determined by the active area of the cells in the stack) from its energy density—i.e., the total energy that it can store (given by tank capacity and specific electrolyte energy density). This energy–power decoupling allows a battery to be tailored for many special cases, such as low power and large capacity (residential BIPV) or large power and lower capacity to mitigate power fluctuations (wind farms or PV plants). Another important feature of this battery technology is the absence of self-discharge, as there is no reaction outside the stack. Table 1. VRFB technical specifications. Specifications Value Manufacturer REDT Capacity 60 kWh (5 kW for 12 h) Rated Power 5 kW Maximum Operating Voltage 62 V Minimum Operating Voltage 48 V Electrical connection Three-phase Lifetime +10,000 deep charge/discharge cycles Full cycle (discharge/charge) Energy Efficiency 70%–80% Table 1. VRFB technical specifications. Table 1. VRFB technical specifications. 4 of 13 Energies 2016, 9, 496 Table 2. Battery inverters technical specifications. Specifications Value Manufacturer Ingeteam Model EMS Home 2.4 DC Max Current 50 A DC Operating Voltage Range 48 V–250 V Rated AC Power 2.4 kW Electrical connection single-phase Table 3. PV inverter technical specifications. Specifications Value Manufacturer Ingeteam Model 3Play 12.5TL M PV Power Range 12.9 kW–16.8 kW DC Voltage Range MPPT1 and MPPT2 200 V–820 V Rated Output AC Power 12.5 kW Electrical connection three-phase Table 2. Battery inverters technical specifications. Table 3. PV inverter technical specifications. Another distinct characteristic of this battery is the usage of vanadium as the main electrolyte element. The VRFB exploits the ability of vanadium to exist in four different oxidation states: from V2+ through V5+, dissolved in sulfuric acid (H2SO4). It is used as the positive electrolyte (V5+/V4+) and as the negative electrolyte (V2+/V3+). The usage of only one element allows the restoration of the electrolyte by simply mixing both electrolytes (positive and negative) and then separating them again by recharging the battery. This process (electrochemical reversibility) makes the electrolyte virtually infinite and non-degradable. The only source of electrolyte degradation is the occurrence of a side reaction with elements in air. To avoid this unwanted reaction, the empty space in the electrolyte tanks is filled with a high purity inert gas (Argon). 2.1. Equipment As these batteries are mostly composed of plastic piping and tanks and as the electrolyte is isolated from air, its lifetime is longer than other technologies, reported as 10,000 cycles [11]). In addition, as previously referred to in the introduction, another important feature of this technology is that it allows very deep discharges (close to 100%), so the whole battery capacity can be used. 2.1.1. Determination of the State of Charge The battery voltage is determined by the voltage of the reference cell multiplied by 40, as the stack is formed by 40 cells in series. Figure 4. State of charge versus battery voltage. The battery voltage is determined by the voltage of the reference cell multiplied by 40, as the stack is formed by 40 cells in series. Figure 4. State of charge versus battery voltage. The battery voltage is determined by the voltage of the reference cell multiplied by 40, as the stack is formed by 40 cells in series. 2.1.1. Determination of the State of Charge The VRF battery installed in this demonstrator is equipped with a parallel electrolyte circuit that carries a small part of the electrolyte flow to just one external cell (called the reference cell) that is neither subjected to charge nor discharge. As this cell voltage is only affected by the real electrolyte state, this cell voltage is the actual battery voltage in a rested state (not influenced by charge or discharge). In addition, the state of charge (SOC) is proportional to this voltage, so by knowing the reference cell voltage, one can obtain the real SOC. The function relating SOC with reference cell voltage was obtained experimentally by the battery manufacturer. Figure 4 shows the SOC variation with the battery voltage (determined by the reference cell voltage multiplied by 40, as the stack is formed by 40 cells in series). In order to obtain the SOC value during the battery operation, this graphic was fit to Equation (1) so the SOC is computed using the reference cell voltage measurement. Equation (1) was defined as a piecewise function, computed as a numerical fit of the data within the operating ranges (from 41.8 V to 65.4 V), as no single function could be fitted properly. Each function was chosen as the one that yielded the best numerical fit for their respective ranges (R-Squared ⩾0.99). 5 of 13 Energies 2016, 9, 496 SOC(V) =        0.0031V4 −0.5606V3 + 37.848V2 −1136.7V + 12810, if 41.8V < V ⩽52.4V −0.1544V3 + 25.961V2 −1443.4V + 26577, if 52.4V < V ⩽58.8V 0.0609V3 −11.88V2 + 773.28V −16694, if 58.8V < V ⩽65.4V (1) Figure 4. State of charge versus battery voltage. The battery voltage is determined by the voltage of the reference cell multiplied by 40, as the stack is formed by 40 cells in series. SOC(V) =        0.0031V4 −0.5606V3 + 37.848V2 −1136.7V + 12810, if 41.8V < V ⩽52.4V −0.1544V3 + 25.961V2 −1443.4V + 26577, if 52.4V < V ⩽58.8V 0.0609V3 −11.88V2 + 773.28V −16694, if 58.8V < V ⩽65.4V (1) SOC(V) =        0.0031V4 −0.5606V3 + 37.848V2 −1136.7V + 12810, if 41.8V < V ⩽52.4V −0.1544V3 + 25.961V2 −1443.4V + 26577, if 52.4V < V ⩽58.8V 0.0609V3 −11.88V2 + 773.28V −16694, if 58.8V < V ⩽65.4V (1) Figure 4. State of charge versus battery voltage. 2.2. Instrumentation and Data Acquisition In order to properly implement an EMS, several parameters are measured, allowing the computation of useful variables that enable smooth system operation. Furthermore, to allow a more complete study of each piece of equipment, several additional measurements (other than critical variables already provided by the equipment) are necessary. Table 4 summarizes the monitored variables. For example, regarding the PV system, it is only necessary to know the AC Power given by the inverter. However, to evaluate the efficiency of the PV system or detect problems, more data is necessary (global radiation in the plane of the BIPV system, PV cells temperature, and wind speed). These measurements are made using a weather station on the roof consisting of a pyranometer, a temperature sensor, and an anemometer, as shown in Figure 5. These sensors are read and processed by a control electronics (arduino Leonardo board), which in turn sends the data via a wireless zigbee network to another arduino connected to the main PC. Figure 5. Pyranometer and anemometer installed on the roof. Figure 5. Pyranometer and anemometer installed on the roof. The logged variables regarding the battery and inverters system are: DC power, AC power, battery voltage, current, and state of charge (SOC). Other useful variables are also read to ensure that the installation is healthy and operating within safe conditions (for example battery temperature, piping flow and pressure, electric grid parameters, and safe battery voltage range). The data is 6 of 13 Energies 2016, 9, 496 transmitted with Modbus protocol over TCP, via Ethernet. In order to obtain a more precise SOC value—it being a crucial parameter—an independent digital bench voltmeter was installed to the reference cell. This independent measurement allows a precision improvement from 0.7% (battery control measurement) to 0.1% (multimeter measurement) of the reference cell voltage. Furthermore, a three phase wattmeter (power analyzer) was installed to measure the power exchanged from the BIPV system and battery to the electric grid. In addition, three wattmeters were installed to obtain AC power measurements for each phase/ battery inverter. Table 4. Monitored variables during the demonstrator operation. BIPV: Building Integrated Photovoltaics. BIPV PV inverter Battery Battery Inverter Grid Global Radiation AC Voltage Ref. Cell Voltage AC Voltage AC 3-phase power PV Cell temperature AC Current Temperature AC Current DC Voltage AC Power DC Voltage AC Power DC Current DC Current DC Power DC Power 2.3. Electrical Loads Table 4. 2.3. Electrical Loads The loads used for the tests were simulated using the building to which the demonstrator is connected, as the consumption of all building loads is much larger than the power and capacity of the battery and PV system. The loads were simulated as being equally distributed by the three-phase electric grid. In this way, whenever a load is to be supplied, the power is sent to the building grid and that same power value is subtracted from the wattmeter measurement. This operation yields the power that would be exchanged with the grid if the loads were real and directly connected to the demonstrator. The load profile used is based on real 15 minute interval measurements made during a year of a real domestic installation [16]. This profile was then scaled according to the demonstrator specifications in order to provide a load profile level balanced to the system scale in what concerns battery usage and installed PV power. This method allows the loads to be of a suitable size relative to the real system specifications. 2.2. Instrumentation and Data Acquisition Monitored variables during the demonstrator operation. BIPV: Building Integrated Photovoltaics. BIPV: Building 3. Self-Consumption Maximization Strategy The strategy tested in the demonstrator was the self-consumption maximization strategy. The main objective of this EMS is to maximize the consumption of PV energy produced locally in the building, hence avoiding injecting energy to the electrical grid. This is achieved by giving priority to PV power and either storing the residual load (energy produced by PV system minus the energy consumed by loads) in an ESS or by taking energy stored previously. Thus, the only exceptional case in which there is an exchange of energy with the grid is when the battery can no longer absorb more energy or when the PV + battery system cannot provide enough energy to the required loads. According to the regulation of certain countries (such as Spain, Germany, Portugal, South Africa, Puerto Rico, etc.), the feeding of PV power into the electrical grid is either forbidden or highly restricted, so it is necessary to take measures to avoid this situation [17–21]. Either the excess power should be curtailed—i.e., PV production is limited to a sub-optimal value to avoid producing more power than what is being consumed at the moment—or, ideally, the excess power would be either stored or used by some deferrable loads (for example, a water heater, washing machine, etc.) [16]. In countries where a feed-in tariff (FIT) is paid for renewable electrical energy, the use of a battery could help to maximize profit. It could ensure that no PV power is lost due to regulatory restrictions or technical problems (for example, a sudden power outage) and even delay the sale of energy to a time when it might be purchased at a higher value (such as during peak hours). In a stand-alone system or in one where no PV power is allowed to be sold to the electrical grid (or if the selling price Energies 2016, 9, 496 Energies 2016, 9, 496 7 of 13 is too low), the user can store the energy for later usage, lowering the necessity to buy electricity from the grid. A flowchart of the self-consumption maximization strategy is presented in Figure 6. In the first step, the control system acquires data from PV production, loads consumption, and the battery state of charge (SOC). Then, it computes the difference between the loads consumption and PV production, referred to as residual load (Presidual). 3. Self-Consumption Maximization Strategy If the differential is positive, it means that current PV power is not enough to supply the loads. Hence, Presidual is provided by the battery (if it has enough accumulated energy). If the battery cannot provide the total necessary power (due to a low SOC), the remainder has to be provided by the electric grid. The reverse happens when there is more PV power being generated than consumed in a given moment. In this case, the remainder should be stored in the battery. If the battery cannot absorb all of the excess power (for instance, near maximum SOC), the remaining power must be fed to the grid (considering the case with no active loads management). Figure 6. Flowchart of the self-consumption maximization strategy implemented in the demonstrator. Figure 6. Flowchart of the self-consumption maximization strategy implemented in the demonstrator. This cycle should run at least every second to provide a quick response to any changes in PV production, consumption, or other situations (for example a blackout or a sudden problem in a piece of equipment). In this way, the grid is not largely affected by large fluctuations in PV production that could otherwise be fed to the electric grid with poor quality (high power variations in a short time). With the proper implementation of this EMS, the benefits obtained should be: lower energy exchange with the public grid, higher self-consumed PV power, and a higher load supply with PV power (either directly or by the battery). In order to validate this EMS—tested in the real size VRFB demonstrator—the following merit factors were defined to quantify the obtained improvements: self-consumption ratio, maximum positive (from the grid), and negative (into the grid) peak grid power, respectively given by Equations (2) to (4). This cycle should run at least every second to provide a quick response to any changes in PV production, consumption, or other situations (for example a blackout or a sudden problem in a piece of equipment). In this way, the grid is not largely affected by large fluctuations in PV production that could otherwise be fed to the electric grid with poor quality (high power variations in a short time). 4. Validation of the Self-Consumption Maximization Strategy in the VRFB Demonstrator 4. Validation of the Self-Consumption Maximization Strategy in the VRFB Demonstrator 3. Self-Consumption Maximization Strategy With the proper implementation of this EMS, the benefits obtained should be: lower energy exchange with the public grid, higher self-consumed PV power, and a higher load supply with PV This cycle should run at least every second to provide a quick response to any changes in PV production, consumption, or other situations (for example a blackout or a sudden problem in a piece of equipment). In this way, the grid is not largely affected by large fluctuations in PV production that could otherwise be fed to the electric grid with poor quality (high power variations in a short time). With the proper implementation of this EMS, the benefits obtained should be: lower energy exchange with the public grid, higher self-consumed PV power, and a higher load supply with PV power (either directly or by the battery). In order to validate this EMS—tested in the real size VRFB demonstrator—the following merit factors were defined to quantify the obtained improvements: self-consumption ratio, maximum positive (from the grid), and negative (into the grid) peak grid power, respectively given by Equations (2) to (4). With the proper implementation of this EMS, the benefits obtained should be: lower energy exchange with the public grid, higher self-consumed PV power, and a higher load supply with PV power (either directly or by the battery). In order to validate this EMS—tested in the real size VRFB demonstrator—the following merit factors were defined to quantify the obtained improvements: self-consumption ratio, maximum positive (from the grid), and negative (into the grid) peak grid power, respectively given by Equations (2) to (4). 3. Self-Consumption Maximization Strategy Energies 2016, 9, 496 Self-consumption ratio = R ∞ 0 (PPV(t) + Pgrid(t)) dt R ∞ 0 Ploads(t) dt (2) Maximum positive Pgrid = Max{Pgrid 15min} (3) Maximum negative Pgrid = Min{Pgrid 15min} (4) Self-consumption ratio = R ∞ 0 (PPV(t) + Pgrid(t)) dt R ∞ 0 Ploads(t) dt (2) (2) R 0 Ploads(t) dt Maximum positive Pgrid = Max{Pgrid 15min} (3) Maximum negative Pgrid = Min{Pgrid 15min} (4) Maximum positive Pgrid = Max{Pgrid 15min} (3) Maximum negative Pgrid = Min{Pgrid 15min} (4) Maximum positive Pgrid = Max{Pgrid 15min} (3) Maximum negative Pgrid = Min{Pgrid 15min} (4) and Pgrid 15min is the average value for each 15 minute time interval, given by: and Pgrid 15min is the average value for each 15 minute time interval, given by: Pgrid 15min = 1 900 900 ∑ t=0 Pgrid(t) (5) (5) where t is time in seconds. where t is time in seconds. where t is time in seconds. 4. Validation of the Self-Consumption Maximization Strategy in the VRFB Demonstrator 4.1. Testing Campaign A testing campaign was carried out in the time frame of one week. It was considered a sensible duration for the EMS validation, representing a balance between a short-time test (one day) and a longer period—for instance, one month. Figure 7 shows fifteen minute averages of the main parameters (PV power, loads consumption, and power exchanged with the battery) recorded during the testing of the Self-Consumption Maximization strategy. Figure 8 shows the power exchanged with the battery and the power exchanged with the grid in the same time period. Figure 7. One week display of the PV, loads, and battery power profiles (PPV, Pload, and Pbat, respectively). Figure 7. One week display of the PV, loads, and battery power profiles (PPV, Pload, and Pbat, respectively). Figure 8. Variation of Pbat, Presidual, and state of charge (SOC), one week period. Figure 8. Variation of Pbat, Presidual, and state of charge (SOC), one week period. 9 of 13 Energies 2016, 9, 496 Figure 9 shows the solar radiation on the PV plane (pyranometer) and PV cell temperature in the same time period. Figure 9 shows the solar radiation on the PV plane (pyranometer) and PV cell temperature in the same time period. Figure 9. Week profile of Solar Radiation (PV plane) and PV Cell Temperature. Figure 9. Week profile of Solar Radiation (PV plane) and PV Cell Temperature. Figure 9. Week profile of Solar Radiation (PV plane) and PV Cell Temperature. Figure 10. Variation of available discharge (blue) and charge (red) power, relative to the nominal power with the battery SOC. Figure 9. Week profile of Solar Radiation (PV plane) and PV Cell Temperature. Figure 10. Variation of available discharge (blue) and charge (red) power, relative to the nominal power with the battery SOC. Figure 10. Variation of available discharge (blue) and charge (red) power, relative to the nominal power with the battery SOC. From the graphics shown in Figures 7 and 9, the PV generation (blue dotted line in Figure 7) was mostly regular, with a clean radiation profile with only one day with significant variation (seen in Figure 9 on 18 May 2015). When the PV power is higher than the consumption, the remaining power (residual load positive) is stored in the battery (battery power is negative when charging). Usually, later in the day, when PV power is decreasing, the residual load (negative) is supplied by the battery. 4.1. Testing Campaign However, when comparing Figure 7 with Figure 8, it can be seen that despite the energy stored in the battery being enough to supply the loads, the power that the battery could deliver was not enough. This is due to the ratio of power versus SOC not being constant throughout the major range of SOC. When the battery is partially discharged (lower SOC), the discharge power that it can provide is lower than the nominal power. The reverse situation occurs with the charge power when the battery is almost at full capacity (higher SOC), as shown in Figure 10 . Figures 11 and 12 show that, at the beginning, the residual load (blue line) began to be entirely supplied by the battery (red line). When the battery got below 5% of SOC (purple line), the loads had to be supplied by the grid, as below this value, the battery voltage gets close to the inverter minimum voltage range. To avoid this situation, a safety measure was implemented. In Figure 11, one can see that despite this safety measure, the battery SOC continued to fall, suggesting that the value used to charge the battery is not enough to overcome the consumption of the inverters and the battery internal losses. 10 of 13 Energies 2016, 9, 496 10 of 13 Figure 11. First day of the self-consumption maximization test. Figure 11. First day of the self-consumption maximization test. Figure 12. Display of the first day of the self-consumption maximization test. Figure 11. First day of the self-consumption maximization test. Figure 12. Display of the first day of the self-consumption maximization test. Figure 12. Display of the first day of the self-consumption maximization test. When the PV power began to increase, the residual load also began to increase (positively), so the available power was used to charge the battery. At the end of the day, when PV power decreased, the previously stored energy was used to supply the loads. At the end of the day (after 21 h), some power had to come from the grid, as the battery could not entirely provide the necessary power to the evening load peak. 5. Results To properly assess the validation and to quantify the self-consumption maximization performance, several merit factors were computed and compared with the case in which the demonstrator did not have an ESS. Table 5 shows the comparison summary of the merit factors for the demonstrator with and without storage, evaluating the overall EMS performance for the testing period. Table 6 shows the merit factors computed for each day of the testing period. In terms of total energy, the installation was able to self-consume 100.0% of the PV generation, and it was able to supply 75.6% of the total consumption. Hence, it was necessary to get 24.4% of the energy from the grid. The maximum peak power from the grid was 2.5 kW, without energy injection into the grid. Considering the demonstrator without storage, only 65.2% of the total energy generated by the PV system was self-consumed, and the peak power injected to the grid was 3.3 kW, while the peak power taken from the grid reached 4.2 kW. To decrease the grid contribution, it is possible to increase the installed PV power or decrease the loads power consumption. In order to have a flexible demonstrator system, allowing the testing Energies 2016, 9, 496 Energies 2016, 9, 496 11 of 13 of different EMS that can require larger storage capacity, the total installed battery capacity ended up not being completely used in this test. This occurrence hints at the opportunity to develop and implement strategies that can provide more benefits than an exclusively short-term or long-term EMS. The remaining unused battery capacity can be used as reserve power in case of an unforeseen blackout, to take advantage of electricity price fluctuations, to provide power to neighbor installations, or even to charge electric vehicles. Additionally, a higher available PV power could be stored in the battery for later use, decreasing the grid necessity. The extra capacity relative to the PV system size also raises the important question of how to optimize the battery capacity regarding PV generation and loads consumption. Table 5. Comparison between the full demonstrator and a virtual one without storage. Table 5. Comparison between the full demonstrator and a virtual one without storage. Merit Factor Storage No Storage Self-consumption ratio (%) 100.0 65.2 Maximum positive peak Pgrid (kW) 2.5 4.2 Maximum negative peak Pgrid (kW) 0.0 3.3 Table 6. Comparison between demonstrator results with and without storage. 5. Results Date Self Consumption Ratio (%) Maximum Positive Peak Pgrid (kW) Maximum Negative Peak Pgrid (kW) 14/05 storage 100.0 1.2 0.0 no storage 67.5 3.2 3.2 15/05 storage 100.0 2.5 0.0 no storage 61.6 3.1 3.3 16/05 storage 100.0 1.4 0.0 no storage 63.5 4.3 3.2 17/05 storage 100.0 2.1 0.0 no storage 66.1 2.6 3.2 18/05 storage 100.0 2.6 0.0 no storage 66.4 3.2 3.2 19/05 storage 100.0 1.2 0.0 no storage 65.5 3.4 3.4 20/05 storage 100.0 1.2 0.0 no storage 66.9 2.5 3.3 This test yielded several useful results for the future operation of this technology demonstrators • The Vanadium Redox Flow technology has the ability to decouple power and capacity but the available power at extreme SOC varies. Figure 10 shows the variation of discharge (blue) and charge (red) power with the SOC. This fact also reveals the importance of analyzing these systems as power sources/sinks and not only in terms of global energy. • The Vanadium Redox Flow technology has the ability to decouple power and capacity but the available power at extreme SOC varies. Figure 10 shows the variation of discharge (blue) and charge (red) power with the SOC. This fact also reveals the importance of analyzing these systems as power sources/sinks and not only in terms of global energy. • The system formed by the VFRB and inverters has achieved an overall average efficiency of 57.7% during the testing period. The efficiency is penalized by the battery operating voltage range being bellow the ideal voltage range of the inverters. The used inverters have a working voltage ranging from 48 V to 300 V, although their efficiency is higher at the top end of this range [15]. • The inverters consumption and battery internal losses are not entirely supplied during the safety procedure. Although the VRF battery is not damaged by an over-discharge, a voltage below the working range can turn the battery inverters off (the battery inverters are powered by the battery and require a minimum of 45 V), hence disturbing the normal operation of the installation. Energies 2016, 9, 496 12 of 13 6. Conclusions The test results of the use of a VRFB as the storage system for the testing of a self-consumption maximization strategy, implemented in a real-size demonstrator, were presented. The results show that the VRFB is a suitable technology to be combined with BIPV systems. Regarding the EMS testing, the strategy successfully maximized the consumption of locally-produced PV energy. Using the battery to store excess PV power (positive residual load), it was possible to completely avoid the injection of energy into the public electric grid—i.e., 100.0% self-consumption ratio. In addition, the total energy consumed by the loads was 75.6%, supplied by PV generation. A closer analysis of the system operation shows that an increase in PV power could further decrease the necessity of the electrical grid, as a large part of the battery capacity (approximately 65.0%) was unused. The correct performance of the EMS is also shown by the reduction of power exchanged with the grid. The peak power from the grid was reduced in 59.0% in comparison with the case without energy storage. This means that the prosumer—apart from benefiting the whole electrical system—not only reduces their electric bill due to the reduction of grid energy consumption, but also due to the reduction of the utility contracted power tariff. This work also showed the relevance of the equipment operational limits (in this case, the battery inverters DC voltage range) to the EMS, and the non-negligible variation of available charge/discharge power with the battery SOC. In future developments of energy management strategies, all these operational limitations should be taken into account to ensure the proper and safe working mode of the whole system as planned. Acknowledgments: The authors would like to acknowledge the support of this work, developed under the PVCROPS Project, which has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement n◦308408. This work was also supported by the Renewable Energies Chair of the University of Évora (PhD scholarship, author Luis Fialho) and by a PhD scholarship (author Tomás Fartaria) from FCT – Fundação para a Ciência e Tecnologia, Portugal, Grant number SFRH/BD/84396/2012. The authors also would like to acknowledge the support of the European Commission OPENAIRE FP7 Post-Grant Open Access Pilot for supporting the publication of this work. Conflicts of Interest: The authors declare no conflict of interest. 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Acknowledgments This book is a translation of the revised and expanded version of my PhD disser- tation, which was approved by the Institute of History at Humboldt-Universität zu Berlin on 21 January 2015. The German version of the book was published in 2018 by Vandenhoeck & Ruprecht as Elitenbildung und Dekolonisierung. Die Évolués in Belgisch Kongo 1944–1960. For the English translation I expanded and updated sections of the manuscript in light of subsequent publications and criticism. Only authors know just how much other people contribute to the creation of books. This volume would never have seen the light of day without the help and support of many individuals. I would like to expressly thank some of them here. First, I am deeply grateful to Andreas Eckert. As my primary supervisor, he supported and shaped the composition of this study with commitment, generos- ity and inspiration. His trusting, motivating and supportive manner was crucial to the success of this project and to my subsequent progress as a scholar. My thanks also go to Gabriele Metzler, who took on the second review of my disser- tation with enthusiastic interest. The suggestions made by both these scholars were particularly valuable as I reworked my initial study. I also owe a debt of gratitude to Alexander Nützenadel, who suggested that the German book be pub- lished in the “Kritische Studien zur Geschichtswissenschaft” series. I am indebt- ed to the editors for including this work in their programme. Their constructive suggestions in the reviews and the stimulating support of Dieter Gosewinkel were of much benefit to the final German-language text, whose publication was facilitated by the genial and professional stewardship of Daniel Sander at Vandenhoeck & Ruprecht. The translation of this work was funded by Geisteswissenschaften Interna- tional – Translation Funding for Work in the Humanities and Social Sciences from Germany, a joint initiative of the Fritz Thyssen Foundation, the German Fed- eral Foreign Office, the collecting society VG WORT and the Börsenverein des Deutschen Buchhandels (German Publishers & Booksellers Association). I’m es- pecially thankful for the jury’s recognition in awarding my book the Special Prize for an outstanding work in the humanities.Wilma Kähmer at Vandenhoeck & Ru- precht was of great help when it came to applying for financial support, while Anke Simon at the Börsenverein des Deutschen Buchhandels made the funding process a smooth one. OpenAccess. © 2021 Daniel Tödt, published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International License. https://doi.org/10.1515/9783110709308-011 Acknowledgments I am grateful to Joël Glasman and the editors of the Africa in Global History series for including my text in their ambitious and exciting pub- lishing programme. The anonymous reviewer provided constructive advice that helped me put the finishing touches to the manuscript. Rabea Rittgerodt at De 370 Acknowledgments Gruyter supported the translation project with tremendous competence, engage- ment and an unfailingly positive attitude. The fact that I could recognize my own language in the English version is down to Alex Skinner, who produced the translation with great professionalism, sensitivity, conscientiousness and elo- quence. The dissertation on which the book is based goes back to my participation in Collaborative Research Centre 640, “Changing Representations of Social Order. Intercultural and Intertemporal Comparisons”, at Humboldt University of Berlin. Its spokesperson Jörg Baberowski, along with Matthias Braun, Xenia Krüger and all the other project participants created an outstanding research environment. Regular visits to the cafeteria with colleagues were an indispensable source of support, the shared office time and daily exchanges with Regina Finsterhölzl a welcome stroke of luck. Michael Pesek and Susann Baller provided important stimulus for my study at an early stage. The student assistants on the “African Modernity” sub-project – Sascha Dannenberg, Josephine Friedrich, Lotte Knote and Anika Stegeman – supported me in a variety of ways. At the Center for Met- ropolitan Studies at the Technical University of Berlin I was fortunate to be able to complete the German book manuscript in the pleasant and supportive setting provided by Dorothee Brantz as well as Elisabeth Asche, Noa Ha and Frederieke Westermann. When revising the English version, I benefited from the active sup- port of Felicitas Hentschke at the International Research Center “Work and Human Lifecycle in Global History” (re: work), who provided a quiet working en- vironment, as well as Daniela Greca and Zeynep Ecem Pulas, who helped me with the acquisition of literature. It was my key supporters during my undergraduate and master’s studies, Stefan Krankenhagen, Rolf Lindner and Silke Strickrodt, who encouraged me to set forth on the doctoral adventure in the first place. I was privileged to pre- sent my research project at many workshops, conferences and colloquia, which provided me with valuable criticism, praise and suggestions. Key decisions along the way were informed by discussions with Dmitri van den Bersselaar, Frederick Cooper, Manfred Hettling, Nancy Rose Hunt, Peter Lambertz, Pedro Monaville and Thomas Mergel. Acknowledgments Joël Glasman supported the project from start to finish with equal parts enthusiasm and critical input. Through his in-depth reading at various points in time, Lasse Heerten helped ensure that I paid sufficient at- tention to global perspectives. Manfred Schmidt and Söhnke Vosgerau read the entire manuscript carefully and made insightful comments; the latter also proof- read my doctoral dissertation. Individual chapters were amended in light of com- ments by Felix Fuhg, Patrick Hege, Lotte Knote, Gesa Trojan, Marlène de Saus- sure and Hannah Schilling. Mike Sigl updated the index for the English version of the book. Christoph Kalter and Marc Buggeln were kind enough to Acknowledgments Acknowledgments 371 give me valuable advice on applying for funding from Geisteswissenschaften In- ternational. The history of the Congo and of Belgium was uncharted territory for me. Many people helped me get my bearings, supported me with useful critique dur- ing the research process and made my stay in Kinshasa an unforgettable expe- rience. Some of them, I am delighted to say, have become colleagues and friends. Kwa Kongo Benz, Saint José Camille Inaka, Peter Lambertz, Sapin Makengele, Cécile Michel, Pedro Monaville, Katrien Pype and Léon Tsambu welcomed me with open arms and excellent contacts. Sebastien Khonde and the residents and employees of the Centre d’Information et d’Animation Missionnaire in Limete made me feel at home and at ease. Filip De Boeck, Susanne Gehrmann, Charles Didier Gondola, Hermelinde Lanza, Antoine Lumenganeso, Jean-Marie Muta- mba-Makombo, Jacob Sabakinu, Dominic Thomas and Charles Tshimanga pro- vided me with valuable leads. The individuals I interviewed gave up a good deal of time for me and showed great openness and hospitality. When I was try- ing to find interview partners who already lived in colonial times, Léon Tsambu took me to the studios of the TV show Sentiments Lipopo, in which more or less older inhabitants of Kinshasa dance to rumba songs. I hadn’t realized I would be live on air. I am grateful to the participants in the programme for their kindly forbearance in view of my clumsy attempts at rumba. My ineptitude may well have prompted the presenter, Jean de la Croix Mobé, to take pity on me and gen- erously allow me access to his father’s private archive. Johan Lagae, Amandine Lauro, Guy Vanthemsche and Jean-Luc Vellut provided me with always instruc- tive exchanges. Acknowledgments The staff of the archives and libraries I visited were helpful and patient: Pierre Dandoy and Alain Gérard at the Archives Africaines in Brussels; Lore Van de Broeck, Sabine Cornelis, Jean Omasombo Tshonda and Anne Welschen at the Musée Royal de l’Afrique Centrale; Jean-Claude Yoka at the Archives natio- nales d’outre-mer in Aix-en-Provence; and Tinne Billet of the Société Multimédia des Auteurs des Arts Visuels (SOFAM). In addition, the staff of the KADOC in Leuven, the Bontinck Library, the Assanef Archive and the Archives Nationales de la République Démocratique du Congo helped ensure the success of my re- search, while Nura Treves facilitated the perusal of several documents. I would like to express my gratitude to the Geschwister Boehringer Ingelheim Stiftung für Geisteswissenschaften in Ingelheim am Rhein and the Center for Met- ropolitan Studies at the Technical University of Berlin for helping cover the print- ing costs of the German edition of the book. My thanks also to the IGK “Work and Human Lifecycle in Global History” (re: work) for covering the costs of proofread- ing and the insertion of illustrations. Open Access was kindly made possible by the Open Access Publication Fund of Humboldt-Universität zu Berlin and by the 372 72 Acknowledgments Translation Prize of the ZEIT-Stiftung Ebelin und Gerd Bucerius awarded by the German Historical Association (VHD). Finally, I would like to thank my parents, who aroused my curiosity and wanderlust early on and have always been there for me with their love and faith in my abilities. I am grateful to my friends, who were of great help during the hectic period of research culminating in this book. No one is more closely connected with this project than my wife Farah. Every line in the book bears the imprint of her encouragement, patience and love. As full time-working pa- rents with two young children, we are thankful for a great care network, includ- ing the team at the Kitaquarium nursery and the parents who co-founded and co-manage it. We are also grateful to Elza Hamoniaux, a fantastic babysitter, and to my mother whose visits facilitated both travel and writing retreats. It must be a disappointment to Noam and Elias that this largely picture-free vol- ume makes for a poor bedtime story. Acknowledgments I wrote the final pages of my dissertation shortly after Noam was born, and she was by my side in her pram when I defend- ed my thesis. Elias was born during the production of the translation, and he has accompanied me ever since as my second lucky charm. I am deeply grateful to them for reminding me every day that the present and future are more important than the past. The book is dedicated to Hesdy Olieberg, a close friend. After meeting some 25 years ago on a basketball court on Fuerteventura, he broadened my perspec- tive on the Black Atlantic and always provided a safe harbour in Amsterdam as I travelled around the world. He was my role model until his very last breath.
https://openalex.org/W4384132315
https://www.qeios.com/read/LLRAAG/pdf
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Review of: "Identifying Psychological Distress Patterns during the COVID-19 Pandemic using an Intersectional Lens"
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Qeios, CC-BY 4.0 · Review, July 13, 2023 Qeios ID: LLRAAG · https://doi.org/10.32388/LLRAAG Review of: "Identifying Psychological Distress Patterns during the COVID-19 Pandemic using an Intersectional Lens" Panagiotis Pelekasis Potential competing interests: No potential competing interests to declare. Potential competing interests: No potential competing interests to declare. 1) The abstract is not very clear. By reading the results it is not clear which differences were noted. For example, it is not clear weather males or females had higher distress. 1) The abstract is not very clear. By reading the results it is not clear which differences were noted. For example, it is not clear weather males or females had higher distress. 2) Psychological distress was not assessed through a single instrument, as it should have, such as the HADS. Instead, several questions from different instruments were used. This instrument was developed for the study purpose and was not validated, which consists a major weakness of the study. 2) Psychological distress was not assessed through a single instrument, as it should have, such as the HADS. Instead, several questions from different instruments were used. This instrument was developed for the study purpose and was not validated, which consists a major weakness of the study. 3) The study has no other major weaknesses. Yet, it investigates a matter that is currently of low interest, since the pandemic has ended. It would be a good and interested paper if published earlier. It is obvious by the absence of references from 2022 and 2023 that the paper was written in the previous years, but for some reason not published. I believe that currently this paper is unfortunately of low interest. 3) The study has no other major weaknesses. Yet, it investigates a matter that is currently of low interest, since the pandemic has ended. It would be a good and interested paper if published earlier. It is obvious by the absence of references from 2022 and 2023 that the paper was written in the previous years, but for some reason not published. I believe that currently this paper is unfortunately of low interest. Qeios ID: LLRAAG · https://doi.org/10.32388/LLRAAG 1/1
https://openalex.org/W4246690928
https://www.researchsquare.com/article/rs-154602/latest.pdf
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How Flushable Wet Wipes Are Causing Sewer Blockages – and An Approach to Prevent That
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Keywords: License:   This work is licensed under a Creative Commons Attribution 4.0 International License. License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Version of Record: A version of this preprint was published at Scientific Reports on April 12th, 2021. See the published version at https://doi.org/10.1038/s41598-021-86971-z. Abstract The authors have requested that this preprint be withdrawn due to erroneous posting. The authors have requested that this preprint be withdrawn due to erroneous posting. EDITORIAL NOTE: The full text of this preprint has been withdrawn by the authors while they make corrections to the work. Therefore, the authors do not wish this work to be cited as a reference. Questions should be directed to the corresponding author. Page 1/2 Page 1/2 Full Text The authors have withdrawn this preprint from Research Square. Page 2/2
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Exposição a agentes químicos e a Saúde do Trabalhador
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Exposição a agentes químicos e a Saúde do Trabalhador Exposure to chemicals and the Workers’ Health Mina Kato1 Eduardo Garcia Garcia2 Victor Wünsch Filho3 Mina Kato1 Eduardo Garcia Garcia2 Victor Wünsch Filho3 1 Editora associada 2 Editor executivo 3 Editor convidado e membro do Conselho Editorial A relevância do tema deste volume da RBSO é amplamente reconhecida. A Organização Internacional do Trabalho (OIT) estima em 35 milhões anuais os casos de doenças relacionadas ao trabalho por exposição a substâncias químicas com a ocorrência de 439.000 mortes, incluindo, entre outras causas relaciona- das, 36.000 óbitos por pneumoconioses, 35.500 óbitos por doenças respira- tórias crônicas, 30.700 óbitos por doenças cardiovasculares e 315.000 óbitos por câncer (ILO, 2004). A Organização Mundial da Saúde, por sua vez, estima que esses cânceres provoquem uma perda anual de 1,4 milhão de anos de vida saudável e que as intoxicações agudas por produtos químicos sejam responsá- veis por outros 7,5 milhões de anos de vida saudável perdidos pela população mundial (WHO, 2002). Certamente, há muito por conhecer, discutir e fazer a respeito da origem, do controle e da prevenção desses problemas, que não são recentes, embora estejam ganhando amplitude pela inclusão constante de novas substâncias no mercado e pelo aumento contínuo de seu uso. Neste nú- mero, são apresentados cinco artigos, um ensaio e duas resenhas relacionados ao tema. No primeiro dos artigos, intitulado Características da exposição ocupacio- nal a poeiras em marmorarias da cidade de São Paulo, são reportadas altas concentrações de poeira originadas pelas ferramentas utilizadas no setor de acabamento a seco. As concentrações de sílica chegaram a ser muitas vezes superiores ao valor do limite de exposição ocupacional recomendado, mas foi apontada a possibilidade de se reduzir significativamente as concentrações ambientais com a adoção de um processo de acabamento a úmido, uma medi- da de proteção coletiva dos trabalhadores. Uma das conseqüências possíveis da exposição a poeiras contendo sílica é uma pneumoconiose – a silicose. As pneumoconioses podem ser causadas por outros compostos de origem mineral (asbestos, carvão, berílio) ou vegetal (algodão, sisal). Mesmo conhecidas desde a Antiguidade, ainda acometem os trabalhadores do século XXI. Em 2006, foram registrados na Previdência So- cial, através das Comunicações de Acidentes de Trabalho (CATs), 56 casos de pneumoconiose relacionados a poeiras contendo sílica e 10 pneumoconioses por outros agentes ou não especificadas (MPAS, 2008). Rev. bras. Saúde ocup., São Paulo, 32 (116): 06-10, 2007 Exposição a agentes químicos e a Saúde do Trabalhador Exposure to chemicals and the Workers’ Health É importante observar que os casos registrados na Previdência se referem àqueles trabalhadores que estão inseridos em meios com conhecimento e acesso ao sistema da Previdên- cia, não representando a totalidade dos casos incidentes no país. Em seu compêndio sobre as doenças dos trabalhadores (De Morbis Ar- tificum Diatriba), originalmente publicado em 1700, Ramazzini comentou sobre a qualidade da poeira a que os mineradores estavam expostos – tão fina que ultrapassava a bexiga de um boi que estava pendurada na oficina de um lapidário (RAMAZZINI, 1999). Plínio, “O Velho” (23 a 79 d.C.), um estudioso romano, já relatava o uso de bexigas de animais como uma tenta- tiva de se proteger contra inalação de poeiras e fumos de chumbo (OSHA, 6 6 Rev. bras. Saúde ocup., São Paulo, 32 (116): 06-10, 2007 2007; RAMAZZINI, 1999), demonstrando a preocupação em impedir ou reduzir o contato do trabalhador com as substâncias químicas por meio de proteção individual. Portanto, a noção de proteção pelo uso de equipamentos de proteção individual (EPIs) não é nova, mas, mesmo atualmente, pode ser difícil de obter-se. É o que ilustra o ensaio A contaminação por agrotóxicos e os Equipamentos de Proteção Individual (EPIs). Os autores reportam que os EPIs, neste caso a vestimenta de proteção na aplicação de agrotóxicos, além de não prote- gerem integralmente o trabalhador contra o produto químico, ainda agravaram os riscos, pois se tornaram fontes de contaminação. Discutem, também, a possibilidade dos EPIs apresentarem lacunas funcionais na concepção, no projeto, no uso, na manutenção, no armazenamento e no descarte. Assim, esses equipamentos deveriam ser continuamente avaliados por meio de estudos de desenvolvimento tecnológico para melhoria da sua funcionali- dade e não ocasionarem, paradoxalmente, danos à saúde humana. Os primeiros agrotóxicos utilizados foram os de sais inorgânicos. Ainda se utilizam no mundo compostos de arsênio, cobre, chumbo e mercúrio, cujos efeitos tóxicos são conhecidos desde a Antiguidade (EPA, 1986; DECC, 2008). Plantas e seus extratos, como o das folhas de tabaco, foram também utilizados na agricultura para combater insetos. Mas os agrotóxicos organossintéticos começaram a ser produzidos e utilizados em larga escala a partir da Segunda Guerra Mundial, com a descoberta da propriedade inseticida do DDT em 1939, apesar de sintetizado em 1874. O sucesso inicial do DDT foi observado no controle de vetores de doenças transmissíveis, como malária e tifo epidêmico. Exposição a agentes químicos e a Saúde do Trabalhador Exposure to chemicals and the Workers’ Health Surgiram, porém, críticas à utilização do inseticida e preocupação com os seus impactos ao ambiente a partir da identificação de inúmeros casos de intoxicação aguda e do aumento de seu uso indiscriminado na agricultura, com conseqüente acúmulo de resíduos no ambiente e na cadeia alimentar, assim como a constatação da ativação de resistência entre os organismos alvo e o risco de câncer em animais, incluindo o ser humano (D´AMATO et al., 2002). O livro de Rachel Carson, Primavera Silenciosa, de 1962, tornou pública a preocupação ecológica com o DDT e condenou a liberação de grandes quantidades da substância e seus congê- neres no ambiente sem o conhecimento integral de seus efeitos nocivos (LEAR, 2002). Os agrotóxicos também estão focalizados no artigo intitulado Incidência de suicídios e uso de agrotóxicos por trabalhadores rurais em Luz (MG), Brasil. Em 2005, o Sistema Nacional de Informações Tóxico-Farmacológicas (SINITOX) registrou 84.456 casos de intoxicações em 28 dos 34 Centros de Informação e Assistência Toxicológica em atividade no país. Desses casos, 15.804 foram intoxicações atribuídas às tentativas de suicídio, dentre os quais 14% aos agrotóxicos de uso agrícola e 11% aos raticidas, superados apenas pelos casos provocados por medica- mentos (59%). Dos 6.827 casos de intoxicação atribuídos à circunstância ocupacional, 37% foram causados por animais peçonhentos, 25% por agrotóxicos de uso agrícola e 13% por produtos químicos industriais. Entre os 477 óbitos registrados, os agrotóxicos foram a causa mais freqüente (33%), seguida por medicamentos (18%), raticidas (11%), drogas de abuso (11%) e animais peçonhentos (9%). O suicídio respondeu por 54% dos óbitos e a faixa etária produtiva, de 20 a 59 anos, respondeu por 68% (325 casos) do total dos óbitos (SINITOX, 2005). De acordo com o artigo de Minas Gerais, a fácil disponibilidade dos produtos poderia ser uma das razões para a escolha em caso de suicídios. A necessidade de aprofundamento da discussão sobre o papel da exposição a esses produtos na indução ao suicídio também se mostrou relevante no estudo. A dificuldade de se usar uma vestimenta confortável e protetora, seja por questões financeiras ou ergonômicas ou por inadequação do produto, além do despreparo dos aplicadores de agrotóxicos no que diz respeito ao uso e ao conhecimento dos riscos são apontados como os determinantes das intoxicações. Rev. bras. Saúde ocup., São Paulo, 32 (116): 06-10, 2007 Exposição a agentes químicos e a Saúde do Trabalhador Exposure to chemicals and the Workers’ Health No ambiente de trabalho de laboratórios clínicos, são observados também ou- tros tipos de fatores de risco à saúde – agentes biológicos, posturas inadequadas e movimentos repetitivos, entre outros. Para este tipo de atividade de prestação de serviços na área de saúde, delineou-se uma área específica de atuação dentro da Saúde do Trabalhador: a Biossegurança. A Biossegurança, apesar de ter recebido algumas definições que restringem seu campo de ação, é considera- da, na Saúde do Trabalhador, parte integrante da Segurança e da Higiene do Trabalho, que se preocupa com os trabalhadores da área de saúde e afins, em cujos ambientes de trabalho estão presentes não somente os fatores de riscos biológicos, mas outros que podem diretamente agravar a saúde ou podem ser desencadeadores de aciden- tes biológicos (VIEIRA & LAPA, 2006). Por longo tempo, o modelo da unicausalidade das doenças dominou o pensamento na área da saúde. Essa forma de pensar dificultava aceitar as doenças multifatoriais ou multicausais e também doenças cujos sintomas eram inespecíficos e difíceis de serem mensurados objetivamente. O modelo unicausal era reducionista e funcio- nal para estudos experimentais, mas impossibilitava explicar a complexidade da realidade. Em contrapartida, o modelo multicausal é complexo e difícil de representar, necessitando-se para tanto utilizar modelos matemáticos complexos. Atualmente, mesmo com essa concepção sendo hegemônica e com o presente desenvolvimento tec- nológico, os aspectos legais da previdência social e trabalhista preservam ainda abordagens reducionistas, como, por exemplo, na procura em estabelecer o nexo causal e o julgamento da insalubridade do ambiente, mesmo quando há exposição a múltiplos agentes (FRANCO & KATO, 1997; SANTOS et al., 2004). Os resultados do artigo Dados sociodemográficos e condições de trabalho de pintores expostos a solventes em uma universidade pública da cidade do Rio de Janeiro fazem parte de um estudo mais amplo que avaliou altera- ções neuropsicológicas que podem estar relacionadas à exposição a solventes orgânicos. Embora os efeitos agudos dos solventes orgânicos sobre o sistema nervoso central fossem conhecidos há muito tempo, o estabelecimento da relação causal entre os efeitos neuropsicológicos em trabalhadores expostos a solventes em longo prazo ocorreu apenas no final do século passado (RAMOS et al., 2004). O artigo mostra que os pintores entrevistados referi- ram uma fonte de renda paralela, uma questão importante quando se busca traçar a relação entre o ambiente de trabalho e os efeitos sobre a saúde. Exposição a agentes químicos e a Saúde do Trabalhador Exposure to chemicals and the Workers’ Health Uma vez que um acidente de grande porte não pode ser reproduzido ou testado experimentalmente, os dados do estudo e a análise de risco realizada são elementos preciosos para fundamentar medidas de emergência e de prevenção de ocorrências futuras. Ainda, permitem o planejamento de políticas públicas com o objetivo de controlar fatores desencadeadores deste tipo de acidente. O desenvolvimento e a aplicação de modelos matemáticos e de novos softwares para a análise de riscos favo- recem a idéia de que as avaliações dos fatores de risco devem ser quantitativas – as concentrações de poeiras no ar ambiente, o nível de contaminação do solo por agrotóxicos, a incidência de radiação, o calor nos ambientes de trabalho etc. Porém, para realizar uma avaliação quantitativa que represente as condições que se deseja estudar é necessário realizar inicialmente uma boa avaliação qualitativa. Até mesmo para ponderar a necessidade das mensurações, a avaliação qualitativa é imprescindível (PUGAS et al., 2001). O manual Avaliação qualitativa de riscos químicos: princípios básicos para o controle das substâncias nocivas à saúde em fundições, apresentado por resenha neste volume, é um instrumento colocado à disposição dos que atuam na área da Saúde do Trabalhador em pequenas e médias empresas. O texto aborda as fundições, mas deriva de um sistema de gerenciamento de risco desenvolvido e padronizado pelo Programa sobre Segurança e Saúde no Trabalho e no Meio Ambiente (SafeWork) da Organização Internacional do Trabalho, a partir do Health and Safety Executive (HSE). O programa, implementado desde 1998, no Reino Unido, tem o objetivo de apoiar esforços na prevenção de exposições a agentes de risco ocupacional por meio de tomada de decisões baseadas em análise qualitativa, sem necessidade de utilizar sempre avaliações quantitativas (PAPP, EIJKEMANS & VICKERS, 2004). Obviamente haverá situações em que avaliações quantitativas do ambiente serão indispensáveis ou desejáveis, porém, o custo e a complexidade dessas avaliações podem dificultar a sua realização. O artigo Avaliação dos riscos associados ao uso do xilol em laboratórios de anatomia patológica e citologia con- tém uma avaliação baseada nas informações dos trabalhadores. Na conclusão, os autores enfatizam a necessidade de educar o trabalhador com base numa visão prevencionista em relação aos riscos ocupacionais e ambientais provenientes do uso daquele agente químico. Provavelmente, o desconhecimento dos trabalhadores não se res- tringe apenas ao agente químico. Exposição a agentes químicos e a Saúde do Trabalhador Exposure to chemicals and the Workers’ Health Mas certamente as causas vão além, passando pelo modelo de produção agrícola adotado, pelas estratégias de difusão dessa tecnologia (sem evidenciar os riscos à saúde e ao ambiente e, ainda, sem considerar o despreparo do usuário e a ausência de recursos para o controle), pela indu- ção ao uso excessivo por vendedores e propagandas, pelas relações e condições precárias de trabalho, além dos determinantes socioeconômicos predominantes no meio rural, como a baixa escolaridade, moradias inadequadas e difícil acesso à atenção à saúde (GARCIA, 2001). Reconhecendo a complexidade da questão da exposição a agentes químicos, na segunda metade do século passado a área da Saúde do Trabalhador passou a operar com abordagens multi e interdisciplinares para solução de problemas, preconizando que a substituição, ou a modificação do processo, ou a intervenção na fonte do risco (equipamentos de proteção coletiva – EPCs) são mais eficazes que os EPIs, que estabelecem barreiras de exposição sem redução da emissão (SANTOS et al., 2004). A alteração no processo de produção com a introdução da umi- dificação descrita no artigo Características da exposição ocupacional a poeiras em marmorarias da cidade de São Paulo é um exemplo. Aliás, Ramazzini, há mais de trezentos anos, já relatava a adoção de EPC em minas subter- râneas – um sistema de ventilação exaustora – quando descreveu a doença dos mineiros (RAMAZZINI, 1999). No universo da Saúde do Trabalhador, entende-se como fonte de emissão um equipamento ou uma situação dentro do processo de produção no local onde o trabalhador executa suas atividades, geralmente demarcada pelos muros das plantas industriais. Para a área ambiental, acrescem-se como fontes geradoras de agentes poluidores os produtos finais e secundários e os resíduos gerados no processo. Assim, tanto o trabalhador quanto a população geral sofrem os efeitos do processo de produção num ambiente transformado pela produção no entorno da fonte 7 Rev. bras. Saúde ocup., São Paulo, 32 (116): 06-10, 2007 ou como consumidores dos produtos gerados nesse processo. Portanto, as questões do ambiente saudável estão intimamente ligadas às da Saúde do Trabalhador. No artigo Acidente químico com dióxido de enxofre em um populoso distrito de uma grande metrópole, os autores estudaram um acidente ambiental a partir de um modelo matemático de dispersão e propuseram alguns cenários possíveis de exposição da população do entorno. Rev. bras. Saúde ocup., São Paulo, 32 (116): 06-10, 2007 Exposição a agentes químicos e a Saúde do Trabalhador Exposure to chemicals and the Workers’ Health O pintor não estaria exposto somente às condições avaliadas, também deve ter sido exposto em seus outros ambientes de trabalho, que podem ser ainda mais precários por serem trabalhos temporários e sem normas contratuais que incluam questões de proteção à saúde dos trabalhadores. Rev. bras. Saúde ocup., São Paulo, 32 (116): 06-10, 2007 8 As investigações das causas que levam o trabalhador a assumir uma segunda atividade, transpõe o âmbito dos fatores de risco imediatamente ligados ao ambiente de trabalho e passa-se para o nível de fatores sociais e eco- nômicos do grupo social ao qual esses trabalhadores pertencem. Por meio de um modelo multicausal, é possível estudar estes fatores em seus diferentes níveis de proximidade em relação ao trabalhador. Para reforçar o fato de que a complexidade da Saúde do Trabalhador é inerente à realidade do ser humano e que, para a compreensão e gestão do risco ocupacional de forma integral, somente a avaliação da exposição a um determinado agente químico não é suficiente, este volume da RBSO inclui a resenha do livro A desmedida do capital, da socióloga francesa Danièle Linhart. A coletânea é formada por diversos artigos que versam sobre as transformações na organização e na gestão do trabalho na empresa moderna, analisando os modelos organi- zacionais na França nos últimos 40 anos. A autora da resenha remete-nos à realidade brasileira, questionando os paralelos desta com a experiência francesa analisada. O conjunto dos trabalhos aqui apresentados traz à tona um questionamento fundamental: o que realmente se alcançou na Saúde do Trabalhador no tocante à exposição a agentes químicos? A comunicação dos riscos e de medidas de proteção, já levantada como necessidade por Ramazzini, ainda não atinge todos os trabalhadores. Ainda se confere um valor excessivo às medidas individuais de proteção, que não condizem com o seu papel efetivo na garantia da proteção integral à saúde do trabalhador. Por outro lado, as medidas de proteção coletiva, algumas já sugeridas e utilizadas há mais de trezentos anos, ainda são recebidas com relutância por parte daque- les que podem modificar o processo de produção. Os trabalhadores ainda executam suas atividades em condições precárias, não apenas no que diz respeito à segurança e à higiene do trabalho, mas também no que tange às ga- rantias de um trabalho digno e decente. Convidamos a todos a refletir sobre esses aspectos com a leitura dos textos aqui publicados. Referências D’AMATO, C.; TORRES, J. P. M.; MALM, O. DDT (dicloro difenil tricloroetano): toxicidade e contaminação ambiental - uma revisão. Quím. Nova, São Paulo, v. 25, n. 6, p. 995-1002, 2002. DECC. Department of Environment and Climate Change. New South Wales Government. Contaminated Agricultural Land. Australia, 2008. Available from: http://www.environment.nsw.gov.au/mao/ contaminatedagriland.htm. Access: 06 mar. 2008. EPA. Environmental Protection Agency. Lead arsenate EPA Pesticide Fact Sheet 12/86. EPA, 1986. Available from: http://pmep.cce.cornell.edu/profiles/insect-mite/fenitrothion-methylpara/lead-arsenate/insect-prof-leadars. html. Access: 06 mar. 2008. FRANCO, T.; KATO, M. Os riscos ambientais e os indivíduos. In: FRANCO T. (org.) Trabalho, riscos industriais e meio ambiente: rumo ao desenvolvimento sustentável? Salvador: EDUFBA – CRh FFCH UFBA, 1997. p. 215-242. GARCIA, E. G. Segurança e saúde no trabalho rural: a questão dos agrotóxicos. São Paulo: Fundac ILO. International Labor Office. Safe work and safety culture. The ILO report for word day for safety and health at work 2004. ILO, 2004. Available from: http://www.ilo.org/public/english/protection/safework/worldday/ products04/report04_eng.pdf . Access: 07 abr. 2008. LEAR, L. Introduction. In: CARSON, R. Silent Spring. New York: Mariner Books / Houghton Mifflin Co., 2002 p. X-XIX. MPAS. Ministério da Previdência Social. Informações estatísticas de acidentes do trabalho – Bases de dados do Anuário Estatístico de Acidentes de Trabalho, edição 2008 – Disponível em: http://creme.dataprev.gov.br/AEAT/ Inicio.htm. Acesso em: 06 mar. 2008. OSHA. Occupational Safety & Health Administration. Office of Training and Education - Industrial Hygiene. Available from: http://www.osha.gov/dcsp/ote/library/industrial_hygiene/industrial_hygiene.html. Access: 06 mar. 2008. PAPP, E. M.; EIJKEMANS, G.; VICKERS C. Reducing worker exposure by using the occupational risk management toolbox. GOHNET News letter, n. 7, 1-3, Summer, 2004. Available from: http://www.who.int/ occupational_health/publications/newsletter/gohnet7e.pdf. Access: 20 mar. 2008. PUGAS, E. et al. Risco ocupacional: Caracterização básica, avaliação qualitativa e priorização. Rev. bras. Saúde ocup., São Paulo, v. 26, n. 99/100, p. 9-30, 2001. RAMAZZINI, B. As doenças dos trabalhadores. Trad. Raimundo Estrela. 2.ed. São Paulo: Fundacentro, 1999. 9 Rev. bras. Saúde ocup., São Paulo, 32 (116): 06-10, 2007 RAMOS, A.; JARDIM, S. R.; SILVA FILHO, J. F. Solvent-related chronic toxic encephalopathy as a target in the worker’s mental health research. An. Acad. Bras. Ciênc., v. 76, n. 4, p.757-769, 2004. SANTOS, A. M. A. et al. Introdução à Higiene Ocupacional. São Paulo: Fundacentro, 2004. SINITOX. Sistema Nacional de Informações Tóxico-Farmacologicas. Ministério da Saúde. Casos registrados de intoxicação humana e envenenamento. Brasil, 2005. Uma análise. Disponível em: http://www.fiocruz.br/ sinitox/2005/umanalise2005.htm. Acesso em: 06 mar. 2008. VIEIRA, V. M.; LAPA, R. Riscos em laboratório: prevenção e controle. Rev. bras. Saúde ocup., São Paulo, 32 (116): 06-10, 2007 Referências Cadernos de Estudos Avançados, Rio de Janeiro, v. 3, n. 1, p. 25-43, 2006. WHO. Word Health Organization. The world health report 2002. Reducing risks, promoting health WHO. Word Health Organization. The world health report 2002. Reducing risks, promoting healthy life. WHO, 2002. Available from: http://www.who.int/whr/2002/en/. Access: 07 abr. 2008. 2002. Available from: http://www.who.int/whr/2002/en/. Access: 07 abr. 2008. 10 10
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ERROR: type should be string, got "https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. Correspondence: Maurus Borne (maurus.borne@kit.edu) Correspondence: Maurus Borne (maurus.borne@kit.edu) Abstract. Since its launch by the European Space Agency in 2018, the Aeolus satellite has been using the first Doppler\nwind lidar in space to acquire three-dimensional atmospheric wind profiles around the globe. Especially in the tropics, these\nmeasurements compensate for the currently limited number of other wind observations, making an assessment of the quality of\nAeolus wind products in this region crucial for numerical weather prediction. To evaluate the quality of the Aeolus L2B wind products across the tropical Atlantic Ocean, 20 radiosondes corresponding to Aeolus overpasses were launched from the islands\n5\nof Sal, Saint Croix and Puerto Rico during August-September 2021 as part of the Joint Aeolus Tropical Atlantic Campaign. During this period, Aeolus sampled winds within a complex environment with a variety of cloud types in the vicinity of the\nInter-tropical Convergence Zone and aerosol particles from Saharan dust outbreaks. On average, the validation for Aeolus\nRaleigh-clear revealed a random error of 3.8 – 4.3 ms–1 between 2–16 km and 4.3 – 4.8 ms–1 between 16–20 km, with a 5 systematic error of -0.5±0.2 ms–1. For Mie-cloudy, the random error between 2–16 km is 1.1 – 2.3 ms–1 and the systematic\n10\nerror is -0.9 ±0.3 ms–1. Below clouds or within dust layers, the quality of Rayleigh-clear measurements can be degraded when\nthe useful signal is reduced. In these conditions, we also noticed an underestimation of the L2B estimated error. Gross outliers\nwhich we define with large deviations from the radiosonde but low error estimates account for less than 5% of the data. These\noutliers appear at all altitudes and under all environmental conditions; however, their root-cause remains unknown. Finally, systematic error of -0.5±0.2 ms–1. For Mie-cloudy, the random error between 2–16 km is 1.1 – 2.3 ms–1 and the systematic\n10\nerror is -0.9 ±0.3 ms–1. Below clouds or within dust layers, the quality of Rayleigh-clear measurements can be degraded when\nthe useful signal is reduced. In these conditions, we also noticed an underestimation of the L2B estimated error. Gross outliers\nwhich we define with large deviations from the radiosonde but low error estimates account for less than 5% of the data. These\noutliers appear at all altitudes and under all environmental conditions; however, their root-cause remains unknown. Validation of Aeolus L2B products over the tropical Atlantic using\nradiosondes Maurus Borne 1, Peter Knippertz 1, Martin Weissmann 2, Benjamin Witschas 3, Cyrille Flamant 4,\nRosimar Rios-Berrios 5, and Peter Veals 6 1Institute of Meteorology and Climate Research, Karlsruhe Institute of Technology (KIT), 76131 Karlsruhe, Germany\n2Universität Wien, Institut für Meteorologie und Geophysik, Wien, Austria g\np y\n3Deutsches Zentrum für Luft- und Raumfahrt e.V. (DLR), Institut für Physik der Atmosphäre, 82234 Oberpfaffenhofen,\nGermany\n4 3Deutsches Zentrum für Luft- und Raumfahrt e.V. (DLR), Institut für Physik der Atmosphäre, 82234 Oberpfaffenhofen,\nGermany 4Laboratoire Atmosphères, Milieux, Observations Spatiales (LATMOS), UMR 8190, CNRS, Sorbonne Université and\nUniversité Paris Saclay, Paris, France\n5N i\nl C\nf\nA\nh i R\nh B\nld\nCO USA 4Laboratoire Atmosphères, Milieux, Observations Spatiales (LATMOS), UMR 8190, CNRS, Sorbonne Université and\nUniversité Paris Saclay, Paris, France\n5 5National Center for Atmospheric Research, Boulder, CO, USA 5National Center for Atmospheric Research, Boulder, CO, USA\n6 p\n6Department of Atmospheric Sciences, University of Utah, Salt Lake City, Utah, USA 6Department of Atmospheric Sciences, University of Utah, Salt Lake City, Utah, USA https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. 1\nIntroduction In situ measurements derived from aircraft reports, ground stations or radiosondes\nare not globally distributed and lead to a lack of observations in the aforementioned regions. To address these deficiencies, the European Space Agency (ESA) deployed the Atmospheric Dynamics Mission Aeolus in To address these deficiencies, the European Space Agency (ESA) deployed the Atmospheric Dynamics Mission Aeolus in\n2018, the first satellite capable of measuring atmospheric winds around the globe from space with a homogeneous space-time\n30\nwind coverage and altitude-resolved profiles up to 30 km height (Reitebuch, 2012). The instrument carries a direct detection\nDoppler wind lidar called ALADIN (Atmospheric LAser Doppler INstrument) that emits short ultraviolet (UV) pulses at 355\nnm along the Line Of Sight (LOS) of the instrument. The Doppler shift of the backscatter signal is detected by a dual-channel\nreceiver consisting of the following elements: a Fizeau interferometer analysing the Doppler shift of the narrowband particle 2018, the first satellite capable of measuring atmospheric winds around the globe from space with a homogeneous space-time\n30\nwind coverage and altitude-resolved profiles up to 30 km height (Reitebuch, 2012). The instrument carries a direct detection\nDoppler wind lidar called ALADIN (Atmospheric LAser Doppler INstrument) that emits short ultraviolet (UV) pulses at 355\nnm along the Line Of Sight (LOS) of the instrument. The Doppler shift of the backscatter signal is detected by a dual-channel\nreceiver consisting of the following elements: a Fizeau interferometer analysing the Doppler shift of the narrowband particle backscatter signal (cloud droplets and aerosols or ice crystals) using the fringe imaging technique (McKay, 2002), referred to\n35\nas the Mie channel, and a dual Fabry-Pérot interferometer detecting the Doppler-shifted frequency of the Rayleigh-Brillouin\nbackscatter spectrum (air molecules) using the double-edge technique (Flesia and Korb, 1999), called the Rayleigh channel. The processing algorithm also distinguishes between retrievals originating from \"cloudy\" or \"clear\" atmospheric conditions,\nresulting in Rayleigh-clear and Mie-cloudy observation types. The two channels complement each other, as Mie-cloudy winds backscatter signal (cloud droplets and aerosols or ice crystals) using the fringe imaging technique (McKay, 2002), referred to\n35\nas the Mie channel, and a dual Fabry-Pérot interferometer detecting the Doppler-shifted frequency of the Rayleigh-Brillouin\nbackscatter spectrum (air molecules) using the double-edge technique (Flesia and Korb, 1999), called the Rayleigh channel. The processing algorithm also distinguishes between retrievals originating from \"cloudy\" or \"clear\" atmospheric conditions,\nresulting in Rayleigh-clear and Mie-cloudy observation types. Correspondence: Maurus Borne (maurus.borne@kit.edu) Finally, we confirm the presence of an orbital-dependent bias of up to 2.5 ms–1 observed with both radiosondes and European Centre\n15\nfor Medium-Range Weather Forecasts model equivalents. The results of this study contribute to a better characterization of\nthe Aeolus wind product in different atmospheric conditions and provide valuable information for further improvement of the\nwind retrieval algorithm. we confirm the presence of an orbital-dependent bias of up to 2.5 ms–1 observed with both radiosondes and European Centre\n15\nfor Medium-Range Weather Forecasts model equivalents. The results of this study contribute to a better characterization of\nthe Aeolus wind product in different atmospheric conditions and provide valuable information for further improvement of the\nwind retrieval algorithm. 1 1 1\nIntroduction According to the World Meteorological Organisation (WMO), wind is the most critical atmospheric variable lacking in the\n20\ncurrent Global Observing System (GOS) (Baker et al., 2014). Especially in the Southern Hemisphere (SH), over the oceans\nand near equatorial regions, numerical weather prediction (NWP) models require additional wind observations with sufficient\ncoverage in time and space to identify key atmospheric dynamics (Stoffelen et al., 2005; Straume et al., 2020). Before the\nlaunch of Aeolus in 2018, satellite wind observations in these regions were only available for a limited number of tropospheric According to the World Meteorological Organisation (WMO), wind is the most critical atmospheric variable lacking in the\n20\ncurrent Global Observing System (GOS) (Baker et al., 2014). Especially in the Southern Hemisphere (SH), over the oceans\nand near equatorial regions, numerical weather prediction (NWP) models require additional wind observations with sufficient\ncoverage in time and space to identify key atmospheric dynamics (Stoffelen et al., 2005; Straume et al., 2020). Before the\nlaunch of Aeolus in 2018, satellite wind observations in these regions were only available for a limited number of tropospheric layers and were mainly provided by atmospheric motion vectors (AMVs) estimated from tracking cloud and water vapour\n25\nfeatures (Bormann et al., 2003; Folger and Weissmann, 2014), or by scatterometer measurements of surface winds (Naderi\net al., 1991; Portabella and Stoffelen, 2009). In situ measurements derived from aircraft reports, ground stations or radiosondes\nare not globally distributed and lead to a lack of observations in the aforementioned regions. dd\nh\nd fi i\ni\nh\nd\nl\nd h\nh i\ni\ni i\nl\ni layers and were mainly provided by atmospheric motion vectors (AMVs) estimated from tracking cloud and water vapour\n25\nfeatures (Bormann et al., 2003; Folger and Weissmann, 2014), or by scatterometer measurements of surface winds (Naderi\net al., 1991; Portabella and Stoffelen, 2009). In situ measurements derived from aircraft reports, ground stations or radiosondes\nare not globally distributed and lead to a lack of observations in the aforementioned regions. To address these deficiencies the European Space Agency (ESA) deployed the Atmospheric Dynamics Mission Aeolus in layers and were mainly provided by atmospheric motion vectors (AMVs) estimated from tracking cloud and water vapour\n25\nfeatures (Bormann et al., 2003; Folger and Weissmann, 2014), or by scatterometer measurements of surface winds (Naderi\net al., 1991; Portabella and Stoffelen, 2009). 1\nIntroduction The two channels complement each other, as Mie-cloudy winds can compensate for gaps in Rayleigh-clear measurements, especially in cloudy and aerosol-loaded regions. Various NWP\n40\ncentres have demonstrated the added value of assimilating Aeolus winds through significant improvements in model fields\nand model background information, especially in tropical regions, the upper tropical troposphere and the lower stratosphere\n(Rennie et al., 2021; Martin et al., 2022a, b; Garrett et al., 2022). can compensate for gaps in Rayleigh-clear measurements, especially in cloudy and aerosol-loaded regions. Various NWP\n40\ncentres have demonstrated the added value of assimilating Aeolus winds through significant improvements in model fields\nand model background information, especially in tropical regions, the upper tropical troposphere and the lower stratosphere\n(Rennie et al., 2021; Martin et al., 2022a, b; Garrett et al., 2022). For an optimal use of the Aeolus wind observations in NWP models, an assessment of the data quality is essential. To achieve\nthis, several scientific and technical studies are carried out in the framework of Calibration/Validation (Cal/Val) activities\n45\norganised by ESA. For wind validation, several reference products have been used such as ground-based remote sensing\nobservations (Belova et al., 2021; Guo et al., 2021; Iwai et al., 2021; Abril-Gago et al., 2022), in situ measurements (Baars\net al., 2020; Chen et al., 2021; Ratynski et al., 2022), airborne measurements (Lux et al., 2020; Witschas et al., 2020; Bedka\net al., 2021; Witschas et al., 2022) or NWP model equivalents (Martin et al., 2021; Zuo et al., 2022). For an optimal use of the Aeolus wind observations in NWP models, an assessment of the data quality is essential. To achieve\nthis, several scientific and technical studies are carried out in the framework of Calibration/Validation (Cal/Val) activities\n45\norganised by ESA. For wind validation, several reference products have been used such as ground-based remote sensing\nobservations (Belova et al., 2021; Guo et al., 2021; Iwai et al., 2021; Abril-Gago et al., 2022), in situ measurements (Baars\net al., 2020; Chen et al., 2021; Ratynski et al., 2022), airborne measurements (Lux et al., 2020; Witschas et al., 2020; Bedka\net al., 2021; Witschas et al., 2022) or NWP model equivalents (Martin et al., 2021; Zuo et al., 2022). this, several scientific and technical studies are carried out in the framework of Calibration/Validation (Cal/Val) activities\n45\norganised by ESA. The contribution\n70\nof the radiosondes in JATAC is complementary to other instruments as they provide accurate wind measurements throughout\nthe troposphere up to the lower stratosphere, which is not probed by many other instruments and provides an almost unique\ndata set for validating the Aeolus winds at this altitude. This article is structured as follows: Section 2 describes the instruments and data while section 3 details the quality con the warm and moist Caribbean, where heavy rainfall events and tropical cyclones frequently affect the area. The contribution\n70\nof the radiosondes in JATAC is complementary to other instruments as they provide accurate wind measurements throughout\nthe troposphere up to the lower stratosphere, which is not probed by many other instruments and provides an almost unique\ndata set for validating the Aeolus winds at this altitude. This article is structured as follows: Section 2 describes the instruments and data while section 3 details the quality con-\ntrol and co-location criteria used for the validation study. Section 4 deals with the quantification of errors, their dependency\n75\non temporal and spatial distance between the compared observations as well as on the presence of clouds and dust. For this\npurpose, we use the Satellite Application Facility for supporting NoWCasting and very short range forecasting (SAFNWC,\nAlonso Lasheras et al. (2005)) satellite-based meteorological Cloud Type (CT) product and the Copernicus Atmosphere Mon-\nitoring Service (CAMS) dust mixing ratio reanalysis. Furthermore, the section includes a case study illustrating the different\nbehaviour of Rayleigh-clear and Mie-cloudy winds under different environmental conditions Finally, section 5 summarises\n80 trol and co-location criteria used for the validation study. Section 4 deals with the quantification of errors, their dependency\n75\non temporal and spatial distance between the compared observations as well as on the presence of clouds and dust. For this\npurpose, we use the Satellite Application Facility for supporting NoWCasting and very short range forecasting (SAFNWC,\nAlonso Lasheras et al. (2005)) satellite-based meteorological Cloud Type (CT) product and the Copernicus Atmosphere Mon-\nitoring Service (CAMS) dust mixing ratio reanalysis. Furthermore, the section includes a case study illustrating the different behaviour of Rayleigh-clear and Mie-cloudy winds under different environmental conditions. Finally, section 5 summarises\n80\nthe main results and provides recommendations for improving the Aeolus wind retrieval algorithm. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. lies\" (Weiler et al., 2021a), which reduced the systematic and random errors in the Rayleigh channel. One phenomenon that\nstill needs to be explored is the sensitivity of Aeolus wind quality to the presence of aerosols and clouds, potentially affecting lies\" (Weiler et al., 2021a), which reduced the systematic and random errors in the Rayleigh channel. One phenomenon that\nstill needs to be explored is the sensitivity of Aeolus wind quality to the presence of aerosols and clouds, potentially affecting\nkey parameters such as the signal levels or scattering ratio (SR) used to calculate the Horizontal Line of Sight (HLOS) winds\n55\nand the associated error estimate (EE). The tropical Atlantic during the boreal summer, spanning from West Africa to the\nCaribbean, is the ideal place to explore these dependencies, with a wide range of atmospheric aerosols (Saharan dust aerosols,\nsea salt aerosols, biomass combustion aerosols) and convective cloud types associated with the West African Monsoon (WAM) lies\" (Weiler et al., 2021a), which reduced the systematic and random errors in the Rayleigh channel. One phenomenon that\nstill needs to be explored is the sensitivity of Aeolus wind quality to the presence of aerosols and clouds, potentially affecting key parameters such as the signal levels or scattering ratio (SR) used to calculate the Horizontal Line of Sight (HLOS) winds\n55\nand the associated error estimate (EE). The tropical Atlantic during the boreal summer, spanning from West Africa to the\nCaribbean, is the ideal place to explore these dependencies, with a wide range of atmospheric aerosols (Saharan dust aerosols,\nsea salt aerosols, biomass combustion aerosols) and convective cloud types associated with the West African Monsoon (WAM)\ncirculation and the Inter Tropical Convergence Zone (ITCZ). key parameters such as the signal levels or scattering ratio (SR) used to calculate the Horizontal Line of Sight (HLOS) winds\n55\nand the associated error estimate (EE). The tropical Atlantic during the boreal summer, spanning from West Africa to the\nCaribbean, is the ideal place to explore these dependencies, with a wide range of atmospheric aerosols (Saharan dust aerosols,\nsea salt aerosols, biomass combustion aerosols) and convective cloud types associated with the West African Monsoon (WAM)\ncirculation and the Inter Tropical Convergence Zone (ITCZ). 1\nIntroduction For wind validation, several reference products have been used such as ground-based remote sensing\nobservations (Belova et al., 2021; Guo et al., 2021; Iwai et al., 2021; Abril-Gago et al., 2022), in situ measurements (Baars\net al., 2020; Chen et al., 2021; Ratynski et al., 2022), airborne measurements (Lux et al., 2020; Witschas et al., 2020; Bedka\net al., 2021; Witschas et al., 2022) or NWP model equivalents (Martin et al., 2021; Zuo et al., 2022). Several anomalies in the Aeolus data have already been detected and improvements in the processing chain and the in-\n50\nstrument have been made accordingly. These include the implementation of a bias correction in both channels related to the\norbital-dependent temperature variations of ALADIN’s M1 mirror (Weiler et al., 2021b) and the correction of \"pixel anoma- Several anomalies in the Aeolus data have already been detected and improvements in the processing chain and the in-\n50\nstrument have been made accordingly. These include the implementation of a bias correction in both channels related to the\norbital-dependent temperature variations of ALADIN’s M1 mirror (Weiler et al., 2021b) and the correction of \"pixel anoma- Several anomalies in the Aeolus data have already been detected and improvements in the processing chain and the in-\n50\nstrument have been made accordingly. These include the implementation of a bias correction in both channels related to the\norbital-dependent temperature variations of ALADIN’s M1 mirror (Weiler et al., 2021b) and the correction of \"pixel anoma- 2 For this purpose, ESA organized the Joint Aeolus Tropical Atlantic Campaign (JATAC) in the period July to September\n60\n2021, which deployed sophisticated airborne lidar instruments over Cabo Verde (German Aerospace Center (DLR), Labo-\nratoire ATmosphères, Milieux, Observations Spatiales (LATMOS)) and the Virgin Islands (National Aeronautics and Space\nAdministration (NASA)) but also ground-based instruments such as radiosondes (Karlsruhe Institute of Technology (KIT),\nUniversity of Oklahoma, University of Utah) and Doppler lidar systems (Leibniz Institute for Tropospheric Research (TRO- For this purpose, ESA organized the Joint Aeolus Tropical Atlantic Campaign (JATAC) in the period July to September\n60\n2021, which deployed sophisticated airborne lidar instruments over Cabo Verde (German Aerospace Center (DLR), Labo-\nratoire ATmosphères, Milieux, Observations Spatiales (LATMOS)) and the Virgin Islands (National Aeronautics and Space\nAdministration (NASA)) but also ground-based instruments such as radiosondes (Karlsruhe Institute of Technology (KIT),\nUniversity of Oklahoma, University of Utah) and Doppler lidar systems (Leibniz Institute for Tropospheric Research (TRO- POS), National Observatory of Athens (NOA)). In this study, we validate Aeolus wind products using radiosondes launched\n65\nfrom western Puerto Rico, northern St. Croix and Sal airport on Cabo Verde. The semi-arid island of Sal is located over the\ntropical East Atlantic off the West African coast, near the northern boundary of the WAM. Rain events are relatively sporadic\nthere, as most synoptic and mesoscale precipitation systems propagate south of the island. The region is exposed to mineral\ndust plumes emanating from Saharan dust outbreaks. In contrast, the islands of St. Croix and Puerto Rico are located within POS), National Observatory of Athens (NOA)). In this study, we validate Aeolus wind products using radiosondes launched\n65\nfrom western Puerto Rico, northern St. Croix and Sal airport on Cabo Verde. The semi-arid island of Sal is located over the\ntropical East Atlantic off the West African coast, near the northern boundary of the WAM. Rain events are relatively sporadic\nthere, as most synoptic and mesoscale precipitation systems propagate south of the island. The region is exposed to mineral\ndust plumes emanating from Saharan dust outbreaks. In contrast, the islands of St. Croix and Puerto Rico are located within the warm and moist Caribbean, where heavy rainfall events and tropical cyclones frequently affect the area. 2.1\nALADIN and Aeolus wind products Aeolus is the second Earth Explorer Core mission and measures global atmospheric wind profiles from a 320 km high sun-\nsynchronous dusk-dawn orbit. It carries the ALADIN instrument (Schillinger et al. (2003)), which is a direct-detection high-\n85 85 3 3 The L1B product comprises the geolocated and observation data as well as optical information (SNR, useful signal, scattering ratio , etc.). The wind product called L2B contains the final horizontal projection of the LOS wind speed profiles of\n105\nthe Rayleigh and Mie channels, where all necessary calibration and instrument corrections have been performed (Dabas et al.,\n2008). This product is suitable for the assimilation in NWP models and scientific research. The L2B product also provides scene\nclassification based upon the backscatter ratio corresponding to the wind originating from a ’cloudy’ or ’clear’ atmospheric\nregion, resulting in Rayleigh-clear, Rayleigh-cloudy, Mie-clear and Mie-cloudy observation types. Throughout the processing scattering ratio , etc.). The wind product called L2B contains the final horizontal projection of the LOS wind speed profiles of\n105\nthe Rayleigh and Mie channels, where all necessary calibration and instrument corrections have been performed (Dabas et al.,\n2008). This product is suitable for the assimilation in NWP models and scientific research. The L2B product also provides scene\nclassification based upon the backscatter ratio corresponding to the wind originating from a ’cloudy’ or ’clear’ atmospheric\nregion, resulting in Rayleigh-clear, Rayleigh-cloudy, Mie-clear and Mie-cloudy observation types. Throughout the processing\nchain, the L1B and L2B processors are continuously updated into different baseline versions to account for revisions and\n110 chain, the L1B and L2B processors are continuously updated into different baseline versions to account for revisions and\n110\nidentified problems. This leads to different HLOS wind observations and quality in different time periods. In this study, data from the near-real-time version Baseline product 12 (L2bP 3.50) are used. We evaluate all observation\ntypes and corresponding Error Estimates (EEs) of the L2B product except Mie-clear observations as the Mie signal should is\nusually weak in clear sky conditions. Additionally, two L1B products are used, namely the scattering ratio (SR) and the useful chain, the L1B and L2B processors are continuously updated into different baseline versions to account for revisions and\n110\nidentified problems. This leads to different HLOS wind observations and quality in different time periods. In this study, data from the near-real-time version Baseline product 12 (L2bP 3.50) are used. We evaluate all observation\ntypes and corresponding Error Estimates (EEs) of the L2B product except Mie-clear observations as the Mie signal should is\nusually weak in clear sky conditions. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. spectral-resolution wind lidar with a Nd:YAG laser transmitter that operates at an ultraviolet wavelength of 354.8 nm. It points\nat 35 ◦with and angle of ∼10° from the zonal direction. ALADIN consists of a two-channel receiver that allows the instrument to measure wind speed from molecular backscatter\n(Rayleigh channel) and particle backscatter (Mie channel). The Rayleigh channel relies on the double-edge technique (Flesia\nand Korb, 1999) using a sequential Fabry-Perot interferometer, where the Doppler shift of the backscattered molecular spectrum\n90\nis retrieved from the signal intensities that are transmitted through two band-pass filters A and B. The final Rayleigh response\nis computed from a contrast-function between both filter signals. For Mie winds, the computation is based on a fringe-imaging\ntechnique (McKay, 2002), in which the Fizeau interferometer forms a linear interference fringe on the detector from the\nnarrowband particle backscatter signal. The lateral displacement of the interference fringe is then used to calculate the Doppler 90 shift. 95\nTo ensure a sufficient Signal-to-Noise Ratio (SNR), the wind measurements are averaged vertically and horizontally into\nsingle observations. Vertical sampling is performed within 24 vertical elevation bins with a resolution that can vary from\n0.25 km at lower elevations to 2 km at higher elevations. They are defined by the Range Bin Settings (RBS) and can vary\ngeographically and between the respective detection channel (Rayleigh and Mie). Horizontally, the measurements are averaged shift. 95\nTo ensure a sufficient Signal-to-Noise Ratio (SNR), the wind measurements are averaged vertically and horizontally into\nsingle observations. Vertical sampling is performed within 24 vertical elevation bins with a resolution that can vary from\n0.25 km at lower elevations to 2 km at higher elevations. They are defined by the Range Bin Settings (RBS) and can vary\ngeographically and between the respective detection channel (Rayleigh and Mie). Horizontally, the measurements are averaged over 87 km and 10 km integration lengths for Rayleigh and Mie channels, respectively, owing to the lower signal levels of the\n100\nRayleigh measurements. The data products are processed through a multi-stage processing chain, with each level containing different information\n(Reitebuch et al., 2018; Tan et al., 2008). In this study, the Level1B (L1B) and Level2B (L2B) products are of particular\ninterest. Additionally, two L1B products are used, namely the scattering ratio (SR) and the useful signal. The SR represents the ratio between the total (molecular and particulate) and the molecular backscatter coefficients. 115\nIt is strictly equal to or greater than one and describes the contribution of the particles to the backscattered signal. Note that\nthe SRs of the L2B products are not used, as some SRs were manually set to one during the processor baseline to eliminate a\ncross-talk correction, which had detrimental effects on the wind quality. The useful signal represents the returned signal levels\nper observation and comprises corrections for the solar background, the dark current and the detection chain offset (DCO). signal. The SR represents the ratio between the total (molecular and particulate) and the molecular backscatter coefficients. 115\nIt is strictly equal to or greater than one and describes the contribution of the particles to the backscattered signal. Note that\nthe SRs of the L2B products are not used, as some SRs were manually set to one during the processor baseline to eliminate a\ncross-talk correction, which had detrimental effects on the wind quality. The useful signal represents the returned signal levels\nper observation and comprises corrections for the solar background, the dark current and the detection chain offset (DCO). signal. The SR represents the ratio between the total (molecular and particulate) and the molecular backscatter coefficients. 115\nIt is strictly equal to or greater than one and describes the contribution of the particles to the backscattered signal. Note that\nthe SRs of the L2B products are not used, as some SRs were manually set to one during the processor baseline to eliminate a\ncross-talk correction, which had detrimental effects on the wind quality. The useful signal represents the returned signal levels\nper observation and comprises corrections for the solar background, the dark current and the detection chain offset (DCO). We apply an additional range correction and signal normalization that takes into account the different range bin thickness and\n120 4 4 At the end of each assimilation cycle, the feedback files with the Aeolus winds\nand their model equivalents can be retrieved from the Meteorological Archival and Retrieval System (MARS). These reports\ncontain information on the assimilated observations, their model background (short-range forecast) and analysis equivalents as\nwell as various quality control flags.. In this study, background equivalents of Aeolus observations are used as an additional\nreference to validate Aeolus HLOS winds. Note that only Rayleigh-clear and the Mie-cloudy winds are in operational use for\n130\nNWP. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. distances between the instruments and the height bins. Due to the sequential implementation of the Fizeau and the Fabry-Perot\ninterometers, signal from Mie scattering can leak into the Rayleigh channel signal. This optical \"cross-talk\" can cause biases,\nespecially in the case of strong Mie returns, as the Rayleigh-channel assumes pure molecular signal in the processing chain. distances between the instruments and the height bins. Due to the sequential implementation of the Fizeau and the Fabry-Perot\ninterometers, signal from Mie scattering can leak into the Rayleigh channel signal. This optical \"cross-talk\" can cause biases,\nespecially in the case of strong Mie returns, as the Rayleigh-channel assumes pure molecular signal in the processing chain. Along with many other NWP centers, the data were assimilated in the European Centre for Medium-Range Weather Fore-\ncasts (ECMWF) Integrated Forecasting System (IFS) by means of the operational four-dimensional ensemble-variational (4D-\n125\nEnVar) data assimilation scheme (4D-EnVar). At the end of each assimilation cycle, the feedback files with the Aeolus winds\nand their model equivalents can be retrieved from the Meteorological Archival and Retrieval System (MARS). These reports\ncontain information on the assimilated observations, their model background (short-range forecast) and analysis equivalents as\nwell as various quality control flags.. In this study, background equivalents of Aeolus observations are used as an additional\nreference to validate Aeolus HLOS winds. Note that only Rayleigh-clear and the Mie-cloudy winds are in operational use for\n130\nNWP. distances between the instruments and the height bins. Due to the sequential implementation of the Fizeau and the Fabry-Perot\ninterometers, signal from Mie scattering can leak into the Rayleigh channel signal. This optical \"cross-talk\" can cause biases,\nespecially in the case of strong Mie returns, as the Rayleigh-channel assumes pure molecular signal in the processing chain. interometers, signal from Mie scattering can leak into the Rayleigh channel signal. This optical \"cross-talk\" can cause biases,\nespecially in the case of strong Mie returns, as the Rayleigh-channel assumes pure molecular signal in the processing chain. Along with many other NWP centers, the data were assimilated in the European Centre for Medium-Range Weather Fore-\ncasts (ECMWF) Integrated Forecasting System (IFS) by means of the operational four-dimensional ensemble-variational (4D-\n125\nEnVar) data assimilation scheme (4D-EnVar). different research components of JATAC. Between the 7 and 28th of September 2021, a total of 37 radiosondes were launched\nfrom Sal airport in Cape Verde, 9 of them corresponding to Aeolus overflights. The launches were coordinated by the Karlsruhe\n135\nInstitute of Technology (KIT) with local support from the JATAC team. This was accomplished using the DFM-09 (GRAW)\nlight weather radiosondes, which measure air pressure, air temperature, relative humidity, wind speed and wind direction. The\nvertical resolution depends on the ascent speed, which varies with the amount of helium in the balloon, but can generally be\nestimated at about 5 ms–1. Most of the radiosondes launched at Sal were ingested into the Global Telecommunication System\n(GTS).\n140 As for Sal, these\n145\nmeasurements were performed with the radiosonde instrument DFM-09 (GRAW). Lastly, 32 launches were conducted from\nthe University of Puerto Rico at Mayagüez (UPRM) campus between 26 August and 14 September 2021, 7 of which could be\nused for the validation of Aeolus. All launches were performed with iMet-4 radiosondes from the International Met System. As with DFM-09, the iMet-4 radiosondes provide measurements of wind speed, wind direction, temperature, humidity and air pressure. The radiosonde data also underwent a quality control check using the Atmospheric Sounding Processing Envi-\n150\nronment (ASPEN) software (Martin and Suhr, 2021) developed by the Earth Observing Laboratory at the National Center for\nAtmospheric Research (NCAR). A summary of the radiosonde launches and weather events sampled at UPRM was provided\nby Rios-Berrios et al. (2023). The total number of radiosonde profiles corresponding to Aeolus overpasses thus amounts to 20, of which 12 correspond to air pressure. The radiosonde data also underwent a quality control check using the Atmospheric Sounding Processing Envi-\n150\nronment (ASPEN) software (Martin and Suhr, 2021) developed by the Earth Observing Laboratory at the National Center for\nAtmospheric Research (NCAR). A summary of the radiosonde launches and weather events sampled at UPRM was provided\nby Rios-Berrios et al. (2023). The total number of radiosonde profiles corresponding to Aeolus overpasses thus amounts to 20 of which 12 correspond to The total number of radiosonde profiles corresponding to Aeolus overpasses thus amounts to 20, of which 12 correspond to\nascending and 8 to descending orbits of Aeolus. An overview of the launches from the different sites can be found in Table 1,\n155\nalong with other co-location parameters fully discussed in Section 3.1. ascending and 8 to descending orbits of Aeolus. An overview of the launches from the different sites can be found in Table 1,\n155\nalong with other co-location parameters fully discussed in Section 3.1. ascending and 8 to descending orbits of Aeolus. An overview of the launches from the different sites can be found in Table 1,\n155\nalong with other co-location parameters fully discussed in Section 3.1. 2.2\nRadiosondes Table 1. Overview of Aeolus overflights and associated radiosonde profiles. Table 1. Overview of Aeolus overflights and associated radiosonde profiles. Week day\nStart and stop time\nOrbit node\nCo-location radius\nNumber of profiles\nSal\nTuesday\n07:28 – 07:29 UTC\nDescending\n50 km\n3\nThursday\n19:23 – 19:24 UTC\nAscending\n180 km\n3\nFriday\n19:36 – 19:37 UTC\nAscending\n280 km\n3\nSaint Croix\nMonday\n10:17 - 10:18 UTC\nDescending\n90 km\n3\nWednesday\n22:12 – 22:13 UTC\nAscending\n160 km\n3\nThursday\n22:25 – 22:26 UTC\nAscending\n340 km\n1\nPuerto Rico\nTuesday\n10:29 – 10:30 UTC\nDescending\n160 km\n2\nThursday\n22:25 – 22:26 UTC\nAscending\n100 km\n2 Week day\nStart and stop time\nOrbit node\nCo-location radius\nNumber of profiles During the campaign, radiosondes were launched from three different locations over the tropical Atlantic and coordinated by\ndifferent research components of JATAC. Between the 7 and 28th of September 2021, a total of 37 radiosondes were launched from Sal airport in Cape Verde, 9 of them corresponding to Aeolus overflights. The launches were coordinated by the Karlsruhe\n135\nInstitute of Technology (KIT) with local support from the JATAC team. This was accomplished using the DFM-09 (GRAW)\nlight weather radiosondes, which measure air pressure, air temperature, relative humidity, wind speed and wind direction. The\nvertical resolution depends on the ascent speed, which varies with the amount of helium in the balloon, but can generally be\nestimated at about 5 ms–1. Most of the radiosondes launched at Sal were ingested into the Global Telecommunication System (GTS). 140 5 5 2.3\nEUMETSAT SAFNWC Cloud type product The Satellite Application Facility for supporting NoWCasting and very short range forecasting (SAFNWC; Alonso Lasheras\net al., 2005) developed a number of satellite-based meteorological products distributed by the European Organisation for the\nExploitation of Meteorological Satellites (EUMETSAT). Among others, they provide the Cloud Type (CT) product (Derrien\n160\nand Le Gléau, 2005), which is a detailed scenery classification of clouds based on different main classes. The Satellite Application Facility for supporting NoWCasting and very short range forecasting (SAFNWC; Alonso Lasheras\net al., 2005) developed a number of satellite-based meteorological products distributed by the European Organisation for the Exploitation of Meteorological Satellites (EUMETSAT). Among others, they provide the Cloud Type (CT) product (Derrien\n160\nand Le Gléau, 2005), which is a detailed scenery classification of clouds based on different main classes. The baseline data originate from the Spinning Enhanced Visible and Infrared Imager (SEVIRI) operated onboard the second\ngeneration METEOSAT geostationary satellites (MSG). Multispectral thresholding techniques (Saunders and Kriebel, 1988;\nDerrien et al., 1993; Stowe et al., 1999) are subsequently applied in the NWCSAF software to process the SEVIRI/MSG images Exploitation of Meteorological Satellites (EUMETSAT). Among others, they provide the Cloud Type (CT) product (Derrien\n160\nand Le Gléau, 2005), which is a detailed scenery classification of clouds based on different main classes. The baseline data originate from the Spinning Enhanced Visible and Infrared Imager (SEVIRI) operated onboard the second\ngeneration METEOSAT geostationary satellites (MSG). Multispectral thresholding techniques (Saunders and Kriebel, 1988;\nDerrien et al., 1993; Stowe et al., 1999) are subsequently applied in the NWCSAF software to process the SEVIRI/MSG images into the various NWC products. The product is available with a temporal resolution of 15 minutes and a nadir spatial resolution\n165\nof 3 km, compared to 11 km at the edge of the field of view. In this study, CT is used to identify the cloud type and cloud cover along the Aeolus tracks and to assess the quality of\nthe Aeolus wind products relative to the presence of clouds. More specifically, we identify the pixels closest to each track of\nAeolus and determine the average percentage of cloud cover at each altitude based on a cloud classification. According to this classification, a measurement bin is considered as cloudy, if it is situated within or below a cloud. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. The radiosondes launched on the Virgin Islands were organised by National Aeronautics and Space Administration (NASA)’s\nConvective Processes Experiment-Aerosols and Winds (CPEX-AW) campaign component of JATAC, with the University of\nUtah conducting the launches on Saint Croix and the University of Oklahoma conducting the launches from Puerto Rico. On Saint Croix, launches were conducted from Carambola between 19 August 2021 and 14 September 2021. Altogether 73\nlaunches were conducted, of which a total of seven radiosondes were used to validate Aeolus in this study. As for Sal, these\n145\nmeasurements were performed with the radiosonde instrument DFM-09 (GRAW). Lastly, 32 launches were conducted from The radiosondes launched on the Virgin Islands were organised by National Aeronautics and Space Administration (NASA)’s\nConvective Processes Experiment-Aerosols and Winds (CPEX-AW) campaign component of JATAC, with the University of\nUtah conducting the launches on Saint Croix and the University of Oklahoma conducting the launches from Puerto Rico. On Saint Croix, launches were conducted from Carambola between 19 August 2021 and 14 September 2021. Altogether 73\nlaunches were conducted, of which a total of seven radiosondes were used to validate Aeolus in this study. As for Sal, these\n145\nmeasurements were performed with the radiosonde instrument DFM-09 (GRAW). Lastly, 32 launches were conducted from\nthe University of Puerto Rico at Mayagüez (UPRM) campus between 26 August and 14 September 2021, 7 of which could be\nused for the validation of Aeolus. All launches were performed with iMet-4 radiosondes from the International Met System. As with DFM-09, the iMet-4 radiosondes provide measurements of wind speed, wind direction, temperature, humidity and ,\ng\np\ng\nlaunches were conducted, of which a total of seven radiosondes were used to validate Aeolus in this study. As for Sal, these\n145\nmeasurements were performed with the radiosonde instrument DFM-09 (GRAW). Lastly, 32 launches were conducted from\nthe University of Puerto Rico at Mayagüez (UPRM) campus between 26 August and 14 September 2021, 7 of which could be\nused for the validation of Aeolus. All launches were performed with iMet-4 radiosondes from the International Met System. As with DFM-09, the iMet-4 radiosondes provide measurements of wind speed, wind direction, temperature, humidity and launches were conducted, of which a total of seven radiosondes were used to validate Aeolus in this study. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. 3.1\nCo-location criteria For the comparison of Aeolus against radiosonde profiles, several steps are required to fit the radiosonde wind measurements\nto the Aeolus measurement grid and to co-locate them in time and space. To ensure vertical consistency, the high-resolution radiosonde measurements are vertically averaged within the 24 range bins\n190\nas specified in the Aeolus L2B product. Subsequently, the radiosondes total horizontal wind speed VRS and direction φRS are\nprojected to the Aeolus HLOS (HLOSRS) using the azimuth angle φAEOLUS also specified in the L2B product, in accordance\nto\nHLOSRS = VRS × cos(φAEOLUS – φRS)\n(1) To ensure vertical consistency, the high-resolution radiosonde measurements are vertically averaged within the 24 range bins\n190\nas specified in the Aeolus L2B product. Subsequently, the radiosondes total horizontal wind speed VRS and direction φRS are\nprojected to the Aeolus HLOS (HLOSRS) using the azimuth angle φAEOLUS also specified in the L2B product, in accordance\nto\nHLOSRS = VRS × cos(φAEOLUS – φRS)\n(1) (1) Moreover, we have chosen co-location radii of up to 340 km, as we assume typical variations in zonal wind to be of a larger\n195\nscale. In fact, during boreal summer, African Easterly Waves (AEWs) and tropical disturbances dominate the tropospheric zonal\nwind variability over the tropical Atlantic, which generally have a horizontal wavelength of 2000-5000 km with a periodicity\nof 2-7 days (Belanger et al., 2016). Section 4.3.2 discusses the error dependencies related to co-location aspects in more detail. Moreover, we have chosen co-location radii of up to 340 km, as we assume typical variations in zonal wind to be of a larger\n195\nscale. In fact, during boreal summer, African Easterly Waves (AEWs) and tropical disturbances dominate the tropospheric zonal\nwind variability over the tropical Atlantic, which generally have a horizontal wavelength of 2000-5000 km with a periodicity\nof 2-7 days (Belanger et al., 2016). Section 4.3.2 discusses the error dependencies related to co-location aspects in more detail. Moreover, we have chosen co-location radii of up to 340 km, as we assume typical variations in zonal wind to be of a larger\n195\nscale. In fact, during boreal summer, African Easterly Waves (AEWs) and tropical disturbances dominate the tropospheric zonal\nwind variability over the tropical Atlantic, which generally have a horizontal wavelength of 2000-5000 km with a periodicity\nof 2-7 days (Belanger et al., 2016). Section 4.3.2 discusses the error dependencies related to co-location aspects in more detail. 2.4\nCAMS Dust products The dust-aerosol mixing ratio is thereby averaged along each track and projected onto Rayleigh-clear and\nMie-cloudy measurement bins to obtain an estimate of the dust concentration for each observation. 185 Mie-cloudy measurement bins to obtain an estimate of the dust concentration for each observation. 185 Mie-cloudy measurement bins to obtain an estimate of the dust concentration for each observation. 185 2.3\nEUMETSAT SAFNWC Cloud type product This refers to following\n170\nclasses for altitudes above 16 km (very high clouds), between 7 and 16 km (very high and high cloud types), between 3 and 7\nkm (very high, high, mid-level, low and fractional cloud types) and finally below 3 km (very high, high, mid-level, low, very\nlow and fractional cloud types). classification, a measurement bin is considered as cloudy, if it is situated within or below a cloud. This refers to following\n170\nclasses for altitudes above 16 km (very high clouds), between 7 and 16 km (very high and high cloud types), between 3 and 7\nkm (very high, high, mid-level, low and fractional cloud types) and finally below 3 km (very high, high, mid-level, low, very\nlow and fractional cloud types). classification, a measurement bin is considered as cloudy, if it is situated within or below a cloud. This refers to following\n170\nclasses for altitudes above 16 km (very high clouds), between 7 and 16 km (very high and high cloud types), between 3 and 7\nkm (very high, high, mid-level, low and fractional cloud types) and finally below 3 km (very high, high, mid-level, low, very\nlow and fractional cloud types). 6 6 2.4\nCAMS Dust products The fourth generation of ECMWF Global Atmospheric Composition Reanalysis (EAC4) (Inness et al., 2019) is produced by\n175\nthe Copernicus Atmosphere Monitoring Service (CAMS) with the main objective of global aerosol monitoring. EAC4 relies on\nECMWF’s IFS, which has been extended to predict and assimilate aerosols (Rémy et al., 2019), trace gases (Flemming et al.,\n2015; Huijnen et al., 2019) and greenhouse gases. The IFS meteorological and atmospheric composition models are combined\nwith data assimilation from satellite products using the 4D-Var data assimilation scheme in CY42R1. In particular, CAMS assimilates the Aerosol Optical Depth (AOD) at 550 nm derived from MODIS and the Polar Multi-Sensor Aerosol Optical\n180\nProperties (PMAp). Reanalysis outputs are provided on three-dimensional time-consistent fields interpolated on 25 pressure\nlevels, a horizontal resolution of about 80 km and a sub-daily time resolution of 6 hours. assimilates the Aerosol Optical Depth (AOD) at 550 nm derived from MODIS and the Polar Multi-Sensor Aerosol Optical\n180\nProperties (PMAp). Reanalysis outputs are provided on three-dimensional time-consistent fields interpolated on 25 pressure\nlevels, a horizontal resolution of about 80 km and a sub-daily time resolution of 6 hours. assimilates the Aerosol Optical Depth (AOD) at 550 nm derived from MODIS and the Polar Multi-Sensor Aerosol Optical\n180\nProperties (PMAp). Reanalysis outputs are provided on three-dimensional time-consistent fields interpolated on 25 pressure\nlevels, a horizontal resolution of about 80 km and a sub-daily time resolution of 6 hours. Similar to the SAFNWC CT, the dust-aerosol mixing ratio is used to assess the quality of the Aeolus wind products in\npresence of dust. The dust-aerosol mixing ratio is thereby averaged along each track and projected onto Rayleigh-clear and Properties (PMAp). Reanalysis outputs are provided on three-dimensional time-consistent fields interpolated on 25 pressure\nlevels, a horizontal resolution of about 80 km and a sub-daily time resolution of 6 hours. Similar to the SAFNWC CT, the dust-aerosol mixing ratio is used to assess the quality of the Aeolus wind products in\npresence of dust. The dust-aerosol mixing ratio is thereby averaged along each track and projected onto Rayleigh-clear and\nMie-cloudy measurement bins to obtain an estimate of the dust concentration for each observation. 185 Similar to the SAFNWC CT, the dust-aerosol mixing ratio is used to assess the quality of the Aeolus wind products in\npresence of dust. 3.2\nStatistical metrics Different metrics were used to validate and estimate the systematic and random error of Aeolus wind products. The bin-to-bin\n200\nwind speed difference between Aeolus and radiosonde along the HLOS is defined as Different metrics were used to validate and estimate the systematic and random error of Aeolus wind products. The bin-to-bin\n200\nwind speed difference between Aeolus and radiosonde along the HLOS is defined as\nΔdiffHLOS = (HLOSAEOLUS – HLOSRS)\n(2) Different metrics were used to validate and estimate the systematic and random error of Aeolus wind products. The bin-to-bin\n200\nwind speed difference between Aeolus and radiosonde along the HLOS is defined as\nΔdiffHLOS = (HLOSAEOLUS – HLOSRS)\n(2) wind speed difference between Aeolus and radiosonde along the HLOS is defined as\nΔdiffHLOS = (HLOSAEOLUS – HLOSRS)\n(2) (2) 7 https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. Thus, the bias μ is defined as the total mean difference μ = 1\nN\nN\nX\ni=1\nΔdiffHLOS (3) with the Mean Absolute Difference (MADI) yielding\n205 MADI = 1\nN\nN\nX\ni=1\n|ΔdiffHLOS| MADI = 1\nN\nN\nX\ni=1\n|ΔdiffHLOS| (4) and N the total number of data points. and N the total number of data points. and N the total number of data points. Additionally, we calculated the standard deviation of the difference STD =\nv\nu\nu\nt 1\ni – 1\nN\nX\ni=1\n(HLOSAEOLUS – HLOSRS)2 (5) and the scaled median absolute deviation (SMAD)\n210 SMAD = 1.4826 × median\n\u0000\f\fΔdiffHLOS – median(ΔdiffHLOS)\n\f\f\u0001 (6) The SMAD is equivalent to the standard deviation for a normal distribution of errors, but is often used in Aeolus validation\nstudies as it is less sensitive to individual outliers with very large differences than the standard deviation. Since the number of data points varies greatly depending on the measurement channel and height, we define the uncertainty\nof the mean bias εμ as\n215 (7) (7) https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. study is comparably homogeneous, we estimate the representativeness error for our comparison to be in the range of 1.5 ms–1\nto 2.5 ms–1. The radiosonde observation error σRS is estimated to be 0.7 ms–1 based on Dirksen et al. (2014). study is comparably homogeneous, we estimate the representativeness error for our comparison to be in the range of 1.5 ms–1\nto 2.5 ms–1. The radiosonde observation error σRS is estimated to be 0.7 ms–1 based on Dirksen et al. (2014). The representativeness and radiosonde observations errors also need to be considered when comparing the differences be-\ntween Aeolus and radiosonde observations with the expected error provided in the Aeolus data product (EEAeolus). To account\nfor this, we add the the radiosonde observation error and an estimated representativeness error of 2 ms–1 to achieve the total\nexpected error for the comparison (EEtot) as follows: The representativeness and radiosonde observations errors also need to be considered when comparing the differences be-\ntween Aeolus and radiosonde observations with the expected error provided in the Aeolus data product (EEAeolus). To account\nfor this, we add the the radiosonde observation error and an estimated representativeness error of 2 ms–1 to achieve the total\nexpected error for the comparison (EEtot) as follows: 230 EEtot =\nq\nEE2\nAeolus + σ2\nRS + σ2rep (9) 3.3\nRepresentativeness The difference between Aeolus and radiosonde observations is the sum of the Aeolus observation error, the radiosonde ob-\nservation error and the error arising from spatial and temporal displacement of the observations and different observation\ngeometries. The latter is usually referred to as representativeness error (Weissmann et al., 2005). As the three error components\n220\ncan be assumed to be uncorrelated, the standard deviation of the Aeolus HLOS winds observation error (σAeolus) can therefore\nbe calculated as The difference between Aeolus and radiosonde observations is the sum of the Aeolus observation error, the radiosonde ob-\nservation error and the error arising from spatial and temporal displacement of the observations and different observation servation error and the error arising from spatial and temporal displacement of the observations and different observation\ngeometries. The latter is usually referred to as representativeness error (Weissmann et al., 2005). As the three error components\n220\ncan be assumed to be uncorrelated, the standard deviation of the Aeolus HLOS winds observation error (σAeolus) can therefore\nbe calculated as geometries. The latter is usually referred to as representativeness error (Weissmann et al., 2005). As the three error components\n220\ncan be assumed to be uncorrelated, the standard deviation of the Aeolus HLOS winds observation error (σAeolus) can therefore\nbe calculated as σAeolus =\nq\nσ2tot – σ2\nRS – σ2rep (8) where σtot is the standard deviation of the total difference between Aeolus and radiosonde observations (STD), σRS is the standard deviation of the radiosonde observation error and σrep is the standard deviation of the representativeness error. Martin\n225\net al. (2021) estimated that the representativeness error for the comparison of Aeolus and radiosonde observations in mit-\nlatitudes is about 2.5 ms–1 based on high-resolution model simulations. As the wind fields in the area of the present validation standard deviation of the radiosonde observation error and σrep is the standard deviation of the representativeness error. Martin\n225\net al. (2021) estimated that the representativeness error for the comparison of Aeolus and radiosonde observations in mit-\nlatitudes is about 2.5 ms–1 based on high-resolution model simulations. As the wind fields in the area of the present validation 8 8 3.4\nQuality control\n235 Quality control (QC) is an important step in the evaluation of Aeolus wind errors. The aim is to check for the validity of the\nmeasurements and discard nonphysical wind results from the analysis process. The QC we apply here is based on the existing\nquality control recommendations (Rennie et al., 2020) from the Aeolus Data Science and Innovation Cluster (DISC), and\nprimarily rely on the HLOS wind error estimate (EE) in the L2B product and the validity flags. The Rayleigh channel EE is based on the uncertainty of the SNR spectrometer response and takes into account error propaga-\n240\ntion arising from the sensitivity of the Fabry-Perot interferometer, Poisson noise in the useful signal and the solar background. Ultimately, the Rayleigh EE is proportional to the inverse squared root of the useful signal on the detector. Future baseline\nversions will include additional noise terms, such as noise related to atmospheric temperature and pressure, or cross-talk con-\ntamination. In contrary, the Mie EE is determined from the accuracy of the fringe peak position using the solution covariance of the Lorentzian fitting algorithm based on four characteristics of the signal shape, i.e the peak position, height, width and\n245\noffset. Following the default QC flags, all Aeolus wind products with a validity flag of 0, EE above 8 ms–1 for Rayleigh and 4\nms–1 for Mie, are omitted. Nevertheless, the used QC might not be enough and the data algorithm may contain gross errors in\nthe wind estimate that have not been flagged as invalid. These errors are usually due to non-Gaussian error sources, such as of the Lorentzian fitting algorithm based on four characteristics of the signal shape, i.e the peak position, height, width and\n245\noffset. Following the default QC flags, all Aeolus wind products with a validity flag of 0, EE above 8 ms–1 for Rayleigh and 4\nms–1 for Mie, are omitted. Nevertheless, the used QC might not be enough and the data algorithm may contain gross errors in\nthe wind estimate that have not been flagged as invalid. These errors are usually due to non-Gaussian error sources, such as instrument/transmission failure, or to a misrepresentation of the measurements in space and time. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. 4.1\nStatistical comparison of Aeolus with radiosonde observations and model winds In this section, the L2B HLOS winds (L2bP 3.50) from Aeolus are compared statistically with radiosonde observations and\n260\nmodel winds. This includes a comparison with the ECMWF model equivalents (subsection 4.1.1), an overview of systematic\nand random differences with respect to Cal/Val sites and orbital nodes (subsection 4.1.2), and finally the identification of an\norbital- and altitude-dependent bias in the Rayleigh-clear channel (subsection 4.1.3). In this section, the L2B HLOS winds (L2bP 3.50) from Aeolus are compared statistically with radiosonde observations and\n260\nmodel winds. This includes a comparison with the ECMWF model equivalents (subsection 4.1.1), an overview of systematic\nand random differences with respect to Cal/Val sites and orbital nodes (subsection 4.1.2), and finally the identification of an\norbital- and altitude-dependent bias in the Rayleigh-clear channel (subsection 4.1.3). The present study relies on a total of 384 Rayleigh-clear and 59 Mie-cloudy bin pairs, of which ∼60% and ∼53% are\nfrom ascending orbits, respectively, with the majority of observations obtained from the Caribbean launch sites (∼56% for\n265\nRayleigh-clear and ∼64% for Mie-cloudy). Rayleigh-cloudy bin pairs are also available, but only in a very small number (16\ncounts), which makes a statistical analysis difficult. 3.4\nQuality control\n235 Since the two aforementioned\n250\nQC are not sufficient to remove these gross errors, an additional QC parameter is used, namely the modified Z-score (Lux et al.,\n2022b; Witschas et al., 2022; Iglewicz and Hoaglin, 1993). The modified Z-score Zm,i is defined as instrument/transmission failure, or to a misrepresentation of the measurements in space and time. Since the two aforementioned\n250\nQC are not sufficient to remove these gross errors, an additional QC parameter is used, namely the modified Z-score (Lux et al.,\n2022b; Witschas et al., 2022; Iglewicz and Hoaglin, 1993). The modified Z-score Zm,i is defined as (10) and describes the median deviations between each wind speed difference normalized with the SMAD. The modified Z-score\nsignificantly influences small data sets, such as those used in this study. Following literature recommendations (Lux et al.,\n255\n2022b; Witschas et al., 2022; Sandbhor and Chaphalkar, 2019; Tripathy et al., 2013), we discard wind observations with a\nmodified Z-score greater than 3 as a final QC. significantly influences small data sets, such as those used in this study. Following literature recommendations (Lux et al.,\n255\n2022b; Witschas et al., 2022; Sandbhor and Chaphalkar, 2019; Tripathy et al., 2013), we discard wind observations with a\nmodified Z-score greater than 3 as a final QC. 9 The generally good agreement between radiosonde and model equivalent\nshows that the co-location parameters used in this study are reliable, as most of the systematic and random errors seem to be\nspecific to the Aeolus Rayleigh-clear data. This stresses the need to identify the underlying potential error sources of Rayleigh- in the model equivalent is smaller than for Aeolus Mie-cloudy winds, with biases of 0.4 ± 0.3 ms–1 and –0.9 ± 0.3 ms–1,\n280\nrespectively. For Rayleigh-cloudy, the STD is larger at 6.6 ms–1 with a bias of 1.0 ± 1.4 ms–1, but given the small statistical\nsample size, there is a risk of a large margin of error. The generally good agreement between radiosonde and model equivalent\nshows that the co-location parameters used in this study are reliable, as most of the systematic and random errors seem to be\nspecific to the Aeolus Rayleigh-clear data. This stresses the need to identify the underlying potential error sources of Rayleigh- clear observations with respect to the presence of clouds and dust aerosols, which are frequent in the region of interest. It is\n285\nalso worth noting that this good agreement indicates that the model equivalent is a robust reference for validating the Aeolus\nwinds in the tropical Atlantic. clear observations with respect to the presence of clouds and dust aerosols, which are frequent in the region of interest. It is\n285\nalso worth noting that this good agreement indicates that the model equivalent is a robust reference for validating the Aeolus\nwinds in the tropical Atlantic. 4.1.1\nComparative analysis with the ECMWF model equivalents 4.1.1\nComparative analysis with the ECMWF model equivalents Figure 1. (a) Aeolus HLOS Rayleigh-clear (blue), Mie-cloudy (red) and Rayleigh-cloudy (orange) wind products plotted against radiosonde\nmeasurements projected along the HLOS for the 20 radiosonde profiles. The gross errors (crosses) are determined using the modified Z-score\nwith a threshold of 3. (b) Aeolus HLOS model equivalents from the ECMWF feedback files plotted against radiosonde measurements. The\ndashed lines are located at the ±10 ms–1 and ±20 ms–1 wind speed difference between two measurements. Figure 1. (a) Aeolus HLOS Rayleigh-clear (blue), Mie-cloudy (red) and Rayleigh-cloudy (orange) wind products plotted against radiosonde\nmeasurements projected along the HLOS for the 20 radiosonde profiles. The gross errors (crosses) are determined using the modified Z-score\nwith a threshold of 3. (b) Aeolus HLOS model equivalents from the ECMWF feedback files plotted against radiosonde measurements. The\ndashed lines are located at the ±10 ms–1 and ±20 ms–1 wind speed difference between two measurements. 10 https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. Figure 1 shows a scatter plot of the radiosonde HLOS (HLOSRS) against Aeolus L2B (HLOSAEOLUS) Rayleigh-clear (blue), Figure 1 shows a scatter plot of the radiosonde HLOS (HLOSRS) against Aeolus L2B (HLOSAEOLUS) Rayleigh-clear (blue),\nMie-cloudy (red) and Rayleigh-cloudy (orange) wind products (a) as well as against Aeolus ECMWF model equivalents\n270\n(HLOSECMWF) (b). Since Rayleigh-cloudy wind observations are not assimilated at ECMWF, they are not displayed in Fig. 1b. The × symbol represent the gross errors rejected with a Z-score threshold of 3 (∼3.5%, ∼4.8% and ∼6.7% of the\ntotal Rayleigh-clear, Mie-cloudy and Rayleigh-cloudy data points, respectively). The dashed lines represent the ±10 ms–1\nand ±20 ms–1 difference between two measurements. The Aeolus model equivalent HLOSECMWF for Rayleigh-clear shows a Mie-cloudy (red) and Rayleigh-cloudy (orange) wind products (a) as well as against Aeolus ECMWF model equivalents\n270\n(HLOSECMWF) (b). Since Rayleigh-cloudy wind observations are not assimilated at ECMWF, they are not displayed in Fig. 1b. The × symbol represent the gross errors rejected with a Z-score threshold of 3 (∼3.5%, ∼4.8% and ∼6.7% of the\ntotal Rayleigh-clear, Mie-cloudy and Rayleigh-cloudy data points, respectively). The dashed lines represent the ±10 ms–1\nand ±20 ms–1 difference between two measurements. The Aeolus model equivalent HLOSECMWF for Rayleigh-clear shows a much better agreement with the radiosonde measurements HLOSRS with a STD of 2.1 ms–1 (Fig. 1b) compared to the Aeolus\n275\nHLOSAEOLUS Rayleigh-clear observations, which have a larger spread and a STD of 4.8 ms–1 (Fig. 1a). The systematic\ndifference of the model equivalent is also smaller with a bias of 0.1 ± 0.1 ms–1 compared to –0.5 ± 0.2 ms–1 for the Aeolus\nobservations. In contrast, the Mie-cloudy winds of both Aeolus model equivalents and HLOSAEOLUS behave similarly with\nrespect to the radiosonde measurements, with STD of 2.93 ms–1 and 2.9 ms–1, respectively. Again, the systematic difference in the model equivalent is smaller than for Aeolus Mie-cloudy winds, with biases of 0.4 ± 0.3 ms–1 and –0.9 ± 0.3 ms–1,\n280\nrespectively. For Rayleigh-cloudy, the STD is larger at 6.6 ms–1 with a bias of 1.0 ± 1.4 ms–1, but given the small statistical\nsample size, there is a risk of a large margin of error. 4.1.2\nSystematic and random errors using radiosondes An overview of the bias and random differences of both channels can be found in Table 2. In terms of systematic errors,\nRayleigh-clear shows a relatively small negative bias of –0.5 ± 0.2 ms–1, on average, which is below ESA’s specification of\n290\n0.7 ms–1 (Ingmann and Straume, 2016). This bias is, however, the result of a large heterogeneity with respect to the Cal/Val\nsites and orbital nodes, with compensating biases of –1.5±0.6 ms–1 and 0.6±0.4 ms–1 for the descending and ascending nodes\non Sal, respectively, compared to negative biases of –1.0±0.3 ms–1 (ascending) and –0.6±0.4 ms–1 (descending) in the Virgin\nIsland. As for random differences, Rayleigh-clear has an average STD of 4.8 ms–1, which varies only marginally between the Rayleigh-clear shows a relatively small negative bias of –0.5 ± 0.2 ms–1, on average, which is below ESA’s specification of\n290\n0.7 ms–1 (Ingmann and Straume, 2016). This bias is, however, the result of a large heterogeneity with respect to the Cal/Val\nsites and orbital nodes, with compensating biases of –1.5±0.6 ms–1 and 0.6±0.4 ms–1 for the descending and ascending nodes\non Sal, respectively, compared to negative biases of –1.0±0.3 ms–1 (ascending) and –0.6±0.4 ms–1 (descending) in the Virgin\nIsland. As for random differences, Rayleigh-clear has an average STD of 4.8 ms–1, which varies only marginally between the Cal/Val sites and orbital nodes, ranging from 4.1 ms–1 to 5.3 ms–1. The overall SMAD is found to be slightly below at 4.3 ms–1. 295\nFor comparison with the ESA recommendation for random errors, we derived the random errors for Aeolus observations\nconsidering also the representativeness errors for the comparison and radiosonde observation errors according to Eq. 8 (table\n3). The random error at 2–16 km altitude of 3.8 – 4.3 ms–1 exceeds the threshold of 2.5 ms–1, while at 16–20 km altitude it\namounts to 4.3 – 4.8 ms–1, also exceeding the ESA threshold of 3 ms–1. The quality of Rayleigh-clear measurements primarily depends on the signal accumulation, which can vary with the thickness of the RBS and the horizontal accumulation length\n300\nas well as with the atmospheric path signal. The latter has been decreasing in recent years as a result of initial instrumental depends on the signal accumulation, which can vary with the thickness of the RBS and the horizontal accumulation length\n300\nas well as with the atmospheric path signal. 4.1.2\nSystematic and random errors using radiosondes The latter has been decreasing in recent years as a result of initial instrumental 11 https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. Table 2. Overview of the mean bias and uncertainty (μ, σμ; ms–1), Standard deviation (STD; ms–1), Scaled Median Absolute Deviation\n(SMAD; ms–1) and counts (COUNT) for the Rayleigh-clear and Mie-cloudy channels, orbital nodes and the different radiosonde locations. Due to the small amount of available data, Rayleigh-cloudy is not shown here. Table 2. Overview of the mean bias and uncertainty (μ, σμ; ms–1), Standard deviation (STD; ms–1), Scaled Median Absolute Deviation\n(SMAD; ms–1) and counts (COUNT) for the Rayleigh-clear and Mie-cloudy channels, orbital nodes and the different radiosonde locations. Due to the small amount of available data, Rayleigh-cloudy is not shown here. Region\nOrbital node\nRayleigh-clear\nMie-cloudy\nμ\nSTD\nSMAD\nCOUNT\nμ\nSTD\nSMAD\nCOUNT\nAscending\n0.6±0.4\n4.9\n4.4\n112\n-1±0.9\n2.9\n3.5\n15\nSal\nDescending\n-1.5±0.6\n4.6\n4.8\n55\n-1.6±0.8\n2.2\n2.1\n6\nAll\n-0.1±0.3\n4.9\n4.5\n167\n-1.2±0.7\n2.7\n3.2\n21\nAscending\n-1.0±0.3\n4.1\n3.7\n119\n-0.6±0.7\n2.9\n3.7\n16\nSCRX/PR\nDescending\n-0.6±0.4\n5.3\n4.3\n98\n-1.0±0.5\n2.9\n2.5\n22\nAll\n-0.8±0.3\n4.7\n4.3\n217\n-0.8±0.4\n2.9\n2.5\n38\nAscending\n-0.2±0.3\n4.6\n4.2\n231\n-0.8±0.6\n2.9\n3.3\n31\nSal/SCRX/PR\nDescending\n-0.9±0.4\n5.0\n4.6\n153\n-1.1±0.4\n2.8\n2.2\n28\nAll\n-0.5±0.2\n4.8\n4.3\n384\n-0.9±0.3\n2.9\n2.6\n59 misalignment, laser-induced contamination, as well as the wavefront error of the 1.5 m telescope. The solar background noise,\nwhich varies along the orbit and season, can also affect the quality of the Rayleigh-clear measurements. ESA’s specification and more uniform across regions and orbital nodes with a slightly larger bias in the descending orbits\n305\nand over Sal. Concerning the random differences, the measurements exhibit a total random error of 1.1 – 2.3 ms–1, which is\nbelow ESA’s 2–16 km recommendation, as most Mie-cloudy measurements are located underneath 16 km altitude. As with the\nbias, the STD and SMAD of Mie-cloudy are also quite independent of orbital and regional dependence. The overall accuracy\nof Mie-cloudy depends on the signal accumulation, the classification algorithm and the quality of the calibration data. The accuracy of Mie-cloudy winds is higher than that of Rayleigh-clear winds as particle backscatter is usually stronger than that\n310\nof clear air in addition to the fact that Mie backscatter is not subject to broadening induced by Rayleigh-Brillouin scattering\n(Witschas et al., 2012). https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. Table 3. Overview of the total systematic (μ, σμ; ms–1) and random (σAeolus; ms–1) errors derived according to Eq. 8 for Rayleigh-clear\nand Mie-cloudy winds for altitudes ranges 2–16km and 16–20km, as well as the corresponding ESA’s error recommendations. The random\nerror σAeolus was computed for a representativeness error σrep ranging from 1.5 ms–1 to 2.5 ms–1. For Mie-cloudy, only the altitude range\n2–16km is shown for the random error, as Mie-cloudy does not sample sufficiently above 16km. Rayleigh-clear\nMie-cloudy\nσAeolus 2–16km\nσAeolus 16–20km\nμ\nσAeolus 2–16km\nμ\nAscending\n3.4 – 3.9\n4.0 – 4.4\n-0.2±0.3\n1.1 – 2.3\n-0.8±0.6\nDescending\n4.3 – 4.7\n4.4 – 4.9\n-0.9±0.4\n0.5 – 2.1\n-1.1±0.4\nAll\n3.8 – 4.3\n4.3 – 4.8\n-0.5±0.2\n1.1 – 2.3\n-0.9±0.3\nESA\n2.5\n3\n0.7\n2.5\n0.7 The difference in results is caused by the different altitudes at which the data are sampled, as the The difference in results is caused by the different altitudes at which the data are sampled, as the aircraft only samples the lower\n10 km portion of the troposphere, which is shown to be more noisy owing to the abundance of dust aerosols in this region. 320\nFor Mie-cloudy, the random error gives 2.9 ± 0.3 ms–1, which is similar to our radiosonde-based results as most Mie-cloudy\nscattering occurs at lower levels. 320 Comparing the results of different Cal/Val studies is tricky as the influence of geographical regions, atmospheric conditions,\ndecreasing laser energy, product baseline and quality control procedures on the result can be significant and must be considered. accuracy of Mie-cloudy winds is higher than that of Rayleigh-clear winds as particle backscatter is usually stronger than that\n310\nof clear air in addition to the fact that Mie backscatter is not subject to broadening induced by Rayleigh-Brillouin scattering\n(Witschas et al., 2012). Comparing the results of different Cal/Val studies is tricky as the influence of geographical regions, atmospheric conditions,\ndecreasing laser energy, product baseline and quality control procedures on the result can be significant and must be considered. In this analysis, comparisons are only made with statistics derived from AVATAR-T airborne-based measurements (Witschas\n315\net al., 2022; Lux et al., 2022b), as these were carried out in the framework of the same JATAC campaign. The statistical analysis\nof AVATAR-T shows systematic errors of –0.1 ± 0.3 ms–1 for Rayleigh-clear and –0.7 ± 0.2 ms–1 for Mie-cloudy, which are\nslightly smaller than for radiosondes. However, the random error of 7.1 ± 0.3 ms–1 for Rayleigh-clear is significantly higher. In this analysis, comparisons are only made with statistics derived from AVATAR-T airborne-based measurements (Witschas\n315\net al., 2022; Lux et al., 2022b), as these were carried out in the framework of the same JATAC campaign. The statistical analysis\nof AVATAR-T shows systematic errors of –0.1 ± 0.3 ms–1 for Rayleigh-clear and –0.7 ± 0.2 ms–1 for Mie-cloudy, which are\nslightly smaller than for radiosondes. However, the random error of 7.1 ± 0.3 ms–1 for Rayleigh-clear is significantly higher. 12 4.1.3\nOrbital bias in the Rayleigh-clear channel Figure 2 shows vertical profiles of the differences between Aeolus Rayleigh-clear observations and radiosonde measurements\nprojected along HLOS (O-RS; solid lines), and the corresponding ECMWF model equivalents (O-B; dotted lines) for both\n325\nascending (red) and descending (blue) orbits over Sal (a), PR and SCRX (b). The shading represents the bias uncertainty σμ. HLOS winds from the descending track are multiplied by -1 to conform with the sign convention of the model coordinate\nsystem. The vertical profiles illustrate the presence of an ascending/descending bias visible in both the O-B and O-RS profiles,\nreaching up to 2.5 ms–1 around 8 km altitude in both regions. The differences below 5 km altitude could be related to the greater amount of dust in Cabo Verde during this period, while above 17 km the differences could partly be related to the lack\n330\nof descending orbit data over Sal (Fig. 2a). This altitude- and orbit-dependent bias was already described by Borne et al. (2023)\nusing first-guess departure statistics over West Africa. greater amount of dust in Cabo Verde during this period, while above 17 km the differences could partly be related to the lack\n330\nof descending orbit data over Sal (Fig. 2a). This altitude- and orbit-dependent bias was already described by Borne et al. (2023)\nusing first-guess departure statistics over West Africa. using first-guess departure statistics over West Africa. This latitude consistent bias caused the zonal winds in the ECMWF analysis to accelerate in the morning and weaken in\nthe evening, affecting the African Easterly Jet (AEJ) and Tropical Easterly Jet (TEJ) in particular. Correcting this bias with This latitude consistent bias caused the zonal winds in the ECMWF analysis to accelerate in the morning and weaken in\nthe evening, affecting the African Easterly Jet (AEJ) and Tropical Easterly Jet (TEJ) in particular. Correcting this bias with a temperature-dependent approach helped to improve the representation of winds in the analysis and forecast fields (Borne\n335\net al., 2023). However, the cause of this bias remains unknown, as it has not been proven to be related to temperature, nor has\nany dependence on wind speed, SNR or useful signal been found (not shown here). Here, as both the O-B and O-RS profiles\nare very close to each other, with deviations below 0.5 ms–1, the existence of this bias can be confirmed observationally with\nradiosondes. As highlighted by Horányi et al. 4.1.3\nOrbital bias in the Rayleigh-clear channel Differences (dots) and average differences (lines) between ascending (red) and descending (blue) winds between Aeolus observa-\ntions (O) and radiosonde HLOS wind measurements (RS, solid line) along with ECMWF model equivalents (B, dotted line) over Sal (a) and\nPuerto Rico - Saint Croix (PR/SCRX, (b). The shadow represents the bias uncertainty σμ. To comply with the sign convention of the model\ncoordinate system, the HLOS winds from the descending orbit are multiplied by -1. Figure 2. Differences (dots) and average differences (lines) between ascending (red) and descending (blue) w Figure 2. Differences (dots) and average differences (lines) between ascending (red) and descending (blue) winds between Aeolus observa-\ntions (O) and radiosonde HLOS wind measurements (RS, solid line) along with ECMWF model equivalents (B, dotted line) over Sal (a) and\nPuerto Rico - Saint Croix (PR/SCRX, (b). The shadow represents the bias uncertainty σμ. To comply with the sign convention of the model\ncoordinate system, the HLOS winds from the descending orbit are multiplied by -1. 4.1.3\nOrbital bias in the Rayleigh-clear channel (2015), biases of the order of 1 ms–1 can already deteriorate forecast quality. a temperature-dependent approach helped to improve the representation of winds in the analysis and forecast fields (Borne\n335\net al., 2023). However, the cause of this bias remains unknown, as it has not been proven to be related to temperature, nor has\nany dependence on wind speed, SNR or useful signal been found (not shown here). Here, as both the O-B and O-RS profiles\nare very close to each other, with deviations below 0.5 ms–1, the existence of this bias can be confirmed observationally with\nradiosondes. As highlighted by Horányi et al. (2015), biases of the order of 1 ms–1 can already deteriorate forecast quality. a temperature-dependent approach helped to improve the representation of winds in the analysis and forecast fields (Borne\n335\net al., 2023). However, the cause of this bias remains unknown, as it has not been proven to be related to temperature, nor has\nany dependence on wind speed, SNR or useful signal been found (not shown here). Here, as both the O-B and O-RS profiles\nare very close to each other, with deviations below 0.5 ms–1, the existence of this bias can be confirmed observationally with\nradiosondes. As highlighted by Horányi et al. (2015), biases of the order of 1 ms–1 can already deteriorate forecast quality. 13 Figure 2. Differences (dots) and average differences (lines) between ascending (red) and descending (blue) winds between Aeolus observa-\ntions (O) and radiosonde HLOS wind measurements (RS, solid line) along with ECMWF model equivalents (B, dotted line) over Sal (a) and\nPuerto Rico\nSaint Croix (PR/SCRX (b) The shadow represents the bias uncertainty\nTo comply with the sign convention of the model Figure 2. Differences (dots) and average differences (lines) between ascending (red) and descending (blue) winds between Aeolus observa-\ntions (O) and radiosonde HLOS wind measurements (RS, solid line) along with ECMWF model equivalents (B, dotted line) over Sal (a) and\nPuerto Rico - Saint Croix (PR/SCRX, (b). The shadow represents the bias uncertainty σμ. To comply with the sign convention of the model\ncoordinate system, the HLOS winds from the descending orbit are multiplied by -1. Figure 2. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. Rayleigh-clear and Rayleigh-cloudy Figure 3 shows the absolute difference between Aeolus and radiosonde measurement points |ΔdiffHLOS| as a function of EEtot\n(a), altitude (b), co-location radius (c) and co-location time (d) for the Rayleigh-clear (blue) and Rayleigh-cloudy (orange)\nobservation types. The solid and dashed blue lines show the Rayleigh-clear MADI and SMAD, respectively, with each value\ncalculated using a minimum sample size of 40 data points for panels a, b and d. Also shown are outliers (cross symbol +),\n350\nthat we define in this study as values with low EE (< 5 ms–1) and large absolute difference (> 10 ms–1), which are of particular\ninterest as they contribute the most to the wind quality degradation. The Rayleigh-clear outliers account for 13 observations,\ni.e. ∼3.4% of the data points. For Rayleigh-cloudy, no MADI and SMAD are computed due to the lack of data. Figure 3 shows the absolute difference between Aeolus and radiosonde measurement points |ΔdiffHLOS| as a function of EEtot\n(a), altitude (b), co-location radius (c) and co-location time (d) for the Rayleigh-clear (blue) and Rayleigh-cloudy (orange)\nobservation types. The solid and dashed blue lines show the Rayleigh-clear MADI and SMAD, respectively, with each value observation types. The solid and dashed blue lines show the Rayleigh-clear MADI and SMAD, respectively, with each value\ncalculated using a minimum sample size of 40 data points for panels a, b and d. Also shown are outliers (cross symbol +),\n350\nthat we define in this study as values with low EE (< 5 ms–1) and large absolute difference (> 10 ms–1), which are of particular\ninterest as they contribute the most to the wind quality degradation. The Rayleigh-clear outliers account for 13 observations,\ni.e. ∼3.4% of the data points. For Rayleigh-cloudy, no MADI and SMAD are computed due to the lack of data. In general, the MADI and SMAD between Rayleigh-clear and radiosonde wind measurements appear to be proportional to In general, the MADI and SMAD between Rayleigh-clear and radiosonde wind measurements appear to be proportional to\nthe Aeolus EEtot (Fig. 3a), with larger deviations associated with larger EEtots, as expected. However, on average, the mean\n355\nEEtot overestimates the MADI by 1 ms–1 for EEtot values below 6 ms–1 (see grey line). This discrepancy can be attributed to\nthe relatively small amount of data used in the study, as the EE is based on the Gaussian assumption of a large data set. 4.2\nError dependency\n340 In this section we examine the error dependency and associated error sources of the different Aeolus wind products. Firstly,\nwe investigate the error dependency as a function of co-location parameters, such as radius and time difference between two\nmeasurement points, to account for representativeness. Secondly, we explore the error dependency in relation to the presence\nof clouds and dust, as these supposedly influence the quality of Aeolus wind products. 14 Rayleigh-clear and Rayleigh-cloudy For\nRayleigh-cloudy measurements, it is difficult to establish a dependency although the absolute difference appears to be generally\nlarger owing to the large STD of 6.6 ms–1 for this observation type. Considering the altitude error dependency of Rayleigh- the Aeolus EEtot (Fig. 3a), with larger deviations associated with larger EEtots, as expected. However, on average, the mean\n355\nEEtot overestimates the MADI by 1 ms–1 for EEtot values below 6 ms–1 (see grey line). This discrepancy can be attributed to\nthe relatively small amount of data used in the study, as the EE is based on the Gaussian assumption of a large data set. For\nRayleigh-cloudy measurements, it is difficult to establish a dependency although the absolute difference appears to be generally\nlarger owing to the large STD of 6.6 ms–1 for this observation type. Considering the altitude error dependency of Rayleigh- clear (Fig. 3b), a general pattern emerges with MADI and SMAD reaching a minimum of 3 ms–1 and 2 ms–1 respectively on\n360\naverage in the middle troposphere at 10 km, while increasing above and below, with MADIs of 4–5 ms–1 and SMADs of almost\n6 ms–1 at 2.5 km and 19 km altitude. As we will see in the next subsection 4.2.2, this error pattern is inversely proportional\nto the Rayleigh backscattered useful signal, as it directly affects the SNR and thereby the quality of the measurement points. Rayleigh-clear outliers seem to occur at all altitudes and Rayleigh-cloudy measurements are primarily found in the lower clear (Fig. 3b), a general pattern emerges with MADI and SMAD reaching a minimum of 3 ms–1 and 2 ms–1 respectively on\n360\naverage in the middle troposphere at 10 km, while increasing above and below, with MADIs of 4–5 ms–1 and SMADs of almost\n6 ms–1 at 2.5 km and 19 km altitude. As we will see in the next subsection 4.2.2, this error pattern is inversely proportional\nto the Rayleigh backscattered useful signal, as it directly affects the SNR and thereby the quality of the measurement points. Rayleigh-clear outliers seem to occur at all altitudes and Rayleigh-cloudy measurements are primarily found in the lower troposphere, below 6 km. 365\nIn Fig. 3c we examine the error dependency with respect to the co-location radius, which extends up to 340 km, a distance\nthat is large relative to the 100 km specified in ESA’s recommendations. Rayleigh-clear and Rayleigh-cloudy However, the MADI and SMAD for Rayleigh-clear\ndo not increase with radius, but stagnate at an average of 3–4 ms–1 for radii above 100 km, while they are slightly higher\nbelow 100 km, reaching 4–5 ms–1. Furthermore, outliers appear across all co-location radii. This indicates that the use of a troposphere, below 6 km. 365\nIn Fig. 3c we examine the error dependency with respect to the co-location radius, which extends up to 340 km, a distance\nthat is large relative to the 100 km specified in ESA’s recommendations. However, the MADI and SMAD for Rayleigh-clear\ndo not increase with radius, but stagnate at an average of 3–4 ms–1 for radii above 100 km, while they are slightly higher\nbelow 100 km, reaching 4–5 ms–1. Furthermore, outliers appear across all co-location radii. This indicates that the use of a troposphere, below 6 km. 365\nIn Fig. 3c we examine the error dependency with respect to the co-location radius, which extends up to 340 km, a distance\nthat is large relative to the 100 km specified in ESA’s recommendations. However, the MADI and SMAD for Rayleigh-clear\ndo not increase with radius, but stagnate at an average of 3–4 ms–1 for radii above 100 km, while they are slightly higher\nbelow 100 km, reaching 4–5 ms–1. Furthermore, outliers appear across all co-location radii. This indicates that the use of a co-location distance up to 340 km is acceptable for the statistical comparison. Exploring the error dependency with respect to\n370\nthe time difference between the observations (Fig. 3d), there is indication for increasing difference for larger time-differences,\ngoing from 3–4 ms–1 at 0 minutes to 4–6 ms–1 above 30 minutes. There is also an asymmetry of the error dependence, with a\nlarger error magnitude for radiosonde observations preceding the Aeolus passage. Since most radiosondes were launched with\nthe objective of reaching the mid-troposphere during the satellite’s passage, the measurements preceding/following Aeolus of co-location distance up to 340 km is acceptable for the statistical comparison. Exploring the error dependency with respect to\n370\nthe time difference between the observations (Fig. 3d), there is indication for increasing difference for larger time-differences,\ngoing from 3–4 ms–1 at 0 minutes to 4–6 ms–1 above 30 minutes. There is also an asymmetry of the error dependence, with a\nlarger error magnitude for radiosonde observations preceding the Aeolus passage. Rayleigh-clear and Rayleigh-cloudy EEtot (a), altitude (b), co-location radius (c) and co-location time quantities expressed as a function of the absolute difference\nbetween radiosonde HLOS winds (HLOSRS) and Aeolus (HLOSAEOLUS) Rayleigh-clear (blue) and Rayleigh-cloudy (orange) observations. Outliers are defined as values with an EE below 5 ms–1 and absolute difference larger than 10 ms–1 and are represented by the cross symbol\n+. The solid blue lines indicate the MADI while the dotted blue lines represent the SMAD of Rayleigh-clear and each value is computed\nusing a minimum sample size of 40 data points. The grey line in panel a represents the diagonal at intercept 0 with slope 1. Due to the limited\namount of data, no MADI and SMAD are shown for Rayleigh-cloudy. Figure 3. EEtot (a), altitude (b), co-location radius (c) and co-location time quantities expressed as a function of the absolute difference\nbetween radiosonde HLOS winds (HLOSRS) and Aeolus (HLOSAEOLUS) Rayleigh-clear (blue) and Rayleigh-cloudy (orange) observations. Outliers are defined as values with an EE below 5 ms–1 and absolute difference larger than 10 ms–1 and are represented by the cross symbol\n+. The solid blue lines indicate the MADI while the dotted blue lines represent the SMAD of Rayleigh-clear and each value is computed\nusing a minimum sample size of 40 data points. The grey line in panel a represents the diagonal at intercept 0 with slope 1. Due to the limited\namount of data, no MADI and SMAD are shown for Rayleigh-cloudy. +. The solid blue lines indicate the MADI while the dotted blue lines represent the SMAD of Rayleigh-cle\nusing a minimum sample size of 40 data points. The grey line in panel a represents the diagonal at intercept 0\namount of data, no MADI and SMAD are shown for Rayleigh-cloudy. Rayleigh-clear and Rayleigh-cloudy Since most radiosondes were launched with\nthe objective of reaching the mid-troposphere during the satellite’s passage, the measurements preceding/following Aeolus of co-location distance up to 340 km is acceptable for the statistical comparison. Exploring the error dependency with respect to\n370\nthe time difference between the observations (Fig. 3d), there is indication for increasing difference for larger time-differences,\ngoing from 3–4 ms–1 at 0 minutes to 4–6 ms–1 above 30 minutes. There is also an asymmetry of the error dependence, with a\nlarger error magnitude for radiosonde observations preceding the Aeolus passage. Since most radiosondes were launched with\nthe objective of reaching the mid-troposphere during the satellite’s passage, the measurements preceding/following Aeolus of more than 30 minutes correspond mainly to measurements at lower/higher altitudes. The larger MADI and SMADI values for\n375\nthese time differences could hence be an indirect effect of the larger errors found at those altitudes (Fig. 3b). Again, no error\ndependency is observed for outliers, with most occurring below ±40 minutes time differences. more than 30 minutes correspond mainly to measurements at lower/higher altitudes. The larger MADI and SMADI values for\n375\nthese time differences could hence be an indirect effect of the larger errors found at those altitudes (Fig. 3b). Again, no error\ndependency is observed for outliers, with most occurring below ±40 minutes time differences. 15 https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. Figure 3. EEtot (a), altitude (b), co-location radius (c) and co-location time quantities expressed as a function of the absolute difference\nbetween radiosonde HLOS winds (HLOSRS) and Aeolus (HLOSAEOLUS) Rayleigh-clear (blue) and Rayleigh-cloudy (orange) observations. Outliers are defined as values with an EE below 5 ms–1 and absolute difference larger than 10 ms–1 and are represented by the cross symbol\n+. The solid blue lines indicate the MADI while the dotted blue lines represent the SMAD of Rayleigh-clear and each value is computed\nusing a minimum sample size of 40 data points. The grey line in panel a represents the diagonal at intercept 0 with slope 1. Due to the limited\namount of data, no MADI and SMAD are shown for Rayleigh-cloudy. Figure 3. Mie-cloudy Figure 4 shows the same error dependencies as in Fig. 3, but for the Mie-cloudy observation type. For Mie-cloudy, we define\noutliers as values exceeding an absolute error of 6 ms–1 along with EEs inferior to 3 ms–1. With a total of 3 data points, they\n380\naccount for ∼5% of the total Mie-cloudy observations. In panels a, b and d, each MADI and SMAD value is calculated using\na minimum sample size of 15 data points As shown in Fig. 4a, the absolute differences for Mie-cloudy measurements are generally smaller than for Rayleigh-clear,\nwith the largest deviations around 7–8 ms–1, while attaining 13–14 ms–1 for Rayleigh-clear. The MADI and SMAD remain\nbetween 2 and 3 ms–1, indicating an overestimation of the EEtot, especially for increasing EEtot. Regarding the altitude error\n385 16 https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. dependency (Fig. 4b), most of the data are found within the 10-15 km layer, which is probably related to the presence of high-\nlevel clouds, and below 7 km, where low- and mid-level clouds and dust layers are found. Due to the sparseness of Mie-cloudy\ndata, both MADI and SMADI do not show a specific vertical error trend. While MADI and SMAD remain between 2.3 and 2.7\nms–1, respectively, they decrease to 1.8 and 2.3 between 1.5 and 3 km altitude before increasing to almost 3 ms–1 in the lowest\n1 km. Fig. 4c shows that similarly to Rayleigh-clear, Mie-cloudy reveals no error dependency with respect to co-location radii,\n390\nwith the mean absolute error and SMAD mainly ranging from 1.7 to 3.2 ms–1, and outliers found at all radii. Regarding the\nerror dependence on time difference (Fig. 4d), we find that most of the measurement differences occur at time intervals of\nless than ±40 minutes. MADIs and SMADs are generally higher for negative co-location times, corresponding to cases where\nradiosonde observations are sampled before those from Aeolus. Nevertheless, we do not notice a strong relationship between\nco location time and errors\n395 data, both MADI and SMADI do not show a specific vertical error trend. While MADI and SMAD remain between 2.3 and 2.7\nms–1, respectively, they decrease to 1.8 and 2.3 between 1.5 and 3 km altitude before increasing to almost 3 ms–1 in the lowest\n1 km. Fig. 4c shows that similarly to Rayleigh-clear, Mie-cloudy reveals no error dependency with respect to co-location radii,\n390\nwith the mean absolute error and SMAD mainly ranging from 1.7 to 3.2 ms–1, and outliers found at all radii. Regarding the\nerror dependence on time difference (Fig. 4d), we find that most of the measurement differences occur at time intervals of\nless than ±40 minutes. MADIs and SMADs are generally higher for negative co-location times, corresponding to cases where\nradiosonde observations are sampled before those from Aeolus. Nevertheless, we do not notice a strong relationship between\nl\nti\nti\nd\n395 1 km. Fig. 4c shows that similarly to Rayleigh-clear, Mie-cloudy reveals no error dependency with respect to co-location radii,\n390\nwith the mean absolute error and SMAD mainly ranging from 1.7 to 3.2 ms–1, and outliers found at all radii. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. 4.2.2\nCloud type and dust As already mentioned, the accuracy of Rayleigh-clear and, to a lesser extent, Mie-cloudy depends on the signal level and\nSNR. In general, the signal level depends on the range bin thickness, the horizontal accumulation length, the atmospheric\npath signal and the overall signal background level. In addition, Rayleigh-clear winds are sensitive to signal attenuation due\nto atmospheric conditions, with weaker signal return under optically thick clouds and dust-aerosol layers. Mie-cloudy is less\n400\nconcerned as backscatter from particles is stronger, although it is sensitive to weak backscatter, e.g. from dust layers. Because\nof its strong sensitivity on signal levels, the EE of Rayleigh-clear only considers Poisson noise and is therefore inversely\nproportional to the squared root of the useful signal. For Mie-cloudy, this rule of thumb is not true. In this context, we aim to\ninvestigate the quality of the Rayleigh-clear and Mie-cloudy winds and the reliability of the corresponding EE with respect to the presence of clouds and dust. 405 Rayleigh-clear Table 4 describes the error dependency of the Rayleigh-clear observations with respect to the presence of clouds and dust,\nwith cases below 50%, above 50% and above 75% of cloudiness, as well as sub-categories distinguishing the dust mixing\nratio above (Dust) and below (DustNO) 10–8 kgkg–1. Note that SMAD is not used for this analysis as this reliably removes Table 4 describes the error dependency of the Rayleigh clear observations with respect to the presence of clouds and dust,\nwith cases below 50%, above 50% and above 75% of cloudiness, as well as sub-categories distinguishing the dust mixing\nratio above (Dust) and below (DustNO) 10–8 kgkg–1. Note that SMAD is not used for this analysis as this reliably removes\noutliers, which ought to be quantified here. We note that the MADI, the STD, and the EEtot all increase with the amount of\n410\nclouds and dust along the track, presumably due to the reduced return signal. In non-dusty conditions (DustNO), we observe\nthat for low cloud cover (<50%), the MADI (3.3 ± 0.2 ms–1) is significantly lower than the EEtot (4.8 ms–1) with a difference\nof 1.5 ms–1, while for higher cloud cover, the difference between MADI and EEtot is much smaller (1.1 ms–1 and 1.0 ms–1\nfor above 50% and 75% of cloudiness, respectively). This phenomenon is further enhanced at higher dust concentrations, with\n1\n1 NO\ng g\ny\ny\noutliers, which ought to be quantified here. We note that the MADI, the STD, and the EEtot all increase with the amount of\n410\nclouds and dust along the track, presumably due to the reduced return signal. In non-dusty conditions (DustNO), we observe\nthat for low cloud cover (<50%), the MADI (3.3 ± 0.2 ms–1) is significantly lower than the EEtot (4.8 ms–1) with a difference\nof 1.5 ms–1, while for higher cloud cover, the difference between MADI and EEtot is much smaller (1.1 ms–1 and 1.0 ms–1\nfor above 50% and 75% of cloudiness, respectively). This phenomenon is further enhanced at higher dust concentrations, with the MADI reaching even higher values (5.7 ± 0.8 ms–1) than the EEtot (5.8 ms–1) for cloud cover above 75%. This highlights\n415\nhow the EEtot in clear sky conditions is well calibrated, while it is becoming gradually too low with the increasing presence of\nclouds and dust. Regarding the\nerror dependence on time difference (Fig. 4d), we find that most of the measurement differences occur at time intervals of\nless than ±40 minutes. MADIs and SMADs are generally higher for negative co-location times, corresponding to cases where\nradiosonde observations are sampled before those from Aeolus. Nevertheless, we do not notice a strong relationship between Figure 4. Same as for Fig. 3, but for Mie-cloudy. For Mie-cloudy (red), outliers are defined as values having an absolute error above 6 ms–1\nand an EE inferior to 3 ms–1. The MADI and the SMAD values are computed using a minimum sample size of 15 data points. Figure 4. Same as for Fig. 3, but for Mie-cloudy. For Mie-cloudy (red), outliers are defined as values having an absolute error above 6 ms–1\nand an EE inferior to 3 ms–1. The MADI and the SMAD values are computed using a minimum sample size of 15 data points. 17 17 https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. Table 4. Overview of the total Error Estimate (EEtot; ms–1), mean absolute difference and uncertainty (MADI, σμ; ms–1), Standard deviation\n(STD; ms–1) and counts (COUNT) for the Rayleigh-clear measurements under different cloud and dust conditions. This includes three\ncategories of cloud cover (< 25 %, > 50 %, > 75 %) for dust mixing ratios above (Dust) and below (DustNO) 109 kgkg–1 along the track. Table 4. Overview of the total Error Estimate (EEtot; ms–1), mean absolute difference and uncertainty (MADI, σμ; ms–1), Standard deviation\n(STD; ms–1) and counts (COUNT) for the Rayleigh-clear measurements under different cloud and dust conditions. This includes three\ncategories of cloud cover (< 25 %, > 50 %, > 75 %) for dust mixing ratios above (Dust) and below (DustNO) 109 kgkg–1 along the track. Cloud < 50 %\nCloud > 50 %\nCloud > 75 %\nDustNO\nDust\nDustNO\nDust\nDustNO\nDust\nEEtot\n4.8\n5.4\n5.0\n5.6\n5.3\n5.8\nMADI\n3.3±0.2\n4.4±0.6\n3.9±0.5\n5.0±0.5\n4.3±0.7\n5.7±0.8\nSTD\n4.3\n5.0\n5.1\n5.9\n5.6\n6.4\nCOUNT\n234\n28\n64\n52\n38\n24\nFigure 5. Altitude as a function of Rayleigh-clear absolute difference |ΔdiffHLOS| (a,e), EEtot (b,f), normalized useful signal (c,g) and SR\n(d,h), where the colouring is dependent on the percentage of SAF clouds (upper row) and CAMS dust mixing ratio (lower row) along the\ntrack. The cross symbol + stands for outliers and defines values with an EE below 5 ms–1 and an absolute difference of more than 10 ms–1. Panel (a) includes the MADI for each cloud cover percentage, with a minimum sample size of 10 data points used to compute each value. Cloud < 50 %\nCloud > 50 %\nCloud > 75 %\nDustNO\nDust\nDustNO\nDust\nDustNO\nDust\nEEtot\n4.8\n5.4\n5.0\n5.6\n5.3\n5.8\nMADI\n3.3±0.2\n4.4±0.6\n3.9±0.5\n5.0±0.5\n4.3±0.7\n5.7±0.8\nSTD\n4.3\n5.0\n5.1\n5.9\n5.6\n6.4\nCOUNT\n234\n28\n64\n52\n38\n24 Cloud < 50 %\nCloud > 50 %\nCloud > 75 %\nDustNO\nDust\nDustNO\nDust\nDustNO\nDust\nEEtot\n4.8\n5.4\n5.0\n5.6\n5.3\n5.8\nMADI\n3.3±0.2\n4.4±0.6\n3.9±0.5\n5.0±0.5\n4.3±0.7\n5.7±0.8\nSTD\n4.3\n5.0\n5.1\n5.9\n5.6\n6.4\nCOUNT\n234\n28\n64\n52\n38\n24 Figure 5. Rayleigh-clear The larger STD with increasing cloudiness and dust concentration suggests an increasingly perturbed pattern\nof Rayleigh-clear measurements,possibly owing to the lower signal levels or to a cross-talk. Figure 5 puts this phenomenon into perspective, by showing the altitude-dependent absolute difference |ΔdiffHLOS| (a,e), the EEtot (b,f), the normalized useful signal (c,g) and the SR (d,h), where the colouring depends on the percentage of SAF clouds\n420\n(upper row) and the CAMS dust mixing ratio (lower row) along the track. For reference, the values that did not pass the QC are\nshown transparently. In addition, panel 5a includes the MADI of four cloud cover percentage categories, where each MADI is\ncomputed with a minimum sample size of 10 values. The colourings in Fig. 5 are illustrative of the results summarised in Table\n4, with measurements showing generally greater MADI under high cloud cover (red, orange, Fig. 5a) than under lower cloud cover (blue, blue-green). Measurements in the lower troposphere are naturally more strongly affected by cloud cover compared\n425\nto higher levels. The same applies to dust (Fig. 5e), which also occurs mainly in the lower 5 km of the troposphere. As we have already shown in Fig. 3b, the absolute error is higher in the upper and lower troposphere and minimised in\nthe middle troposphere around 10 km altitude. This trend is well reflected in the EEtot in Fig. 5b, which is an indication of As we have already shown in Fig. 3b, the absolute error is higher in the upper and lower troposphere and minimised in\nthe middle troposphere around 10 km altitude. This trend is well reflected in the EEtot in Fig. 5b, which is an indication of 18 https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. density. In the lower troposphere, the return signal is lower due to strong attenuation under clouds and dust layers. Interestingly,\nthe values with high EEtot and smaller useful signal in the mid-troposphere between 5 and 12.5 km in red likely correspond to\nmeasurements sampled under thick clouds, resulting in a strongly attenuated signal. They account for most of the measurements\nwith cloud cover greater than 75 % in this altitude range, while the cloud tops appear to be located between 12.5 and 15 km, as\n435\nthey exhibit a larger normalized useful signal and a SR greater than 1 (Fig. 5d,h). Finally, outliers are found under all types of\ncloud and dust conditions and affect different altitude ranges. They also occur for regular normalized useful signals, with most\nSRs lying around 1, which rules out a cause related to atmospheric particles. Altitude as a function of Rayleigh-clear absolute difference |ΔdiffHLOS| (a,e), EEtot (b,f), normalized useful signal (c,g) and SR\n(d,h), where the colouring is dependent on the percentage of SAF clouds (upper row) and CAMS dust mixing ratio (lower row) along the\ntrack. The cross symbol + stands for outliers and defines values with an EE below 5 ms–1 and an absolute difference of more than 10 ms–1. Panel (a) includes the MADI for each cloud cover percentage, with a minimum sample size of 10 data points used to compute each value. the generally good consistency between the EEtot and the absolute differences. As expected, this tendency fits inversely with\nthe normalized useful signal shown in Fig. 5c, with lower signal in the upper and lower troposphere. Indeed, in the higher\n430\ntroposphere the air is less dense and the thickness of the RB’s is not sufficient to compensate for the decrease in air molecule the generally good consistency between the EEtot and the absolute differences. As expected, this tendency fits inversely with\nthe normalized useful signal shown in Fig. 5c, with lower signal in the upper and lower troposphere. Indeed, in the higher\n430\ntroposphere the air is less dense and the thickness of the RB’s is not sufficient to compensate for the decrease in air molecule i\nthe normalized useful signal shown in Fig. 5c, with lower signal in the upper and lower troposphere. Indeed, in the higher\n430\ntroposphere the air is less dense and the thickness of the RB’s is not sufficient to compensate for the decrease in air molecule 19 Mie-cloudy 4b,\nmost backscatter occurs in two layers, i.e. within 10–15 km and below 7 km altitude. The majority of measurements have\nnormalized useful signals above 5e13 a.u. (Fig. 6c,g), which is overall above the normalized useful signal of the rejected\n450\nmeasurements shown in transparent. Furthermore, the SRs are generally above 1 (Fig. 6d,h), which is characteristic of Mie-\ncloudy measurements. More specifically, measurements sampled above 12.5 km have a cloud cover of more than 75 % along\nthe track and probably correspond to cloud tops, as they have stronger SRs between 1.5 and 3 (Fig. 6d,h). They exhibit good\nquality as well, with an average MADI of 1.5 ms–1 (Fig. 4b). Between 7.5 and 12.5 km altitude, most of the measurements\noccur with cloud cover less than 50 %, with SRs falling below 1.3. In this altitude range, there are also 2 outliers, which\n455\ninterestingly have SRs around 1 and a normalized useful signal in the same order of magnitude as the discarded ones. Their\npresence is unusual, as Mie-cloudy measurements are only obtainable for SRs above 1. Finally, below 7.5 km, the cloud cover\nis mainly above 50 %, while the dust concentration is mainly below 5 × 10–8 kgkg–1, showing that most of the Mie-cloudy\nbackscatter results from clouds and not from dust. As can be seen in Fig. 6g, measurements with high dust concentration\n(brown) are discarded (transparent) with normalized useful signals below 5e13 a.u. Surprisingly, measurements sampled at the\n460\nlower 1 km have the lowest normalized useful signals, mostly below 5e13 a.u. and are not discarded. These, however, tend to\nhave larger SRs between 1 and 2, which can compensate for the low normalized useful signal in the calculation of the EE. They\nalso correspond to the largest MADI scaling up to 4 ms–1 on average (i.e. Fig. 4d) in addition to relatively high EEtots (i.e. Fig. 6b,f). Two measurements also show negative SRs, which is an artifact, due to insufficient background signal corrections. The third outlier in the lower 1 km does not have abnormal characteristics compared to other measurements at this altitude. 465 normalized useful signals above 5e13 a.u. (Fig. 6c,g), which is overall above the normalized useful signal of the rejected\n450\nmeasurements shown in transparent. Furthermore, the SRs are generally above 1 (Fig. 6d,h), which is characteristic of Mie-\ncloudy measurements. Mie-cloudy More specifically, measurements sampled above 12.5 km have a cloud cover of more than 75 % along\nthe track and probably correspond to cloud tops, as they have stronger SRs between 1.5 and 3 (Fig. 6d,h). They exhibit good\nquality as well, with an average MADI of 1.5 ms–1 (Fig. 4b). Between 7.5 and 12.5 km altitude, most of the measurements normalized useful signals above 5e13 a.u. (Fig. 6c,g), which is overall above the normalized useful signal of the rejected\n450\nmeasurements shown in transparent. Furthermore, the SRs are generally above 1 (Fig. 6d,h), which is characteristic of Mie-\ncloudy measurements. More specifically, measurements sampled above 12.5 km have a cloud cover of more than 75 % along\nthe track and probably correspond to cloud tops, as they have stronger SRs between 1.5 and 3 (Fig. 6d,h). They exhibit good\nquality as well, with an average MADI of 1.5 ms–1 (Fig. 4b). Between 7.5 and 12.5 km altitude, most of the measurements normalized useful signals above 5e13 a.u. (Fig. 6c,g), which is overall above the normalized useful signal of the rejected\n450\nmeasurements shown in transparent. Furthermore, the SRs are generally above 1 (Fig. 6d,h), which is characteristic of Mie-\ncloudy measurements. More specifically, measurements sampled above 12.5 km have a cloud cover of more than 75 % along\nthe track and probably correspond to cloud tops, as they have stronger SRs between 1.5 and 3 (Fig. 6d,h). They exhibit good\nquality as well, with an average MADI of 1.5 ms–1 (Fig. 4b). Between 7.5 and 12.5 km altitude, most of the measurements occur with cloud cover less than 50 %, with SRs falling below 1.3. In this altitude range, there are also 2 outliers, which\n455\ninterestingly have SRs around 1 and a normalized useful signal in the same order of magnitude as the discarded ones. Their\npresence is unusual, as Mie-cloudy measurements are only obtainable for SRs above 1. Finally, below 7.5 km, the cloud cover\nis mainly above 50 %, while the dust concentration is mainly below 5 × 10–8 kgkg–1, showing that most of the Mie-cloudy\nbackscatter results from clouds and not from dust. As can be seen in Fig. 6g, measurements with high dust concentration occur with cloud cover less than 50 %, with SRs falling below 1.3. Mie-cloudy Table 5 shows the same as Table 4, but for Mie-cloudy. Due to the limited amount of data for Mie-cloudy winds, the inter-\n440\npretation of the results should be treated with caution. We find that, in contrast to Rayleigh-clear, the EE, MADI and STD\ndecrease with the percentage of cloud cover along the path. This is understandable as clouds provide the strongest backscatter\nsignal required for high quality Mie-cloudy measurements. However, the presence of dust for cloud cover below 50 % leads to\na decrease in EEtot, MADI and STD, while conversely there is an increase of these quantities in more dense cloudy conditions Table 5 shows the same as Table 4, but for Mie-cloudy. Due to the limited amount of data for Mie-cloudy winds, the inter-\n440\npretation of the results should be treated with caution. We find that, in contrast to Rayleigh-clear, the EE, MADI and STD\ndecrease with the percentage of cloud cover along the path. This is understandable as clouds provide the strongest backscatter\nsignal required for high quality Mie-cloudy measurements. However, the presence of dust for cloud cover below 50 % leads to\na decrease in EEtot, MADI and STD, while conversely there is an increase of these quantities in more dense cloudy conditions (>50 %, >75 %). A possible explanation is that in clear-sky conditions, the backscatter from dust layers is strong enough to\n445\nobtain high quality measurements, whereas in cloudy conditions, the attenuation by clouds weakens the backscatter return from\nthe dust. Figure 6 depicts the same as Fig. 5, but for Mie-cloudy. As mentioned in the previous section when discussing in Fig. 4b,\nmost backscatter occurs in two layers, i.e. within 10–15 km and below 7 km altitude. The majority of measurements have (>50 %, >75 %). A possible explanation is that in clear-sky conditions, the backscatter from dust layers is strong enough to\n445\nobtain high quality measurements, whereas in cloudy conditions, the attenuation by clouds weakens the backscatter return from\nthe dust. Figure 6 depicts the same as Fig. 5, but for Mie-cloudy. As mentioned in the previous section when discussing in Fig. 4b,\nmost backscatter occurs in two layers, i.e. within 10–15 km and below 7 km altitude. The majority of measurements have Figure 6 depicts the same as Fig. 5, but for Mie-cloudy. As mentioned in the previous section when discussing in Fig. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. Table 5. Same as table 4, but for Mie-cloudy. Cloud < 50 %\nCloud > 50 %\nCloud > 75 %\nDustNO\nDust\nDustNO\nDust\nDustNO\nDust\nEEtot\n3.7\n3.6\n3.4\n3.5\n3.2\n3.4\nMADI\n2.8±0.5\n2.4±0.2\n1.8±0.3\n2.5±0.4\n1.6±0.3\n2.6±0.6\nSTD\n2.96\n1.53\n1.89\n2.95\n1.68\n3.18\nCOUNT\n11\n9\n16\n23\n8\n13\nFigure 6. Same as Fig. 5 but for Mie-cloudy. Here the cross symbol + defines values with an EE below 3 ms–1 and an absolute difference\nabove 6 ms–1. Table 5. Same as table 4, but for Mie-cloudy. Cloud < 50 %\nCloud > 50 %\nCloud > 75 %\nDustNO\nDust\nDustNO\nDust\nDustNO\nDust\nEEtot\n3.7\n3.6\n3.4\n3.5\n3.2\n3.4\nMADI\n2.8±0.5\n2.4±0.2\n1.8±0.3\n2.5±0.4\n1.6±0.3\n2.6±0.6\nSTD\n2.96\n1.53\n1.89\n2.95\n1.68\n3.18\nCOUNT\n11\n9\n16\n23\n8\n13 Figure 6 Same as Fig 5 but for Mie cloudy Here the cross symbol + defines values with an EE below 3 ms–1 and an absolute difference Figure 6. Same as Fig. 5 but for Mie-cloudy. Here the cross symbol + defines values with an EE below 3 ms–1 and an absolute difference\nabove 6 ms–1. Mie-cloudy In this altitude range, there are also 2 outliers, which\n455\ninterestingly have SRs around 1 and a normalized useful signal in the same order of magnitude as the discarded ones. Their\npresence is unusual, as Mie-cloudy measurements are only obtainable for SRs above 1. Finally, below 7.5 km, the cloud cover\nis mainly above 50 %, while the dust concentration is mainly below 5 × 10–8 kgkg–1, showing that most of the Mie-cloudy\nbackscatter results from clouds and not from dust. As can be seen in Fig. 6g, measurements with high dust concentration occur with cloud cover less than 50 %, with SRs falling below 1.3. In this altitude range, there are also 2 outliers, which\n455\ninterestingly have SRs around 1 and a normalized useful signal in the same order of magnitude as the discarded ones. Their\npresence is unusual, as Mie-cloudy measurements are only obtainable for SRs above 1. Finally, below 7.5 km, the cloud cover\nis mainly above 50 %, while the dust concentration is mainly below 5 × 10–8 kgkg–1, showing that most of the Mie-cloudy\nbackscatter results from clouds and not from dust. As can be seen in Fig. 6g, measurements with high dust concentration (brown) are discarded (transparent) with normalized useful signals below 5e13 a.u. Surprisingly, measurements sampled at the\n460\nlower 1 km have the lowest normalized useful signals, mostly below 5e13 a.u. and are not discarded. These, however, tend to\nhave larger SRs between 1 and 2, which can compensate for the low normalized useful signal in the calculation of the EE. They\nalso correspond to the largest MADI scaling up to 4 ms–1 on average (i.e. Fig. 4d) in addition to relatively high EEtots (i.e. Fig. 6b,f). Two measurements also show negative SRs, which is an artifact, due to insufficient background signal corrections. The third outlier in the lower 1 km does not have abnormal characteristics compared to other measurements at this altitude. 465 20 https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. Figure 7. Overview of the cloud-free case study for a radiosonde launched from Sal airport on 9th September 2021 at 18:45 UTC and the\nascending orbit of Aeolus between 19:23:56-19:24:31 UTC for a co-location radius of 180km. (a) Vertical radiosonde HLOS wind profile\n(black solid line) and projected onto Rayleigh-clear RBS (black stepped line), as well as averaged Rayleigh-clear observations (blue dots),\ncorresponding EE (error bars) and ECMWF model equivalents (Meq, stepped lines). (b) Vertical profile of the Rayleigh-clear EEtot (blue\nline), together with the EEtot of all profiles (grey solid lines) and their average (black solid line). (c), (d) Same as (b), but for normalised\nuseful signal and CAMS dust mixing ratio, respectively. (e) Horizontal map showing the SAFNWC CT at 19:00 UTC and the co-location\nperimeter (white solid line), the Aeolus track (red solid line) and the radiosonde launch site (red cross). Figure 7. Overview of the cloud-free case study for a radiosonde launched from Sal airport on 9th September 2021 at 18:45 UTC and the\nascending orbit of Aeolus between 19:23:56-19:24:31 UTC for a co-location radius of 180km. (a) Vertical radiosonde HLOS wind profile\n(black solid line) and projected onto Rayleigh-clear RBS (black stepped line), as well as averaged Rayleigh-clear observations (blue dots),\ncorresponding EE (error bars) and ECMWF model equivalents (Meq, stepped lines). (b) Vertical profile of the Rayleigh-clear EEtot (blue\nline), together with the EEtot of all profiles (grey solid lines) and their average (black solid line). (c), (d) Same as (b), but for normalised\nuseful signal and CAMS dust mixing ratio, respectively. (e) Horizontal map showing the SAFNWC CT at 19:00 UTC and the co-location\nperimeter (white solid line), the Aeolus track (red solid line) and the radiosonde launch site (red cross). passed over on an ascending orbit between 19:23:56 UTC and 19:24:31 UTC within a co-location radius of 180 km around the\nlaunch site. Figure 7a depicts the corresponding sampled radiosonde HLOS wind profile (black lines) as well as Rayleigh-clear\n(blue) wind measurement points with associated EEtot shown as error bars and ECMWF model equivalents shown as stepped\nlines. The corresponding Rayleigh-clear EEtot, normalized useful signal and CAMS dust mixing ratio profiles are shown in\n475\nblue in Figs. 4.2.3\nCase studies To further investigate the properties of the Aeolus wind errors, this section presents three case studies comparing Aeolus and\nradiosonde wind measurements under three different atmospheric conditions, namely clear sky, high cloud cover and high dust\nconcentration. 470 The first case-study illustrated in Fig. 7 presents a comparison between Aeolus and radiosonde wind measurements collected\nunder clear sky conditions. The radiosonde was launched over Sal Airport at 18:45 UTC on 9 September 2021, and Aeolus 21 7b, 7c and 7d, respectively, along with all other profiles in grey and the average of all profiles in black. Figure (blue) wind measurement points with associated EEtot shown as error bars and ECMWF model equivalents shown as stepped\nlines. The corresponding Rayleigh-clear EEtot, normalized useful signal and CAMS dust mixing ratio profiles are shown in\n475\nblue in Figs. 7b, 7c and 7d, respectively, along with all other profiles in grey and the average of all profiles in black. Figure 22 Beneath the cloud\nbase at 13 km altitude, however, it appears that the Rayleigh-clear measurements follow an irregular pattern, with most of the\nmeasurements and error bars not matching the radiosonde observations, reaching deviations higher than 10 ms–1. Accordingly,\nwe find that the EEtot (Fig. 8b) is larger in this altitude range mainly varying between 5 and 6 ms–1, which also corresponds to a sharp decrease of the normalized useful signal well below the average (Fig. 8c). Nonetheless, the ECMWF model-equivalents\n500\nin Fig. 8a remain fairly accurate relative to the radiosonde measurements. This result mirrors the findings presented in the\nprevious section, namely that the Rayleigh-clear EEtot is systematically underestimated when the normalized useful signal\nis strongly attenuated. It appears that the normalized useful signal further decreases below 2.5 km, presumably as a result\nof the increasing dust concentration at this height (Fig. 8d), which most likely leads to a QC rejection of the Rayleigh-clear sharp decrease of the normalized useful signal well below the average (Fig. 8c). Nonetheless, the ECMWF model-equivalents\n500\nin Fig. 8a remain fairly accurate relative to the radiosonde measurements. This result mirrors the findings presented in the\nprevious section, namely that the Rayleigh-clear EEtot is systematically underestimated when the normalized useful signal\nis strongly attenuated. It appears that the normalized useful signal further decreases below 2.5 km, presumably as a result\nof the increasing dust concentration at this height (Fig. 8d), which most likely leads to a QC rejection of the Rayleigh-clear measurements. 505\nLastly, Fig. 9 examines the influence of dust on the quality of Aeolus. In this case, the radiosonde was launched on 21\nSeptember 2021 at 06:50 UTC for a descending orbit of Aeolus, which passed over a co-location perimeter with a radius of 60\nkm between 07:28:44 UTC and 07:29:07 UTC. As can be seen in Fig. 9e, the atmospheric conditions in the co-location area\nwere completely cloud free at 07:30, with some low level cloud further south of the island. The radiosonde profile shown in Fig. measurements. 505\nLastly, Fig. 9 examines the influence of dust on the quality of Aeolus. In this case, the radiosonde was launched on 21\nSeptember 2021 at 06:50 UTC for a descending orbit of Aeolus, which passed over a co-location perimeter with a radius of 60\nkm between 07:28:44 UTC and 07:29:07 UTC. As can be seen in Fig. These high-clouds appear to be located between 13 km and 16 km altitude, as three Mie-cloudy (red) and two Rayleigh-\ncloudy (orange) measurements are found in this range, and where the normalized useful signal is found to have a maximum. In\nthis altitude range, all Rayleigh-clear, Rayleigh-cloudy and Mie-cloudy measurements exhibit good quality, with radiosonde co-location region between 07:28:32 UTC and 07:28:55 UTC, i.e. during the descending node. As can been seen in panel 8e,\n490\nwhich corresponds to SAFNWC CT at 07:30 UTC, Aeolus overpasses a variety of high clouds, mainly high semitransparent\nclouds. These high-clouds appear to be located between 13 km and 16 km altitude, as three Mie-cloudy (red) and two Rayleigh-\ncloudy (orange) measurements are found in this range, and where the normalized useful signal is found to have a maximum. In\nthis altitude range, all Rayleigh-clear, Rayleigh-cloudy and Mie-cloudy measurements exhibit good quality, with radiosonde co-location region between 07:28:32 UTC and 07:28:55 UTC, i.e. during the descending node. As can been seen in panel 8e,\n490\nwhich corresponds to SAFNWC CT at 07:30 UTC, Aeolus overpasses a variety of high clouds, mainly high semitransparent\nclouds. These high-clouds appear to be located between 13 km and 16 km altitude, as three Mie-cloudy (red) and two Rayleigh-\ncloudy (orange) measurements are found in this range, and where the normalized useful signal is found to have a maximum. In\nthis altitude range, all Rayleigh-clear, Rayleigh-cloudy and Mie-cloudy measurements exhibit good quality, with radiosonde measurements generally within the error bars. Above this cloud cover at 16 km, we only find Rayleigh-clear measurements\n495\nthat also perform well, with an EEtot (Fig. 8b) and normalized useful signal (Fig. 8c) close to average. Beneath the cloud\nbase at 13 km altitude, however, it appears that the Rayleigh-clear measurements follow an irregular pattern, with most of the\nmeasurements and error bars not matching the radiosonde observations, reaching deviations higher than 10 ms–1. Accordingly,\nwe find that the EEtot (Fig. 8b) is larger in this altitude range mainly varying between 5 and 6 ms–1, which also corresponds to a measurements generally within the error bars. Above this cloud cover at 16 km, we only find Rayleigh-clear measurements\n495\nthat also perform well, with an EEtot (Fig. 8b) and normalized useful signal (Fig. 8c) close to average. In this case study, the radiosonde was also launched from\nSal airport, this time at 07:00 UTC on 14 September 2021, with a co-location radius of 60 km. Aeolus passed across the\nco-location region between 07:28:32 UTC and 07:28:55 UTC, i.e. during the descending node. As can been seen in panel 8e,\n490\nwhich corresponds to SAFNWC CT at 07:30 UTC, Aeolus overpasses a variety of high clouds, mainly high semitransparent\nclouds. These high-clouds appear to be located between 13 km and 16 km altitude, as three Mie-cloudy (red) and two Rayleigh-\ncloudy (orange) measurements are found in this range, and where the normalized useful signal is found to have a maximum. In\nthis altitude range, all Rayleigh-clear, Rayleigh-cloudy and Mie-cloudy measurements exhibit good quality, with radiosonde\nmeasurements generally within the error bars. Above this cloud cover at 16 km, we only find Rayleigh-clear measurements\n495\nthat also perform well, with an EEtot (Fig. 8b) and normalized useful signal (Fig. 8c) close to average. Beneath the cloud\nbase at 13 km altitude, however, it appears that the Rayleigh-clear measurements follow an irregular pattern, with most of the\nmeasurements and error bars not matching the radiosonde observations, reaching deviations higher than 10 ms–1. Accordingly,\nwe find that the EEtot (Fig. 8b) is larger in this altitude range mainly varying between 5 and 6 ms–1, which also corresponds to a\nsharp decrease of the normalized useful signal well below the average (Fig. 8c). Nonetheless, the ECMWF model-equivalents\n500\nin Fig. 8a remain fairly accurate relative to the radiosonde measurements. This result mirrors the findings presented in the\nprevious section, namely that the Rayleigh-clear EEtot is systematically underestimated when the normalized useful signal\nis strongly attenuated. It appears that the normalized useful signal further decreases below 2.5 km, presumably as a result\nof the increasing dust concentration at this height (Fig. 8d), which most likely leads to a QC rejection of the Rayleigh-clear\nmeasurements. 505 co-location region between 07:28:32 UTC and 07:28:55 UTC, i.e. during the descending node. As can been seen in panel 8e,\n490\nwhich corresponds to SAFNWC CT at 07:30 UTC, Aeolus overpasses a variety of high clouds, mainly high semitransparent\nclouds. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. 7e shows the SAFNWC CT over the Cape Verde region at 19:00 UTC. In the latter panel, it can be seen that conditions were\npredominantly cloud-free along the Aeolus track (red solid line) and within the co-location radius (white solid line), while\nsome low clouds can be found in the surrounding area. In these clear-sky conditions, it is not surprising to find that most of the\nmeasurements are of the Rayleigh-clear observation type, with no Mie-cloudy and Rayleigh-cloudy measurements (Fig. 7a). 480\nThroughout the atmosphere above 2.5 km, the quality of Rayleigh-clear is very good, with most error bars overlapping with\nradiosonde measurements and ECMWF model equivalents. In general, we found that the EEtot estimate (Fig. 7b) is below\naverage throughout the atmosphere, with a minimum of 3.5 ms–1 at 8 km altitude and a maximum above 5 ms–1 at 17.5 km\nand 2.5 km altitude. This is consistent with to a normalized useful signal (Fig. 7c) close to the average, except between 2.5 and 12.5 km, where it is higher, most likely due to the absence of cloud attenuation. In general, EEtot and normalized useful signal\n485\ndecrease below 5 km, which is accompanied by an increase in the dust mixing ratio. This increase reaches 1.2 kgkg–1 at about\n2 km altitude, below which no measurements are found, presumably filtered out during the QC procedure. Figure 8 shows the same as Fig. 7, but for cloudy conditions. In this case study, the radiosonde was also launched from\nSal airport, this time at 07:00 UTC on 14 September 2021, with a co-location radius of 60 km. Aeolus passed across the 12.5 km, where it is higher, most likely due to the absence of cloud attenuation. In general, EEtot and normalized useful signal\n485\ndecrease below 5 km, which is accompanied by an increase in the dust mixing ratio. This increase reaches 1.2 kgkg–1 at about\n2 km altitude, below which no measurements are found, presumably filtered out during the QC procedure. Figure 8 shows the same as Fig. 7, but for cloudy conditions. In this case study, the radiosonde was also launched from\nSal airport, this time at 07:00 UTC on 14 September 2021, with a co-location radius of 60 km. Aeolus passed across the Figure 8 shows the same as Fig. 7, but for cloudy conditions. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. Figure 8. Same as Fig. 7, but for the case study with high cloud cover. Here, the radiosonde was launched from the Sal airport at 07:00 UTC\non the 14th September 2021, while Aeolus overpassed the co-location area, with radius of 60km, on a descending node between 07:28:32\nand 07:28:55 UTC. In panel a, the red and orange colours represent the averaged Mie-cloudy and Rayleigh-cloudy observations (points),\nrespectively, with the corresponding EE shown as error bars and ECMWF model equivalents (Meq) shown as stepped lines. The SAFNWC\nCT shown in (e) corresponds to 07:30 UTC. Figure 8. Same as Fig. 7, but for the case study with high cloud cover. Here, the radiosonde was launched from the Sal airport at 07:00 UTC\non the 14th September 2021, while Aeolus overpassed the co-location area, with radius of 60km, on a descending node between 07:28:32\nand 07:28:55 UTC. In panel a, the red and orange colours represent the averaged Mie-cloudy and Rayleigh-cloudy observations (points),\nrespectively, with the corresponding EE shown as error bars and ECMWF model equivalents (Meq) shown as stepped lines. The SAFNWC\nCT shown in (e) corresponds to 07:30 UTC. while outliers with EEs of less than 5 ms–1 (Fig. 9b) can be spotted above 15km and below 5km. This error structure is\nsurprising, as both the normalized useful signal and error estimation curves are similar to the one of the cloud-free case study\nin Fig. 7b and 7c. However, in panel 9c, we see that the Rayleigh-clear error pattern coincides with a strong peak in dust\nmixing ratio, reaching more than 2 10–7 kgkg–1 around 3.5 km altitude. The presence of dust seems to affect the quality of\n515\nRayleigh-clear measurements without influencing the normalized useful signal and thus leading to an underestimation of the\nEE. Reason could be linked to a cross-talk. while outliers with EEs of less than 5 ms–1 (Fig. 9b) can be spotted above 15km and below 5km. This error structure is\nsurprising, as both the normalized useful signal and error estimation curves are similar to the one of the cloud-free case study\nin Fig. 7b and 7c. 9e, the atmospheric conditions in the co-location area\nwere completely cloud free at 07:30, with some low level cloud further south of the island. The radiosonde profile shown in Fig. 9a indicates that Aeolus primarily measured in the Rayleigh channel along this orbital segment. Rayleigh-clear measurements\n510\nappear to be consistent with radiosonde wind measurements throughout the mid-troposphere between 5 km and 15 km altitude, 9a indicates that Aeolus primarily measured in the Rayleigh channel along this orbital segment. Rayleigh-clear measurements\n510\nappear to be consistent with radiosonde wind measurements throughout the mid-troposphere between 5 km and 15 km altitude, 23 https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. Figure 9. Same as Figs. 7 and 8 but for the case study with dust. Here, the radiosonde was launched from the Sal airport at 06:50 UTC on\nthe 21th September 2021, while Aeolus overpassed the co-location area, with radius of 60km, on a descending node between 07:28:44 UTC\nand 07:29:07 UTC. The SAFNWC CT shown in (e) corresponds to 07:30 UTC. Figure 9. Same as Figs. 7 and 8 but for the case study with dust. Here, the radiosonde was launched from the Sal airport at 06:50 UTC on\nthe 21th September 2021, while Aeolus overpassed the co-location area, with radius of 60km, on a descending node between 07:28:44 UTC\nand 07:29:07 UTC. The SAFNWC CT shown in (e) corresponds to 07:30 UTC. However, in panel 9c, we see that the Rayleigh-clear error pattern coincides with a strong peak in dust\nmixing ratio, reaching more than 2 10–7 kgkg–1 around 3.5 km altitude. The presence of dust seems to affect the quality of\n515\nRayleigh-clear measurements without influencing the normalized useful signal and thus leading to an underestimation of the\nEE. Reason could be linked to a cross-talk. 515 24 5\nConclusions In this study, we conducted a cross-Atlantic validation of Aeolus wind observations using radiosondes in the scope of the Joint\nAeolus Tropical Atlantic Campaign (JATAC). Of the total 20 radiosonde profiles included in this work, 11 were launched from\n520\nPuerto Rico and St. Croix in the Caribbean and 9 from Sal Airport on Cape Verde between August and September 2021. The\nadvantage of radiosondes is that they provide good vertical coverage, providing 384 Rayleigh-clear bin-to-bin comparisons\nfrom the surface to an altitude of 20 km and 59 Mie-cloudy comparisons, mainly restricted to the presence of clouds and\naerosols. After having applied several Quality Control (QC) and adaptation grid procedures, we quantified the quality of Aeolus Tropical Atlantic Campaign (JATAC). Of the total 20 radiosonde profiles included in this work, 11 were launched from\n520\nPuerto Rico and St. Croix in the Caribbean and 9 from Sal Airport on Cape Verde between August and September 2021. The\nadvantage of radiosondes is that they provide good vertical coverage, providing 384 Rayleigh-clear bin-to-bin comparisons\nfrom the surface to an altitude of 20 km and 59 Mie-cloudy comparisons, mainly restricted to the presence of clouds and\naerosols. After having applied several Quality Control (QC) and adaptation grid procedures, we quantified the quality of 25 https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. Rayleigh-clear, Mie-cloudy and to a lesser extent Rayleigh-cloudy observation types, with respect to co-location aspects as\n525\nwell as atmospheric conditions such as cloud cover and dust concentration. Rayleigh-clear, Mie-cloudy and to a lesser extent Rayleigh-cloudy observation types, with respect to co-location aspects as\n525\nwell as atmospheric conditions such as cloud cover and dust concentration. According to our statistical analysis, the total systematic error of Rayleigh-clear is –0.5 ± 0.2 ms–1, which is in agreement\nwith the ESA recommendation of 0.7 ms–1. The random error was calculated from the standard deviation of the difference\nbetween radiosonde and Aeolus measurements accounting for radiosonde observation errors estimated at 0 7±0 28 ms–1 and Rayleigh-clear, Mie-cloudy and to a lesser extent Rayleigh-cloudy observation types, with respect to co-location aspects as\n525\nwell as atmospheric conditions such as cloud cover and dust concentration. According to our statistical analysis, the total systematic error of Rayleigh-clear is –0.5 ± 0.2 ms–1, which is in agreement\nwith the ESA recommendation of 0.7 ms–1. The random error was calculated from the standard deviation of the difference\nbetween radiosonde and Aeolus measurements, accounting for radiosonde observation errors estimated at 0.7±0.28 ms–1 and between radiosonde and Aeolus measurements, accounting for radiosonde observation errors estimated at 0.7±0.28 ms–1 and\nrepresentativeness errors ranging from 1.5 to 2.5 ms–1. In the altitude range of 2–16 km and 16–20 km, the random error\n530\nis 3.8 – 4.3 ms–1 and 4.3 – 4.8 ms–1, respectively, which is above the ESA-specified values of 2.5 ms–1 and 3 ms–1, respec-\ntively. In general, Rayleigh-clear shows no error dependency with respect to co-location radius, even for distances reaching\n340 km, whilst being more sensitive to co-location time, especially if the radiosonde measurement is ahead of Aeolus’ over-\nflight time, which presumably corresponds to low altitude measurements. In addition, the systematic and random errors are representativeness errors ranging from 1.5 to 2.5 ms–1. In the altitude range of 2–16 km and 16–20 km, the random error\n530\nis 3.8 – 4.3 ms–1 and 4.3 – 4.8 ms–1, respectively, which is above the ESA-specified values of 2.5 ms–1 and 3 ms–1, respec-\ntively. In general, Rayleigh-clear shows no error dependency with respect to co-location radius, even for distances reaching\n340 km, whilst being more sensitive to co-location time, especially if the radiosonde measurement is ahead of Aeolus’ over-\nflight time, which presumably corresponds to low altitude measurements. In addition, the systematic and random errors are height-dependent, with larger errors occurring in the upper troposphere, mainly caused by the reduction in signal return from\n535\ndecreasing air density, and in lower levels, most likely caused by the signal attenuation by clouds and dust. The error estimate\nlikewise follows a similar form to the observed height error dependency, as it is inversely proportional to the squared root\nof the normalized useful signal. In cases where the normalized useful signal is strongly attenuated by clouds or dust, the er-\nror estimate is generally underestimated, with measurements exhibiting non physical features and departures from radiosonde height-dependent, with larger errors occurring in the upper troposphere, mainly caused by the reduction in signal return from\n535\ndecreasing air density, and in lower levels, most likely caused by the signal attenuation by clouds and dust. The error estimate\nlikewise follows a similar form to the observed height error dependency, as it is inversely proportional to the squared root\nof the normalized useful signal. In cases where the normalized useful signal is strongly attenuated by clouds or dust, the er-\nror estimate is generally underestimated, with measurements exhibiting non physical features and departures from radiosonde winds larger than the error estimate. A redefinition of the Rayleigh-clear error estimate could account for this underestimation\n540\nby including other sources of noise, such as detector noise or readout noise, which increase for reduced signal levels. Fur-\nthermore, a cross-talk, i.e. the leakage of the Mie signal into the Rayleigh receiver, could also explain this underestimation,\nespecially in the case of strong Mie returns. However, this supposition was not investigated in the context of this study. Outliers,\ndefined as measurements with small error estimate and large absolute differences, are found under all conditions, i.e. for all co-location radii, co-location times, altitudes as well as cloud and dust cover. Their origin does not appear to be correlated\n545\nwith low signal levels but seem to be inherent to the statistical nature of the error distribution. Taking other terms into account\nwhen defining the error estimate, such as the influence of temperature, pressure or scattering ratio on the Rayleigh response,\ncould certainly contribute to improving the error characterisation. The ECMWF model equivalents of Rayleigh-clear are found\nto have a significantly better agreement with the radiosonde wind measurements compared to the Rayleigh-clear observations. co-location radii, co-location times, altitudes as well as cloud and dust cover. Their origin does not appear to be correlated\n545\nwith low signal levels but seem to be inherent to the statistical nature of the error distribution. Taking other terms into account\nwhen defining the error estimate, such as the influence of temperature, pressure or scattering ratio on the Rayleigh response,\ncould certainly contribute to improving the error characterisation. The ECMWF model equivalents of Rayleigh-clear are found\nto have a significantly better agreement with the radiosonde wind measurements compared to the Rayleigh-clear observations. This is a further confirmation that the co-location parameters used for this validation study are appropriate and that the model\n550\nequivalents provide a suitable reference for validating Aeolus. In addition, we demonstrate the existence of an orbital- and\naltitude-dependent bias in the Rayleigh-clear channel, which is visible with respect to both radiosondes and ECMWF model\nequivalents. This bias has already been documented by Borne et al. (2023) in West Africa using model equivalents and is now\nconfirmed observationally. The underlying cause for this bias, however, remains unknown. In addition, we find that Rayleigh- For Mie-cloudy, the statistical analysis yielded a systematic negative deviation of –0.9±0.3 ms–1 within ESA specifications\nwhen uncertainty is taken into account, and it is consistent across all orbital nodes and Cal/Val sites. The random error between\n2–16 km is 1.1 – 2.3 ms–1, which falls within the ESA recommendations. The general quality of Mie-cloudy winds does not 26 Competing interests. The authors declare that they have no conflict of interest. Author contributions. MB, PK and MW conceptualized the study and developed the methodology. MB, PK, MW and BW carried out the\n580\ninvestigation and validation. PK, MW, PV and RR provided financial support for the project. MB, PV and RR were responsible for data\ncuration. MB conducted the formal analysis and wrote the original draft of the paper. MB, PK, MW, BW, CF, PV and RR reviewed and\nedited the paper. https://doi.org/10.5194/egusphere-2023-742\nPreprint. Discussion started: 3 May 2023\nc⃝Author(s) 2023. CC BY 4.0 License. depend on the co-location radius, while it is more sensitive to temporal differences. The errors appear to be larger at 5 km and\n560\nabout 1 km altitude, typically at the upper and lower limits of the Saharan Air Layer, where clouds frequently occur. According\nto Lux et al. (2022a), the Mie fringe of the Fizeau interferometer can be distorted in the case of strong backscatter gradients, e.g. at cloud edges. Interestingly, Mie-cloudy does not seem to sample within dust layers, as most bins with high dust concentrations\nare rejected by the QC. Furthermore, the systematic and random Mie errors decrease with the percentage of cloud cover, while depend on the co-location radius, while it is more sensitive to temporal differences. The errors appear to be larger at 5 km and\n560\nabout 1 km altitude, typically at the upper and lower limits of the Saharan Air Layer, where clouds frequently occur. According\nto Lux et al. (2022a), the Mie fringe of the Fizeau interferometer can be distorted in the case of strong backscatter gradients, e.g. at cloud edges. Interestingly, Mie-cloudy does not seem to sample within dust layers, as most bins with high dust concentrations\nare rejected by the QC. Furthermore, the systematic and random Mie errors decrease with the percentage of cloud cover, while they increase in the presence of dust. This may be attributed to the generally weak backscatter of dust, increasing the error of\n565\nthe Mie-cloudy winds. Similar to Rayleigh-clear, outliers with small error estimate and large absolute differences can be found\nfor all co-location distance, co-location time, altitude, dust concentrations and cloud cover. An improvement of the Mie EE is\nexpected from an optimisation of the Mie core algorithm, such as the fitting function or the classification algorithm. The presented study determined the error dependencies of the different Aeolus observation types and error estimates with y\ny\nrespect to tropical clouds and dust. The acquired information are valuable to further improve the processing algorithms in order\n570\nto meet the requirements of the mission. respect to tropical clouds and dust. The acquired information are valuable to further improve the processing algorithms in order\n570\nto meet the requirements of the mission. Code availability. The analysis was conducted using the Python language and the code can be provided on request. Code availability. Acknowledgements. We would like to thank Thorsten Fehr for organising the Joint Aeolus Tropical Atlantic Campaign (JATAC), which\n585\nmade this cross-Atlantic study possible. We would also like to thank the German Aerospace Center (DLR), which assisted in the transport\nof radiosonde material and helium tanks from Germany to Sal in Cape Verde. Sincere gratitude also goes to the large teams involved in the\nradiosonde launches. For the Cape Verdean team, our special thanks go to Azusa Takeishi, Tanguy Jonville and Cedric Gacial for their hard\nwork and dedication. The analysis was conducted using the Python language and the code can be provided on request. Forecasts (ECMWF) model-equivalents are available in the ECMWF Meteorological Archival and Retrieval System (MARS) operational\n575\narchive. Dust mixing ratio data is openly available and can be downloaded in the Copernicus Atmosphere Monitoring Service (CAMS) Data\nStore. The NW SAF Cloud Type (CT) product can by obtained using the software package NWC/PPS available from http://www.nwcsaf.org/. The radiosonde data corresponding to launches from Puerto Rico are publicly available at http://dx.doi.org/10.5067/CPEXAW/DATA101. The other radiosonde data can be provided on request. Author contributions. MB, PK and MW conceptualized the study and developed the methodology. MB, PK, MW and BW carried out the\n580\ninvestigation and validation. PK, MW, PV and RR provided financial support for the project. MB, PV and RR were responsible for data\ncuration. MB conducted the formal analysis and wrote the original draft of the paper. MB, PK, MW, BW, CF, PV and RR reviewed and\nedited the paper. 27 References\n590 J., Langland, R.,\net al.: Lidar-measured wind profiles: The missing link in the global observing system, Bulletin of the American Meteorological Society, Baker, W. E., Atlas, R., Cardinali, C., Clement, A., Emmitt, G. D., Gentry, B. M., Hardesty, R. M., Källén, E., Kavaya, M. J., Langland, R.,\net al.: Lidar-measured wind profiles: The missing link in the global observing system, Bulletin of the American Meteorological Society,\n95, 543–564, 2014. 00 95, 543–564, 2014. 600\nBedka, K. M., Nehrir, A. R., Kavaya, M., Barton-Grimley, R., Beaubien, M., Carroll, B., Collins, J., Cooney, J., Emmitt, G. 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English
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Beyond co-localization: inferring spatial interactions between sub-cellular structures from microscopy images
BMC bioinformatics
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cc-by
11,343
RESEARCH ARTICLE Open Access Open Access * Correspondence: ivos@ethz.ch 1Institute of Theoretical Computer Science, ETH Zurich, Universitätstrasse 6, 8092 Zürich, Switzerland © 2010 Helmuth 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: Sub-cellular structures interact in numerous direct and indirect ways in order to fulfill cellular functions. While direct molecular interactions crucially depend on spatial proximity, other interactions typically result in spatial correlations between the interacting structures. Such correlations are the target of microscopy-based co- localization analysis, which can provide hints of potential interactions. Two complementary approaches to co- localization analysis can be distinguished: intensity correlation methods capitalize on pattern discovery, whereas object-based methods emphasize detection power. Results: We first reinvestigate the classical co-localization measure in the context of spatial point pattern analysis. This allows us to unravel the set of implicit assumptions inherent to this measure and to identify potential confounding factors commonly ignored. We generalize object-based co-localization analysis to a statistical framework involving spatial point processes. In this framework, interactions are understood as position co- dependencies in the observed localization patterns. The framework is based on a model of effective pairwise interaction potentials and the specification of a null hypothesis for the expected pattern in the absence of interaction. Inferred interaction potentials thus reflect all significant effects that are not explained by the null hypothesis. Our model enables the use of a wealth of well-known statistical methods for analyzing experimental data, as demonstrated on synthetic data and in a case study considering virus entry into live cells. We show that the classical co-localization measure typically under-exploits the information contained in our data. Conclusions: We establish a connection between co-localization and spatial interaction of sub-cellular structures by formulating the object-based interaction analysis problem in a spatial statistics framework based on nearest- neighbor distance distributions. We provide generic procedures for inferring interaction strengths and quantifying their relative statistical significance from sets of discrete objects as provided by image analysis methods. Within our framework, an interaction potential can either refer to a phenomenological or a mechanistic model of a physico- chemical interaction process. This increased flexibility in designing and testing different hypothetical interaction models can be used to quantify the parameters of a specific interaction model or may catalyze the discovery of functional relations. Beyond co-localization: inferring spatial interactions between sub-cellular structures from microscopy images Jo A Helmuth1,2, Grégory Paul1,2 and Ivo F Sbalzarini1,2* Jo A Helmuth1,2, Grégory Paul1,2 and Ivo F Sbalzarini1,2* Helmuth et al. BMC Bioinformatics 2010, 11:372 http://www.biomedcentral.com/1471-2105/11/372 Helmuth et al. BMC Bioinformatics 2010, 11:372 http://www.biomedcentral.com/1471-2105/11/372 Background The actual physical or che- mical interaction between sub-cellular objects can be of short temporal duration and they can quickly separate thereafter. In such situations, high thresholds can increase the detection power, but only at the expense of increased false-positive rates. When interactions take place over long distances, the choice of threshold impli- citly determines a range limit of the analysis. Apart from fixing the interaction scale a priori, using a hard distance threshold also implies a binary distinction of pair-wise distances: either they are below the threshold and hence the objects are assumed to interact - or they don’t. A co-localization percentage thus corresponds to an indirect measure for the preference of “interaction” over “non-interaction”. This preference reflects the strength of the interaction. However, it also depends on the frequency of possible distances that the population of objects can assume. y f Such inference, however, entails a trade-off between the objectives of pattern discovery and statistical detec- tion power. According to these objectives, two comple- mentary approaches to co-localization analysis can be distinguished: Intensity correlation methods capitalize on pattern discovery [1], whereas object-based methods [2] emphasize detection power. Intensity correlation meth- ods quantify correlations in the intensities of different color channels on individual pixels. Intensity correlation methods are straightforward to implement and use. The results, however, may be difficult to interpret since inter- actions need to be inferred from correlations in intensity space, which is sensitive to the blurring and noise inher- ent to microscopic imaging systems [3]. Object-based methods quantify the spatial relationships between sets of discrete objects. This requires reducing the image to a set of geometric objects using, e.g., image segmentation or fitting of structure models. Object-based approaches infer interactions from correlations in physical space, which allows constructing intuitive and simple co- localization measures, such as counting the number of overlapping objects [2]. More specifically, the cellular context in which the inter- actions take place is a confounding factor. A high co-loca- lization percentage can, for example, be observed in a cell with densely packed sub-cellular structures of interest, irrespective of their interaction strength. This artifact needs to be considered in statistical tests [7] or corrected for in order to construct an interaction score [8]. Taken together, object-based approaches provide intuitive co-localization measures whose statistical inter- pretation, however, is not straightforward. Background interaction as the collection of all effects that cause sig- nificant (above the level predicted by a null hypothesis) correlations in the positions of the participating objects. interaction as the collection of all effects that cause sig- nificant (above the level predicted by a null hypothesis) correlations in the positions of the participating objects. Over the last decades, advances in fluorescent markers have enabled probing interactions of sub-cellular struc- tures in the microscope, either directly or indirectly. The direct approach relies on experiments that generate a signal upon the proximity required for molecular interaction. Indirect approaches are based on indepen- dently imaging two populations of interest, and A general biological principle states that cellular func- tion results from the combined interactions of sub-cellu- lar structures in space and time. Interactions typically manifest themselves through statistical dependencies in the spatial distributions of the involved structures. Here, we adopt this general definition and we understand Over the last decades, advances in fluorescent markers have enabled probing interactions of sub-cellular struc- tures in the microscope, either directly or indirectly. The direct approach relies on experiments that generate a signal upon the proximity required for molecular interaction. Indirect approaches are based on indepen- dently imaging two populations of interest, and * Correspondence: ivos@ethz.ch 1Institute of Theoretical Computer Science, ETH Zurich, Universitätstrasse 6, 8092 Zürich, Switzerland Helmuth et al. BMC Bioinformatics 2010, 11:372 http://www.biomedcentral.com/1471-2105/11/372 Page 2 of 12 searching for clues of interaction in their spatial distri- butions. This approach is based on the paradigm that spatial proximity (or co-localization) is a hallmark of many types of physical and chemical interactions between sub-cellular structures. If two or more struc- tures interact, their spatial distributions hence appear correlated. The reverse, however, is not necessarily true. Presence or absence of significant co-localization does not imply presence or absence of interaction. The rea- son is that co-localization depends on the specific inter- action mechanism: An unobserved third structure may act as a confounding factor (in the statistical sense), making the observed structures appear co-localized even though they do not interact. Furthermore, one can ima- gine interaction mechanisms that lead to spatial distri- butions with correlations that are not captured by simple co-localization measures. Hence, the interaction has to be statistically inferred from the data. that can be reliably detected. Background Here, we establish a connection between co-localization and the notion of interaction as used in spatial statistics [9], namely the non-independence of the relative positions of objects under study. This is based on modeling the nearest-neighbor distance distribution between the observed objects. These distances are the result of inter- actions, measurement inaccuracies, and the geometry of the domain in which the objects are distributed. This modeling provides generic procedures for inferring interaction strengths and quantifying their statistical sig- nificance. Our approach helps formalizing design deci- sions in co-localization and interaction studies and shows how they translate to biological hypotheses. Stan- dard object-based co-localization analysis is included as a special case, which makes explicit the connections between interaction and co-localization. After develop- ing and characterizing the statistical interaction analysis framework, we exemplify its utility in a biological study of virus entry. The intensity-based approach is limited to interactions on a spatial scale on the order of the resolution of the microscope. While the object-based approach is not necessarily limited to any particular length scale (note that the localization accuracy for an isolated object is not limited by the spatial resolution of the microscope, but rather the signal-to-noise ratio [4-6]), a spatial scale is nevertheless assumed in practice. Many object-based co-localization methods rely on a hard threshold for the distances between objects in order to distinguish between “co-localized” and “not co-localized” for each individual pair of objects [2]. The choice of distance threshold greatly influences the types of interactions Basic scenario: co-localization analysis Basic scenario: co-localization analysis We review the basic concepts of classical object-based co-localization analysis and its interpretation in terms of interactions. Helmuth et al. BMC Bioinformatics 2010, 11:372 http://www.biomedcentral.com/1471-2105/11/372 Helmuth et al. BMC Bioinformatics 2010, 11:372 http://www.biomedcentral.com/1471-2105/11/372 Page 3 of 12 Page 3 of 12 Figure 1 Illustration of co-localization analysis and cellular context. (A) Illustration of co-localization analysis based on nearest neighbor distances (arrows) between point-like objects X = {xi}N i=1 (dots) and circular objects Y = {yj}M j=1 (solid circles). For all distances d, the state density q(d) is proportional to the total length of the d-isoline (dashed lines) in Ω. The expected co-localization in the absence of interactions, Ct 0, is proportional to the area enclosed by the t-isoline (gray region). (B)-(D) Effect of the positioning of the objects Y on q(d), illustrating the influence of the cellular context. Ct = 1 N N  i=1 1(di < t) N→∞ −−−→  t −∞ p(d)dd , (2) (2) where 1(·) is the indicator function and t an applica- tion-specific distance threshold. The form of Eq. 2 implies assumptions about how the objects in X and Y interact. The interaction process is considered to be translation- and rotation-invariant since only the dis- tance between interacting objects is taken into account. Based on this distance only two categories of positions of the objects in X are distinguished: either they are suf- ficiently close to any object in Y to be considered inter- acting, or they are not. Furthermore, objects in X interact with at most one object in Y and they do not experience the presence of any yj unless they cross the distance threshold t. The choice of t reflects an assump- tion about the length scale of the interaction to be detected. Based on this distance only two categories of positions of the objects in X are distinguished: either they are suf- ficiently close to any object in Y to be considered inter- acting, or they are not. Furthermore, objects in X interact with at most one object in Y and they do not experience the presence of any yj unless they cross the distance threshold t. The choice of t reflects an assump- tion about the length scale of the interaction to be detected. Basic scenario: co-localization analysis Inferring interactions from an observed co-localization measure Ct is not trivial since Ct > 0 does not necessa- rily imply any interaction between the objects. This is because spatial correlations can also be caused by con- founding factors, such as the cellular context {Ω, Y}. Even if the objects in X and Y do not interact there is a finite probability that any possible distance in an interval Δd about di is observed. We arbitrarily choose Y as a reference in order to compute the relative frequency of possible distances (state density) as: Figure 1 Illustration of co-localization analysis and cellular context. (A) Illustration of co-localization analysis based on nearest N Figure 1 Illustration of co-localization analysis and cellular context. (A) Illustration of co-localization analysis based on nearest neighbor distances (arrows) between point-like objects X = {xi}N i=1 (dots) and circular objects Y = {yj}M j=1 (solid circles). For all distances d, the state density q(d) is proportional to the total length of the d-isoline (dashed lines) in Ω. The expected co-localization in the absence of interactions, Ct 0, is proportional to the area enclosed by the t-isoline (gray region). (B)-(D) Effect of the positioning of the objects Y on q(d), illustrating the influence of the cellular context. Figure 1 Illustration of co-localization analysis and cellular context. (A) Illustration of co-localization analysis based on nearest neighbor distances (arrows) between point-like objects X = {xi}N i=1 (dots) and circular objects Y = {yj}M j=1 (solid circles). For all distances d, the state density q(d) is proportional to the total length of the d-isoline (dashed lines) in Ω. The expected co-localization in the absence of interactions, Ct 0, is proportional to the area enclosed by the t-isoline (gray region). (B)-(D) Effect of the positioning of the objects Y on q(d), illustrating the influence of the cellular context. Object-based co-localization measures are typically constructed for two sets of objects X = {xi}N i=1 and Y = {yj}M j=1. These objects are located in a bounded region Ω ⊂Rn with boundary ∂Ω and dimensionality n (usually 2 or 3; see Fig. 1). Each object i (j) is repre- sented by a feature vector xi (yj) that comprises infor- mation about the object’s position and, if available, its dimension and shape. These features vectors are extracted from image data by means of image segmenta- tion or fitting of structure models. Basic scenario: co-localization analysis q(d) = lim d→0 Pr(di ∈[d, d + d]|“no interaction”, Y) d . (3) q(d) = lim d→0 Pr(di ∈[d, d + d]|“no interaction”, Y) d . (3) q(d) = q(d) = (3) This density q(d) is determined by the positions, dimensions, and number density of the objects in Y (see Fig. 1). Independent random positions will result in a relatively wide density q(d) (Fig. 1C). With regularly placed objects Y, large distances do not occur (Fig. 1B). Clustering increases the frequency of long distances at the expense of short distances (Fig. 1D). Objects with large surfaces or a large number density give rise to shorter distances. In case there are interactions between the objects in X and Y, some of the possible distances are additionally favored over others, deforming the den- sity q(d) to p(d). Suppose one wishes to investigate the interaction between the objects in X and Y, one can define for each xi the distance to the nearest neighbor (NN) in Y, di = min j  d  xi, yj  . (1) (1) The function d(·) is a suitable distance function in fea- ture space, for example the Euclidean distance between point-like objects or the minimum distance between outlines of more complex objects. A nearest-neighbor distance distribution p(d) can then be estimated from the set of distances D = {di}N i=1. The classical overlap or nearest-neighbor-distance co-localization measure Ct fol- lows by counting [8]: The co-localization measure Ct is, therefore, not suffi- cient to separate the contributions from the cellular context and the interactions. Information about the interactions is only contained in the deviation from an expected base-level in the absence of interactions. This base level, Ct 0, is the co-localization measure that would be observed under the hypothesis H0: “no interaction” Helmuth et al. BMC Bioinformatics 2010, 11:372 http://www.biomedcentral.com/1471-2105/11/372 Page 4 of 12 Page 4 of 12 p (X|, Y) = N i=1 p (xi|, Y) ∝ N i=1 exp (−φ (di)) , (6) (obtained by letting p(d) = q(d) and numerical evalua- tion of the integral in Eq. 2). But how does a certain deviation from the base level Ct 0 relate to interactions between the objects, and what deviations can be consid- ered significant? We address this question in the follow- ing sections by generalizing co-localization analysis to interaction analysis. Generalization: interaction analysis (7) Spatial point process analysis [9-11] is a standard sta- tistical framework for studying the spatial distribution of interacting objects. Our interaction analysis is derived from the general binary Gibbs process with fixed number of objects. Its central component is an effective pair-wise interaction potential F(·). In many applications, “interaction” is an abstraction of the dif- ferent effects that collectively cause an observed spatial pattern. Nevertheless, the mathematical form of the Gibbs process corresponds to physical models of inter- acting objects. The potential associates an energy level with each pair {i, j} of interacting objects. The prob- ability density of the Gibbs process for two sets of interacting objects, X and Y, has the shape of a Boltz- mann distribution: The normalization constant Z (the partition function) renders p(d|q) a true probability density function. The normalization constant Z (the partition function) renders p(d|q) a true probability density function. So far, we have not specified any particular shape for the interaction potential (·), which can be a parametric or non-parametric model. A specific choice constitutes a hypothesis or assumption about the range, strength, and distance dependence of the interaction. These three aspects of the interaction are represented independently in our parameterization: φ(d) = ϵ f d −t σ  . (8) (8)  is the strength, f encodes the shape, s defines the length-scale, and t is a shift along the distance axis of the interaction potential. Using Eqs. 7 and 8 we find the joint probability density of observations D:  is the strength, f encodes the shape, s defines the length-scale, and t is a shift along the distance axis of the interaction potential. Using Eqs. 7 and 8 we find the joint probability density of observations D: p (X, Y) ∝exp ⎛ ⎝− N  i=1 M  j=1   xi, yj  ⎞ ⎠, (4) (4) p  D|q  = Z−N N i=1 q(di) exp  −ϵf di −t σ  . (9) i.e., states with lower energy occur with higher prob- ability. Eq. 4 implies mutual independence of the objects within the same set X or Y, in agreement with the assumptions formulated in the previous section. For nearest-neighbor interactions, the corresponding inter- action potential is given by: (9) This is the central class of models that we use to extend co-localization analysis to interaction analysis. Basic scenario: co-localization analysis Ideally, an interaction score is inde- pendent of the cellular context and reflects variations of the interaction strength in a monotonous fashion. The first step toward constructing such a score is a precise definition of the term interaction strength in the context of an interaction model. (6) where, unlike in Eq. 4, an explicit dependence of the potential on xi is no longer present. The probability of observing a certain xi is propor- tional to exp (-(di)). The probability of observing a cer- tain di, however, also depends on how frequently an arbitrary object x is a distance di away from its NN in the given cellular context. This frequency is given by the state density q(d) as stated in Eq. 3. Straightforward calculations yield: p (d|, Y) = p  d|q  = Z−1q(d) exp (−φ (d)) . (7) Generalization: interaction analysis All interaction models will be formulated as specific instances of such a model.   xi, yj  = φ (di) if yj is NN of xi 0 else , (5) The assumptions underlying the simple overlap co- localization measure can, for example, be formalized in a specific interaction potential. Only two categories of distances (d <t and d ≥t) are distinguished (Eq. 2). This implies a step function for the shape f(z) of the interac- tion potential (d) (taking s = 1): (5) where the function (d) specifies the distance depen- dence of the interaction. Assume a cellular context {Ω,Y} is given. The prob- ability density p(X|Ω,Y) for the potential in Eq. 5 then only depends on the di. An inner sum over all j, as in Eq. 4, is then not required. The mutual independence within X allows factorizing p(X|Ω,Y) into terms that only depend on a single di: φst(d) = ϵ f st(d −t) with f st(z) = −1 if z < 0 0 else . (10) φst(d) = ϵ f st(d −t) with (10) Helmuth et al. BMC Bioinformatics 2010, 11:372 http://www.biomedcentral.com/1471-2105/11/372 Page 5 of 12 Figure 2 Power analysis for a step potential. Minimum Ct (A) and ˆϵ (B) that allows rejecting H0: “no interaction” (a = 0.05) as a function of the base-level Ct 0. In A, the expected value of Ct under H0 is indicated by a dashed line. (C) Statistical power (1 - b) for detecting interactions of a true strength  = 1. Red, green, and blue lines correspond to N = 10, 100, and 1000, respectively, in all three panels. Using the integral definition in Eq. 2, the co-localiza- tion measure Ct can then be expressed as a function of the interaction strength. Inserting Eq. 10 into Eq. 7 and Eq. 2 and solving for  yields an estimator ˆϵ of the model interaction strength: ˆϵ = ˆϵ(N, q) = log  Ct 1 −Ct  −log  Ct 0 1 −Ct 0  . (11) (11) The quantity ˆϵ corrects for the cellular context and, therefore, fulfills our requirements for a valid interaction score. Eq. 11 relates the purely descriptive co-localiza- tion measure Ct to an interaction model between the objects in X and Y. Hypothesis testing and power analysis for the step potential In the parameterization of our interaction model (Eqs. 8 and 9), the presence of an interaction is equivalent to  ≠0. Since ˆϵ is an estimator, it is a random variable. Even if the hypothesis H0: “no interaction” is true, a non-zero ˆϵ can occur with finite probability (ˆϵ ≠0 does not imply  ≠0). Inference about interactions requires finding a critical estimated interaction strength above which one can reject H0 on a prescribed significance level a. Figure 2 Power analysis for a step potential. Minimum Ct (A) and ˆϵ (B) that allows rejecting H0: “no interaction” (a = 0.05) as a function of the base-level Ct 0. In A, the expected value of Ct under H0 is indicated by a dashed line. (C) Statistical power (1 - b) for detecting interactions of a true strength  = 1. Red, green, and blue lines correspond to N = 10, 100, and 1000, respectively, in all three panels. Figure 2 Power analysis for a step potential. Minimum Ct (A) and ˆϵ (B) that allows rejecting H0: “no interaction” (a = 0.05) as a function of the base-level Ct 0. In A, the expected value of Ct under H0 is indicated by a dashed line. (C) Statistical power (1 - b) for detecting interactions of a true strength  = 1. Red, green, and blue lines correspond to N = 10, 100, and 1000, respectively, in all three panels. critical value does, however, not guarantee that it will always be detected by the test (type II error: b). Further- more, a weak interaction may lead to unwanted rejec- tion of H0. The behavior of the test critically depends on the effect size, which quantifies the departure from H0. Here, effect size refers to the true interaction strength  = a > 0. The statistical “power” (1 - b) quan- tities the probability of rejecting H0 when H1: “ = st,  = a“ is true. In Fig. 2C, the detection power for a true strength of a = 1 is shown as a function of Ct 0. As expected from Fig. 2B, the power is low at the extremes of Ct 0, eventually dropping significantly below the recommended value of 0.8, even for N = 100. Weak interactions are harder to detect, requiring larger sample sizes to yield a certain power. Generalization: interaction analysis It builds a bridge between patterns in the data (the cellular context summarized in q and the measure Ct) and functional relationships (interac- tions) between sub-cellular components. Whether an observed estimate ˆϵ is indicative of the actual presence of an interaction, however, has to be addressed using statistical inference as presented in the following section. Hypothesis testing and power analysis for the step potential This critical interaction strength is determined by the distribution of ˆϵ under H0 (null distribution), which depends on the sample size N, q, and the prescribed a. Under H0, CtN is binomially distributed with parameters (Ct 0, N). Hence, the critical Ct can be computed from the (numerically) inverted cumulative distribution func- tion of the binomial distribution. The corresponding cri- tical ˆϵ follows from Eq. 11. The dependence of the critical Ct and ˆϵ on Ct 0 and N is shown in Fig. 2A and 2B. It can be seen that the minimum significant excess over Ct 0 varies only weakly with Ct 0 (Fig. 2A). Obviously, large values of Ct 0 in con- junction with small N do not allow rejecting H0, even if Ct = 1. The critical value of ˆϵ is highest at the two extremes of Ct 0 and lowest for Ct 0 ≈0.4 (Fig. 2B). As for Ct, it can be seen that for large Ct 0 and small N no finite ˆϵ is sufficiently large to allow rejecting H0. In the design of experimental interaction studies, a key objective is to maximize the robustness and reliability of detecting effects of unknown size. Power can be increased by optimizing the experimental design or the subsequent statistical analysis. While increasing the sample size might be possible, controlling the cellular The curves in Fig. 2B show the decision of the statisti- cal test based on the estimated interaction strength ˆϵ. A true interaction with a strength  greater than this Helmuth et al. BMC Bioinformatics 2010, 11:372 http://www.biomedcentral.com/1471-2105/11/372 Page 6 of 12 Figure 3 Power analysis for non-step potentials. (A) Black line: state density q(d) for M = 100 circular objects Y with radius R = 3.57 randomly placed in a square domain of size 200 × 200; R is chosen to yield a circle-covered area fraction of 0.1; Colored lines: resulting distance distribution p(d) for the three potentials shown in B. (B) Plummer potential (Eq. 12) with  = 1 and varying scale parameter. (C) Monte-Carlo estimates of 80%-power isolines in the N-a-plane; dashed lines: tests based on Tst, solid lines: tests based on Tpl. Note that larger kinks in the dashed lines are due to the discreteness of Tst and are statistically significant. Hypothesis testing and power analysis for the step potential Colors in A-C indicate scale parameters of the true potential; red: s = 0.2, green: s = 1.0, and blue: s = 5.0. context is not feasible in most situations. Our analysis is based on the interaction model introduced in the pre- vious section. It allows specifying different shapes f(·) and scales s of the interaction potential. Power could potentially be increased by better modeling the interac- tion potential. In the next section, we thus quantify the influence of alternative model potentials on statistical power. Improving statistical power with non-step interaction potentials Constructing statistical tests as described above requires assuming a specific shape and scale of the interaction potential. In the absence of prior knowledge, however, this model potential can be arbitrarily different from the true potential of the actual biological interactions under observation. Test statistics that are based on a model potential close to the real one may achieve greater power. We quantify the influence of the discrepancy between the model and the true potential by considering a scenario where N objects {xi} are distributed in the square region Ω containing M randomly placed circular objects {yi} with identical radii R. Fig. 3A shows the corresponding state density q(d). The objects in X interact with the objects in Y according to the Plummer potential (with t = 0): φpl(d) = ϵ f pl  d σ  with f pl (z) = −  z2 + 1 −0.5 if z > 0 −1 else . (12) (12) Figure 3 Power analysis for non-step potentials. (A) Black line: state density q(d) for M = 100 circular objects Y with radius R = 3.57 randomly placed in a square domain of size 200 × 200; R is chosen to yield a circle-covered area fraction of 0.1; Colored lines: resulting distance distribution p(d) for the three potentials shown in B. (B) Plummer potential (Eq. 12) with  = 1 and varying scale parameter. (C) Monte-Carlo estimates of 80%-power isolines in the N-a-plane; dashed lines: tests based on Tst, solid lines: tests based on Tpl. Note that larger kinks in the dashed lines are due to the discreteness of Tst and are statistically significant. Colors in A-C indicate scale parameters of the true potential; red: s = 0.2, green: s = 1.0, and blue: s = 5.0. Figure 3 Power analysis for non-step potentials. (A) Black line: state density q(d) for M = 100 circular objects Y with radius R = 3.57 randomly placed in a square domain of size 200 × 200; R is chosen to yield a circle-covered area fraction of 0.1; Colored lines: resulting distance distribution p(d) for the three potentials shown in B. (B) Plummer potential (Eq. 12) with  = 1 and varying scale parameter. (C) Monte-Carlo estimates of 80%-power isolines in the N-a-plane; dashed lines: tests based on Tst, solid lines: tests based on Tpl. Improving statistical power with non-step interaction potentials Note that larger kinks in the dashed lines are due to the discreteness of Tst and are statistically significant. Colors in A-C indicate scale parameters of the true potential; red: s = 0.2, green: s = 1.0, and blue: s = 5.0. Figure 3 Power analysis for non-step potentials. (A) Black line: state density q(d) for M = 100 circular objects Y with radius R = 3.57 Figure 3 Power analysis for non-step potentials. (A) Black line: state densit q(d) for M 100 circ lar objects Y ith radi s R 3 57 This potential has an overall 1 = d-shape, but finite value and slope everywhere. The parameter  again con- trols the interaction strength (potential depth). The parameter s sets the length scale of the interaction (potential range) and allows gradually changing (d) from a step-like shape to a potential that causes signifi- cant attraction toward the objects in Y over large dis- tances (see Fig. 3B). For a set of distances D, distributed according to Eq. 9 with (d) = pl(d), a test for the presence of interactions can thus be constructed based on Tpl = −N i=1 f pl(di/σ) under H0: “no interaction”, where the scale parameter s is assumed to be known. The null-distribution can be approximated by i.i.d. Monte Carlo (MC) samples {Tpl k }K k=1 (see Materials and Methods). An observed value of Tpl is then ranked among the {Tpl k }K k=1. If it ranks higher than ⌈(1 - a)K⌉-th, H0 is rejected on the signifi- cance level a [12]. The statistical power of this test to reject H0 when H1:  = pl,  = a“ is true, can be For such more general potentials, algebraic expres- sions for ˆϵ (such as Eq. 11 for the step potential) can in general not be derived. Statistical tests for the presence of interactions can nevertheless be constructed using a different statistic. Since Eq. 9 describes a member of the exponential family, T = − N  i=1 f di −t σ  , (13) (13) is a sufficient test statistic for  [12]. Helmuth et al. BMC Bioinformatics 2010, 11:372 http://www.biomedcentral.com/1471-2105/11/372 Page 7 of 12 endocytosis and a marker for early endosomes. These dynamic, lipid-bounded organelles are formed by invagi- nations of the plasma membrane. They are the first sort- ing compartment of clathrin-derived cargo [13]. Improving statistical power with non-step interaction potentials However, Ad2 exhibits significant interaction with endo- somes in half of the cells (a = 0.05). Like Ad2, TS1 is known to enter the cell by clathrin- mediated endocytosis, but the mutation inhibits escape from endosomes [14,15]. This should be reflected in a deviation of the empirical distribution of observed dis- tances D from the null distribution p(d) = q(d), which is stronger for TS1 than for Ad2. In our framework, this translates to a non-flat interaction potential between virus centroids and outlines of Rab5-positive endosomes. Fig. 3C shows the number of samples required to reach 80% power as a function of the strength a of the true interaction potential. It can be seen that the power of a test based on the true interaction potential (solid lines) is higher than the power of a test based on a step potential (dashed lines). Moreover, this difference strongly increases with increasing potential range s: for s = 5 (blue lines) using the step model potential requires 4 times more samples. If the true potential is close to a step potential (s = 0.2, red lines), both tests perform comparably well. Moreover, the figure also shows that interactions over longer distances are harder to detect. We therefore conclude that one needs to be careful when assuming a step potential (as implicitly done in traditional co-localization analysis). Controlling power requires prior knowledge about the interaction potential. Such prior knowledge can easily be included in the present framework by choosing t, s, and f(·). Before modeling an interaction potential, we test H0: “(d) = 0” against H1: “(d) ≠0” for each imaged cell using a non-parametric statistical test (see Materials and Methods). This test does not assume any specific shape of the interaction potential, which allows detecting any type of interaction, albeit with reduced power. The results are summarized in Table 1. The fraction of cells for which H0 has to be rejected is significantly higher for TS1 than for Ad2, irrespective of the significance level and despite the on average smaller sample sizes N. However, Ad2 exhibits significant interaction with endo- somes in half of the cells (a = 0.05). Example: virus trafficking These results indicate that the interaction potential is non-zero for many cells. They do not, however, permit any conclusions about the shape or strength of the interac- tion potential, for which, in addition, no prior information is available. We therefore apply a non-parametric estima- tion procedure for the interaction potential to get a sketch of its strength and distance-dependence. Subsequently we can specify and identify parametric potentials. Ignoring, for now, possible variability between cells and virus types, The uptake and intracellular transport of virus particles is a complex process that involves temporary association with membrane receptors and multiple organelles of the endocytic machinery, such as early and late endosomes [13]. In many cases, fluorescence microscopy allows resolving the involved entities as discrete objects. This has previously motivated the use of object-based co- localization measures to quantify association kinetics and unravel infection pathways. Here, we show how the generalized framework of interaction analysis presented above can be applied in a practical experimental situa- tion, and how it enables using a large toolbox of well- known statistical techniques. Improving statistical power with non-step interaction potentials Either fluorescently tagged Adenovirus serotype 2 (Ad2) or its temperature sensitive mutant (TS1) were recorded in the red color channel. Images were taken between 2 and 46 min post infection. The same data have already been used in a previous study [5]. Virus positions and endo- some outlines were extracted from the images as described in the Materials and Methods section. Based on these object representations, the set D of virus-to- nearest-endosome distances and the state density q(d) were computed for each of the imaged cells. estimated with additional MC simulations: For a fixed effect size a > 0, one draws N distances di from p(d), computes Tpl, and conducts the test as described above [12]. This procedure is repeated many times and the fraction of tests rejected serves as an estimator of the power. In order to quantify the influence of the model poten- tial on statistical power, we test H0 against H1 and H2: “ = st,  = a“ on data generated under H1 for varying s (see Fig. 3B for the true interaction potentials under H1). Testing H0 against H2 makes use of the sufficient statistic Tst = −N i=1 f st(di), which is proportional to Ct with t = 0. As opposed to Tpl, this statistic only contains information about the signs of the di and should thus yield a less powerful test. p g Like Ad2, TS1 is known to enter the cell by clathrin- mediated endocytosis, but the mutation inhibits escape from endosomes [14,15]. This should be reflected in a deviation of the empirical distribution of observed dis- tances D from the null distribution p(d) = q(d), which is stronger for TS1 than for Ad2. In our framework, this translates to a non-flat interaction potential between virus centroids and outlines of Rab5-positive endosomes. Before modeling an interaction potential, we test H0: “(d) = 0” against H1: “(d) ≠0” for each imaged cell using a non-parametric statistical test (see Materials and Methods). This test does not assume any specific shape of the interaction potential, which allows detecting any type of interaction, albeit with reduced power. The results are summarized in Table 1. The fraction of cells for which H0 has to be rejected is significantly higher for TS1 than for Ad2, irrespective of the significance level and despite the on average smaller sample sizes N. Table 1 Results of non-parametric statistical tests for interaction in the virus trafficking data Scale parameters ˆσ ∗of potentials as found by maximum-likelihood estimation, and the corresponding maximized pooled log-likelihoods max l* for the different potentials (Eq. 17.) which take into account the smaller number of free parameters. With a difference in log-likelihood of > 103 to second-best fit, the Hermquist potential is by far the best fit. It is also subjectively most similar to the non- parametric potential identified above. Fig. 5 shows an example of an imaged cell, infected with TS1, together with the empirical and estimated distance distributions and the corresponding Hermquist potential. The images of Ad2-infected cells are visually indistinguishable from those of TS1-infected cells and are hence not shown. Despite fitting only one independent parameter (s* is fixed from the estimate over all cells), the estimated model distribution captures the features of the data remarkably well. The parameters of the potentials are found by maxi- mum likelihood estimation (MLE). In order to exclude cell-to-cell variations of the potential range, we do not determine the pairs (k, sk) for each cell separately. Rather, we estimate for a given potential a single scale parameter sk = s* common to all cells, while the inter- action strengths k may vary between cells. The resulting (Ncells+1)-dimensional estimation problem is solved with a nested ML algorithm (see Materials and Methods). The common scale ˆσ ∗and the maximum of the pooled log-likelihood l* for the four potentials are reported in Table 2. As a reference, the values are also given for a step potential with distance threshold t = 0. The estimated interaction strength ˆϵ of the Hermquist potential varies within and between the two groups of infected cells. The within-group variability comprises statistical fluctuations and natural variations between cells. Since virus internalization and transport is a dynamic process, the time at which a cell was imaged (time post infection) is a further source of in-group variability. Fig. 6 shows the estimated interaction strength of a Hermquist potential for all cells infected with Ad2 (blue crosses) and TS1 (red circles) as a func- tion of the time post infection. Throughout the observa- tion period, the interaction strength for TS1 is significantly larger than that for Ad2, confirming the trend reported in Table 1. Furthermore, a temporal maximum of the interaction strength is apparent for TS1, while for Ad2 no significant variation over time can be resolved. Table 1 Results of non-parametric statistical tests for interaction in the virus trafficking data These results indicate that TS1 and Ad2 use different uptake pathways or exhibit signifi- cantly different escape kinetics from Rab5-positive endosomes. The potentials are ranked according to their log-likeli- hood. It can be seen that the step potential is outper- formed by all others. This remains unchanged even if one compares Akaike or Bayesian information criteria, Figure 4 Non-parametric estimate of the interaction potential. The non-parametric estimate of the interaction potential based on all imaged cells. Table 1 Results of non-parametric statistical tests for interaction in the virus trafficking data Table 1 Results of non-parametric statistical tests for interaction in the virus trafficking data #cells p < 0.05 p < 0.01 N Ad2 135 70 (52%) 25 (19%) 180 ± 50 TS1 139 128 (92%) 100 (72%) 157 ± 59 First column: number of cells analyzed; second and third columns: number and percentage of cells for which H0 was rejected on the indicated significance levels; forth column: mean and standard deviation of the observed number of virus particles per cell. We consider a set of 274 two-color fluorescence microscopy images of single HER-911 cells expressing the small GTPase Rab5 tagged with enhanced green fluorescent protein (EGFP), recorded in the green color channel. Rab5 is a regulator of clathrin-mediated Helmuth et al. BMC Bioinformatics 2010, 11:372 http://www.biomedcentral.com/1471-2105/11/372 Page 8 of 12 Table 2 Comparison of estimated scale parameters of interaction potentials for the virus trafficking data ˆσ ∗ max l* rank Hermquist 3.96 -1.2247·105 1 Linear, type 1 4.14 -1.2362·105 2 Linear, type 2 6.61 -1.2427·105 4 Plummer 1.15 -1.2374·105 3 Step (t = 0) -1.2632·105 5 Scale parameters ˆσ ∗of potentials as found by maximum-likelihood estimation, and the corresponding maximized pooled log-likelihoods max l* for the different potentials (Eq. 17.) Table 2 Comparison of estimated scale parameters of interaction potentials for the virus trafficking data we pool all data and estimate a common non-parametric potential φn.p.(d) (see Materials and Methods). The esti- mated ˆφn.p.(d) is shown in Fig. 4. Its shape is notably differ- ent from a step function. The slow decay suggests that viruses interact with endosomes over distances of about 10 pixels (1 μm) from their center. The estimated non-parametric potential serves as a template for the shape of parametric models. Parametric potentials can be identified more robustly from sets of observed distances of individual cells. This allows corre- lating their parameters with co-variates such as the virus type or the time at which a cell was imaged after infec- tion. We consider four different potentials, two that resemble the shape in Fig. 4 (Hermquist and Linear type 1) and two that are generalizations of the step potential with a plateau below d = 0 (Linear type 2 and Plum- mer). For all potentials, we fix the threshold to t = 0. Definitions of the potential shapes f(·) are given in the Materials and Methods section. Conclusions Nearest-endosome-distance isolines (dashed red lines) are shown in the magnified inset. (B) State density q(d) for the shown cell (dashed black line), observed virus-to-nearest-endosome distances (marks and histogram, N = 143), and estimated distance distribution from the model p(d) (solid black line). (C) Estimated Hermquist potential (ˆϵ = 3.90, ˆσ ∗= 3.96) of the interactions between viruses and nearest endosomes. Figure 5 Interaction analysis applied to virus trafficking. Figure 5 Interaction analysis applied to virus trafficking. Interaction analysis for a single cell infected with TS1, imaged 27 min post infection. (A) Imaged endosomes (Rab5-EGFP) with overlaid outlines (solid red lines) and virus centroid positions (blue crosses, virus channel not shown). Nearest-endosome-distance isolines (dashed red lines) are shown in the magnified inset. (B) State density q(d) for the shown cell (dashed black line), observed virus-to-nearest-endosome distances (marks and histogram, N = 143), and estimated distance distribution from the model p(d) (solid black line). (C) Estimated Hermquist potential (ˆϵ = 3.90, ˆσ ∗= 3.96) of the interactions between viruses and nearest endosomes. This limitation can be relaxed by affording more gen- eral shapes of the interaction potential, which naturally extends co-localization analysis to (spatial) co-distribu- tion analysis without requiring any additional assump- tions. The additional flexibility allows capturing information about a wider range of sub-cellular interac- tions. This was demonstrated by statistical power analysis of the classical and generalized measures. Our results highlight that the probability of detecting an interaction strongly depends on the cellular context. We furthermore illustrated the influence of the range of an interaction on its detectability. Test statistics that include knowledge about the shape of the true interaction potential can greatly reduce the number of samples required to achieve a certain target power. Physico-chemical models might provide such prior knowledge. Alternatively, a non-para- metric phenomenological potential can be estimated from the data as demonstrated here. This potential can then serve as a template for the parametric potentials used in subsequent analyses. In addition, the present fra- mework enables comparison of the likelihoods of spatial statistics framework based on nearest-neighbor distance distributions. The present framework provides generic procedures for inferring interaction strengths and quantifying their statistical significance. Standard object-based co-localization analysis is included as a limit case, making explicit the connections between the present framework and more classical approaches. Conclusions (A) Imaged endosomes (Rab5-EGFP) with overlaid outlines (solid red lines) and virus centroid positions (blue crosses, virus channel not shown). Nearest-endosome-distance isolines (dashed red lines) are shown in the magnified inset. (B) State density q(d) for the shown cell (dashed black line), observed virus-to-nearest-endosome distances (marks and histogram, N = 143), and estimated distance distribution from the model p(d) (solid black line). (C) Estimated Hermquist potential (ˆϵ = 3.90, ˆσ ∗= 3.96) of the interactions between viruses and nearest endosomes. Figure 6 Time-resolved interaction analysis of the trafficking of two strains of viruses. Estimated strength of a Hermquist potential (scale s* = 3.96) for the interaction between endosomes and virus particles versus the time post infection. Red circles: TS1; blue crosses: Ad2. The time course of the mean (solid lines) and the ± 1 standard deviation interval (shaded bands) are estimated using a Nadaraya-Watson kernel estimator with bandwidth of 5 min. Figure 6 Time-resolved interaction analysis of the trafficking of two strains of viruses. Estimated strength of a Hermquist potential (scale s* = 3.96) for the interaction between endosomes and virus particles versus the time post infection. Red circles: TS1; blue crosses: Ad2. The time course of the mean (solid lines) and the ± 1 standard deviation interval (shaded bands) are estimated using a Nadaraya-Watson kernel estimator with bandwidth of 5 min. Figure 6 Time-resolved interaction analysis of the trafficking of two strains of viruses. Estimated strength of a Hermquist potential (scale s* = 3.96) for the interaction between endosomes and virus particles versus the time post infection. Red circles: TS1; blue crosses: Ad2. The time course of the mean (solid lines) and the ± 1 standard deviation interval (shaded bands) are estimated using a Nadaraya-Watson kernel estimator with bandwidth of 5 min. dependence of the interaction. We have shown that classical co-localization analysis amounts to estimating the parameters of a step potential. This requires a notion of “inside” and “outside”, either naturally defined by the physical extent of the objects or imposed through the step function’s distance threshold. For point-like objects, or weak correlations between object positions, the choice of distance threshold is arbitrary. Figure 5 Interaction analysis applied to virus trafficking. Interaction analysis for a single cell infected with TS1, imaged 27 min post infection. (A) Imaged endosomes (Rab5-EGFP) with overlaid outlines (solid red lines) and virus centroid positions (blue crosses, virus channel not shown). Conclusions We have introduced a statistical inference framework for robustly estimating interaction parameters from experimentally observed object distributions. Figure 4 Non-parametric estimate of the interaction potential. The non-parametric estimate of the interaction potential based on all imaged cells. This allowed establishing a connection between spatial co-distributions of objects and interaction, by formulat- ing the object-based interaction analysis problem in a Helmuth et al. BMC Bioinformatics 2010, 11:372 http://www.biomedcentral.com/1471-2105/11/372 Page 9 of 12 Figure 5 Interaction analysis applied to virus trafficking. Interaction analysis for a single cell infected with TS1, imaged 27 min post infection. (A) Imaged endosomes (Rab5-EGFP) with overlaid outlines (solid red lines) and virus centroid positions (blue crosses, virus channel not shown). Nearest-endosome-distance isolines (dashed red lines) are shown in the magnified inset. (B) State density q(d) for the shown cell (dashed black line), observed virus-to-nearest-endosome distances (marks and histogram, N = 143), and estimated distance distribution from the model p(d) (solid black line). (C) Estimated Hermquist potential (ˆϵ = 3.90, ˆσ ∗= 3.96) of the interactions between viruses and nearest endosomes. dependence of the interaction. We have shown classical co-localization analysis amounts to estim the parameters of a step potential. This requi notion of “inside” and “outside”, either naturally de by the physical extent of the objects or imposed thr the step function’s distance threshold. For poin objects, or weak correlations between object posit the choice of distance threshold is arbitrary. This limitation can be relaxed by affording more eral shapes of the interaction potential, which natu extends co-localization analysis to (spatial) co-dist tion analysis without requiring any additional assu tions. The additional flexibility allows captu information about a wider range of sub-cellular int tions. This was demonstrated by statistical power an of the classical and generalized measures. Our re Figure 6 Time-resolved interaction analysis of the traffickin two strains of viruses. Estimated strength of a Hermquist pote (scale s* = 3.96) for the interaction between endosomes and v particles versus the time post infection. Red circles: TS1; blue crosses: Ad2. The time course of the mean (solid lines) and the standard deviation interval (shaded bands) are estimated using Nadaraya-Watson kernel estimator with bandwidth of 5 min. Figure 5 Interaction analysis applied to virus trafficking. Interaction analysis for a single cell infected with TS1, imaged 27 min post infection. Conclusions We empirically found that the strengths of the potentials estimated from the projected 2D data may be smaller than those estimated directly on the raw 3D data (data not shown). Although all distances D are systematically reduced by the projection, this effect is overcompensated by the non-linear distortion of q(d), which is strongest for intermediate distances, but negligible for very small and large distances. Besides projection artifacts, errors in the image proces- sing may also influence the estimated co-localization measures. Depending on the accuracy of the image segmentation method used, object sizes can be under- or overestimated, or entire objects can be missed alto- gether. This problem is inherent to all forms of co- localization or distribution analysis. We have assessed the sensitivity of our method with respect to image segmentation errors by successively eroding or dilating the endosomes from the presented case study. The results show that the mean of the estimated strength of the Hermquist potential remains unaffected, yet the variance of the estimate increases for strong erosion when entire endosomes start to be missed (data not shown). This robustness of the present method is due to the state density q(d) correcting for size errors. The classical co-localization measure, naively corrected for the cellular context by subtracting the amount of unspecific co-localization C0, significantly changes when under- or over-estimating object sizes. For strong erosion, leading to very small and frequently All of these limitations could be relaxed by using posi- tion-dependent interaction potentials or allowing for many-body interactions as described by general Gibbs processes. Considering such processes, however, is theo- retically and numerically challenging. The presented fra- mework could also be extended by including additional confounding factors, such as imaging artifacts causing spurious co-localization. Temporal plasticity of interac- tions, cell-to-cell variations, and experiment-to-experi- ment variations could be accounted for through additional co-variates (time, cell index, experiment index) in the statistical model. Already in its present form, the statistical framework can be used to test more general hypotheses, such as “interactions are stronger in strain A than in strain B”. The interpretation of fitted potentials is limited to their relative strengths. In the absence of a mechanistic or physical model of the process that has created the observed spatial pattern, biophysical interpretation of the identified parameter values is difficult or misleading. Conclusions In the present framework, two novel key quantities emerge: (i) the state density q(d), which is the distribu- tion of nearest-neighbor distances expected under the null hypothesis of no interaction, and (ii) the interaction potential (d), which defines the strength and distance Helmuth et al. BMC Bioinformatics 2010, 11:372 http://www.biomedcentral.com/1471-2105/11/372 Page 10 of 12 Page 10 of 12 different hypothetical physico-chemical interaction mod- els directly on the original image data. missing objects, it even drops to a meaningless value of zero (data not shown). Since image segmentation errors are always present in practical applications, we consider the robustness of our method one of its major advantages over classical measures. The present approach enables applying a wide range of established statistical tools for analyzing experimen- tal data, from parameter identification to model selec- tion. This workflow was illustrated by studying the spatial patterns of endosomes and viruses infecting live human cells. In this case study, the experimental data were very well explained using only a single free para- meter per cell. Among the five potentials considered, the step potential (corresponding to the classical co- localization measure) was worst in explaining the data. This highlights the benefit of the present method over classical co-localization analysis. Moreover, the fitted potentials provided additional quantitative readouts that could be used in subsequent machine learning analyses. The presented framework is limited by the same assumptions that also underlie classical co-localization analysis: (i) spatial homogeneity and (ii) isotropy of the interaction within the observation window, and (iii) exclusively nearest-neighbor interactions between objects of different classes. Assumption (i) is, e.g., violated if large areas of the analyzed images do not contain any objects. In this case, estimation of q(d) is not robust. Assumption (iii) imposes limits on admissible distances between objects: If objects X are attracted toward objects Y, the distances between the objects within the set Y need to be larger than the typical interaction range. y For simplicity the case study was done on 2D projec- tions of 3D images. The presented approach, however, is equally applicable in three dimensions without any changes, provided three-dimensional object detection and segmentation is available. Projecting the data into two dimensions alters the estimated potentials (as it also does for any other co-localization measure), since it distorts both the distance data D and the state den- sity q(d). Conclusions This is because the fitted interaction potentials reflect the collection of all intracellular phenomena that lead to the observed point pattern. Interestingly, however, a relation between the steady-state distribution of a diffu- sion process with added deterministic forces and the distribution of the Gibbs process (Eq. 4) exists: If the deterministic force acting between the diffusing objects is given by -∂/∂d, the two distributions become identi- cal (in appropriate units). This fact points a possibility of connecting fitted interaction potentials with biophysi- cal processes. Image acquisition and processing Endosomes and virus particles were imaged with a high- resolution spinning disk confocal microscope (NA 1.35, 100× objective plus additional 1.6× lens, 100 nm pixel Helmuth et al. BMC Bioinformatics 2010, 11:372 http://www.biomedcentral.com/1471-2105/11/372 Page 11 of 12 The parametric tests used in sections “Hypothesis test- ing and power analysis for the step potential” and “Improving statistical power with non-step interaction potentials” followed a simpler protocol. The ranking was directly performed among the scalar test statistics Tst and Tpl, avoiding the detour via U. A priori estimation of the expectation and variance of Tst and Tpl was there- fore not required. size) as described [5]. We acquired z-stacks of 8 images each with a 400 nm z-spacing. Stacks were maximum projected prior to image analysis. Endosome outlines were represented as piece-wise linear closed splines in the focal plane. Outlines were estimated from images using a specialized model-based image analysis techni- que [5], yielding sub-pixel localization accuracy and pre- cision. Virus particles were modeled as points and represented by estimated intensity centroid positions [6]. Prior to distance measurement, relative shifts between virus and endosome positions due to chromatic aberra- tion were corrected using an empirical calibration func- tion [5,16]. The boundary ∂Ω of the region Ω was defined as the cell boundary. An approximation of it was found by low-pass filtering and thresholding of the endosome images. ML estimation of potentials For a given potential , the log-likelihood of its para- meters Θ given the observations Dk in cell k is: l(|Dk, k) = Nk  i=1 pφ(dk,i|qk) = −Nk log  Zk()  + Nk  i=1 log  qk  dk,i  −φ  dk,i;   . (16) (16) Measuring q(d) The state density q(d) was determined from the objects {yi} contained in the region Ω. Positions x in Ω were sampled exhaustively on a uniform Cartesian grid with spacing h = 0.25 pixel. For each x, the dis- tance di to the nearest neighbor in Y was computed. Using this finite sample of distances D = {di}i, an approximation of q(d) was found by Gaussian kernel smoothing density estimation using the MATLAB (The MathWorks, Inc.) function ksdensity.m with default settings. Simultaneous estimation of the common scale s* and independent strengths k of a set of Ncells cells was done by maximizing the pooled log-likelihood: l∗({k}|{Dk}) = Ncells  k=1 l(k|Dk, k) (17) (17) with respect to the parameters {k} = {(ϵk, σ ∗)}. This was done by numerically maximizing (using Nelder- Mead simplex) the sum of maxima maxϵkl((ϵk, σ ∗)|Dk, k) with respect to s*. Test for interaction Greber (University of Zurich) for providing experimental data. JAH further thanks Rajesh Ramaswamy (ETH Zurich) for encouraging comments on early results and Christian Müller (ETH Zurich) for his help with CMA-ES. and Urs F. Greber (University of Zurich) for providing experimental data. JAH further thanks Rajesh Ramaswamy (ETH Zurich) for encouraging comments on early results and Christian Müller (ETH Zurich) for his help with CMA-ES. Implementation Stat Sci 2000, 15:61-78. 11. Diggle PJ: Statistical Analysis of Spatial Point Patterns A Hodder Arnold Publication, 2nd edition 2003. 12. Assunção R: Score test for pairwise interaction parameters of Gibbs point processes. Braz J Probab Stat 2003, 17:169-178. 12. Assunção R: Score test for pairwise interaction parameters of Gibbs point processes. Braz J Probab Stat 2003, 17:169-178. 13. Mellman I, Warren G: The road taken: Past and future foundations of membrane trafic. Cell 2000, 100:99-112. 13. Mellman I, Warren G: The road taken: Past and future foundations of membrane trafic. Cell 2000, 100:99-112. 14. Imelli N, Ruzsics Z, Puntener D, Gastaldelli M, Greber UF: Genetic reconstitution of the human Adenovirus type 2 temperature-sensitive 1 mutant defective in endosomal escape. Virol J 2009, 6(174). 14. Imelli N, Ruzsics Z, Puntener D, Gastaldelli M, Greber UF: Genetic reconstitution of the human Adenovirus type 2 temperature-sensitive 1 mutant defective in endosomal escape. Virol J 2009, 6(174). Additional material p 15. Gastaldelli M, Imelli N, Boucke K, Amstutz B, Meier O, Greber UF: Infectious Adenovirus Type 2 Transport Through Early but not Late Endosomes. Trafic 2008, 9(12):2265-2278. 15. Gastaldelli M, Imelli N, Boucke K, Amstutz B, Meier O, Greber UF: Infectious Adenovirus Type 2 Transport Through Early but not Late Endosomes. Trafic 2008, 9(12):2265-2278. Additional file 1: MATLAB source code. ZIP archive containing the MATLAB source code for potentials, likelihood functions, and statistical tests, as well as sample scripts and sample data at the time of writing. 16. Kozubek M, Matula P: An efficient algorithm for measurement and correction of chromatic aberrations in fluorescence microscopy. J Microsc 2000, 200:206-217. 16. Kozubek M, Matula P: An efficient algorithm for measurement and correction of chromatic aberrations in fluorescence microscopy. J Microsc 2000, 200:206-217. 17. Heikkinen J, Penttinen A: Bayesian smoothing in the estimation of the pair potential function of Gibbs point processes. Bernoulli 1999, 5(6):1119-1136. 17. Heikkinen J, Penttinen A: Bayesian smoothing in the estimation of the pair potential function of Gibbs point processes. Bernoulli 1999, 5(6):1119-1136. • Plummer potential: defined in Eq. 12. 6. Sbalzarini IF, Koumoutsakos P: Feature point tracking and trajectory analysis for video imaging in cell biology. J Struct Biol 2005, 151(2):182-195. Author details 1 1Institute of Theoretical Computer Science, ETH Zurich, Universitätstrasse 6, 8092 Zürich, Switzerland. 2Swiss Institute of Bioinformatics, ETH Zurich, Universitätstrasse 6, 8092 Zürich, Switzerland. Potentials were parameterized as (d) = f((d - t)/s) with interaction strength , length scale s, and threshold t = 0. Their shapes f(·) were defined as: References 1. Costes SV, Daelemans D, Cho EH, Dobbin Z, Pavlakis G, Lockett S: Automatic and quantitative measurement of protein-protein colocalization in live cells. Biophys J 2004, 86(6):3993-4003. 2. Bolte S, Cordelieres FP: A guided tour into subcellular colocalization analysis in light microscopy. J Microsc 2006, 224:213-232. 3. Anlauf E, Derouiche A: A practical calibration procedure for fluorescence colocalization at the single organelle level. J Microsc 2009, 233:225-233. 4. Thompson RE, Larson DR, Webb WW: Precise nanometer localization analysis for individual fluorescent probes. Biophys J 2002, 82(5):2775-2783. 5. Helmuth JA, Burckhardt CJ, Greber UF, Sbalzarini IF: Shape reconstruction of subcellular structures from live cell fluorescence microscopy images. J Struct Biol 2009, 167:1-10. 1. Costes SV, Daelemans D, Cho EH, Dobbin Z, Pavlakis G, Lockett S: Automatic and quantitative measurement of protein-protein colocalization in live cells. Biophys J 2004, 86(6):3993-4003. 2. Bolte S, Cordelieres FP: A guided tour into subcellular colocalization analysis in light microscopy. J Microsc 2006, 224:213-232. 3. Anlauf E, Derouiche A: A practical calibration procedure for fluorescence colocalization at the single organelle level. J Microsc 2009, 233:225-233. 4. Thompson RE, Larson DR, Webb WW: Precise nanometer localization analysis for individual fluorescent probes. Biophys J 2002, 82(5):2775-2783. 5. Helmuth JA, Burckhardt CJ, Greber UF, Sbalzarini IF: Shape reconstruction of subcellular structures from live cell fluorescence microscopy images. J Struct Biol 2009, 167:1-10. • Linear potential, type 2: f 12(z) = ⎧ ⎨ ⎩ 0 if z > 1 −1 if z < 0 −(1 −z) else . (22) (22) • Plummer potential: defined in Eq. 12. Test for interaction The piece-wise linear non-parametric potential φn.p.(d) was defined as a weighted sum of kernel func- tions (·) centered on the support points dp: Following [12], a non-parametric test for interaction was constructed using the distance counts T = (T1, . . . , TL)t , Tl = N i=1 1(tl < di ≤tl+1) , (14) (14) φn.p.(d) = P p=1 wp κ(d −dp) with κ(z) = |z|/h if |z| < h 0 else . (18) (18) in L = 20 equi-sized bins defined by L + 1 strictly increasing thresholds tl that span the entire non-zero range of q(d) for a given cell. First, a Monte Carlo sam- ple {Tk}K k=1 from the null distribution of T was obtained by sampling N = |D| distances di from q(d), computing Tk, and repeating this procedure K times. This sample allowed approximating the expectation E0(T) and co- variance matrix Cov0(T) of the null distribution. The test statistic U was defined as P = 21 support points dp were distributed between -5 and 95 with constant spacing h = 5 pixel. Setting wP = 0 enforced φn.p.(d) = 0 for all d ≥95. Setting  = n.p. the remaining weights were estimated by numerically maximizing (using CMA-ES) the penalized joint log- likelihood [17]: U = (E0(T) −T)t Cov0(T)−1 (E0(T) −T) . (15) (15) pl(|{Dk}) = Ncells  k=1 l(|Dk, k) + P−1  p=1 wp −wp+1 s 2 , (19) Second, T and U were computed for the set D of observed distances. U was then ranked among the {Uk}K k=1 obtained from an additional Monte Carlo sample {Tk}K k=1, generated as described above. If it ranked higher than ⌈(1 - a)K⌉-th, H0 was rejected on the significance level a. (19) k=1 + P−1  p=1 wp −wp+1 s 2 , (19) with respect to Θ = (w1,...,wP-1). Smoothness of n.p. was controlled by the parameter s = 2. The quadratic penalty in with respect to Θ = (w1,...,wP-1). Smoothness of n.p. was controlled by the parameter s = 2. The quadratic penalty in Page 12 of 12 Page 12 of 12 Helmuth et al. BMC Bioinformatics 2010, 11:372 http://www.biomedcentral.com/1471-2105/11/372 Helmuth et al. BMC Bioinformatics 2010, 11:372 http://www.biomedcentral.com/1471-2105/11/372 Helmuth et al. BMC Bioinformatics 2010, 11:372 http://www.biomedcentral.com/1471-2105/11/372 Eq. 19 corresponded to a Gaussian prior with zero mean and standard deviation s on the differences wp - wp+1. and Urs F. Implementation p All software was implemented in MATLAB version 7.9 (The Mathworks, Inc.) and run on a 2.66 GHz Intel Core2 Duo machine. Estimation of two-parameter potentials (Eqs. 12 and 20 to 22) took a few milliseconds per cell. Computation of q(d) took about one second. This time, however, strongly depended on the sampling resolution used. The non-parametric test for interaction took about half a second per cell. The time needed to estimate the common scale parameter for all cells was around ten minutes. A constantly updated version of the developed software is freely available from the web site of the authors http://www.mosaic.ethz.ch/Down- loads. The MATLAB functions, scripts, and sample data at the time of writing are contained in additional file 1. 7. Zhang B, Chenouard N, Olivio-Marin JC, Meas-Yedid V: Statistical Colocalization in Biological Imaging with False Discovery Control. Proceedings of the 2008 IEEE International Symposium on Biomedical Imaging: From Nano to Macro: 14-17 May 2008; Paris, France 2008, 1327-1330. 7. Zhang B, Chenouard N, Olivio-Marin JC, Meas-Yedid V: Statistical Colocalization in Biological Imaging with False Discovery Control. Proceedings of the 2008 IEEE International Symposium on Biomedical Imaging: From Nano to Macro: 14-17 May 2008; Paris, France 2008, 1327-1330. 8. Lachmanovich E, Shvartsman DE, Malka Y, Botvin C, Henis YI, Weiss AM: Co- localization analysis of complex formation among membrane proteins by computerized fluorescence microscopy: application to immunofluorescence co-patching studies. J Microsc 2003, 212(2):122-131. 9. Møller J, Waagepetersen R: Statistical inference and simulation for spatial point processes CRC Press 2004. Colocalization in Biological Imaging with False Discovery Control. Proceedings of the 2008 IEEE International Symposium on Biomedical Imaging: From Nano to Macro: 14-17 May 2008; Paris, France 2008, 1327-1330. 8. Lachmanovich E, Shvartsman DE, Malka Y, Botvin C, Henis YI, Weiss AM: Co- localization analysis of complex formation among membrane proteins by computerized fluorescence microscopy: application to immunofluorescence co-patching studies. J Microsc 2003, 212(2):122-131. 9. Møller J, Waagepetersen R: Statistical inference and simulation for spatial point processes CRC Press 2004. y 8. Lachmanovich E, Shvartsman DE, Malka Y, Botvin C, Henis YI, Weiss AM: Co- localization analysis of complex formation among membrane proteins by computerized fluorescence microscopy: application to immunofluorescence co-patching studies. J Microsc 2003, 212(2):122-131. p g 9. Møller J, Waagepetersen R: Statistical inference and simulation for spatial point processes CRC Press 2004. 10. Stoyan D, Penttinen A: Recent applications of point process methods in forestry statistics. Authors’ contributions JAH and GP developed the theory and analyzed the virus trafficking data. JAH designed, conducted, and analyzed numerical experiments and drafted the manuscript. GP participated in designing and analyzing the numerical experiments and helped in writing the manuscript. IFS participated in designing the theory and numerical experiments, helped writing and editing the manuscript, and coordinated the project. All authors read and approved the final manuscript. • Hermquist potential: • Hermquist potential: f he(z) = −(z + 1)−1 if z > 0 −(1 −z) else . (20) (20) • Linear potential, type 1: • Linear potential, type 1: Received: 10 March 2010 Accepted: 7 July 2010 Published: 7 July 2010 Received: 10 March 2010 Accepted: 7 July 2010 Published: 7 July 2010 f 11(z) = 0 if z > 1 −(1 −z) else . (21) f 11(z) = 0 if z > 1 −(1 −z) else . (21) Acknowledgements g JAH was financed by the ETH Research Commission under grant TH-1007-1. GP was funded through CTI grant 9325.2 PFLS-LS from the Swiss Federal Commission for Technology and Innovation. This project was also supported with a grant from the Swiss SystemsX.ch initiative, grant LipidX-2008/011 to IFS. The authors thank Christoph J. Burckhardt (Harvard University, Cambridge, MA) doi:10.1186/1471-2105-11-372 Cite this article as: Helmuth et al.: Beyond co-localization: inferring spatial interactions between sub-cellular structures from microscopy images. BMC Bioinformatics 2010 11:372.
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Two-phase damage and plate generation in a 2-D model of mantle convection
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Two-phase damage and plate generation in a 2-D model of mantle convection William Landuyt, David Bercovici, Yanick Ricard To cite this version: William Landuyt, David Bercovici, Yanick Ricard. Two-phase damage and plate generation in a 2-D model of mantle convection. Geophysical Journal International, 2008, ￿10.1111/j.1365- 246X.2008.03844￿. ￿hal-00361567￿ To cite this version: William Landuyt, David Bercovici, Yanick Ricard. Two-phase damage and plate generation in a 2-D model of mantle convection. Geophysical Journal International, 2008, ￿10.1111/j.1365- 246X.2008.03844￿. ￿hal-00361567￿ Distributed under a Creative Commons Attribution 4.0 International License S U M M A R Y The formation of narrow, rapidly deforming plate boundaries separating strong plate interiors are integral components of the generation of plate tectonics from mantle convection. The development of narrow plate boundaries requires the interaction of a non-linear rheology and convection. One such non-linear rheology is two-phase damage theory which employs a non- equilibrium relation between interfacial surface energy, pressure and viscous deformation, thereby forming a theoretical model for void generation. Two-phase damage theory was recently extended to allow for deformational work to increase the fineness (reduce the grain size) of the matrix phase. We present results testing two-phase damage theory in a 2-D convectively driven system where we allow for (1) pure void-generating damage, (2) pure fineness-generating damage and (3) combined void- and fineness-generating damage. Pure void-generating damage is found to be unsuccessful at producing plate-like features. Fineness- generating damage is successful at inducing plate-like behaviour in certain circumstances, including increasing viscosity sensitivity to fineness and certain regimes of damage input and healing rate. Cases with combined void- and fineness-generating damage produce significantly more localization than the end-members due to the apparent increase of deformational work input into fineness generation. The interaction of microcracks and grain size reduction in two-phase damage theory suggests a rheological model for shear localization necessary for the formation of plate tectonic boundaries. GJI Tectonics and geodynamics Key words: Planetary tectonics; Dynamics of lithosphere and mantle. Key words: Planetary tectonics; Dynamics of lithosphere and mantle. some of the most challenging features for plate generation studies to explain (Bercovici 2003). Initiation of subduction (King 2001) and the creation of passive ridges (Ricard & Froidevaux 1986; Tackley 2000b) are also vital aspects of plate tectonics to be understood. HAL Id: hal-00361567 https://hal.science/hal-00361567v1 Submitted on 17 Oct 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License doi: 10.1111/j.1365-246X.2008.03844.x Geophys. J. Int. (2008) 174, 1065–1080 Plate generation and two-phase damage theory in a model of mantle convection W. Landuyt,1 D. Bercovici1 and Y. Ricard2 1Department of Geology and Geophysics, Yale University, New Haven, CT, USA. E-mail: william.landuyt@yale.edu 2Laboratoire des Sciences de la Terre, CNRS, Ecole Normale Superieure de Lyon, Lyon, France W. Landuyt,1 D. Bercovici1 and Y. Ricard2 1Department of Geology and Geophysics, Yale University, New Haven, CT, USA. E-mail: william.landuyt@yale.edu 2Laboratoire des Sciences de la Terre, CNRS, Ecole Normale Superieure de Lyon, Lyon, France Accepted 2008 May 6. Received 2008 February 2; in original form 2007 June 1 C⃝2008 The Authors Journal compilation C⃝2008 RAS 1 I N T RO D U C T I O N These models have been able to generate narrow shear zones, but the connection be- tween the assumed damage parameter and the underlying physics of damage creation is often prescribed rather than derived (Bercovici 2003). Two-phase dynamics has been richly studied in geophysics, with many people considering its application to problems in melt- dynamics (McKenzie 1984; Spiegelman 1993a,b; Katz et al. 2006). Two-phase damage theory was developed in order to take a first- principles approach to studying the partitioning of deformational work into both dissipative heating and the generation of surface energy (Bercovici et al. 2001a). The generation of surface energy represents internal damage within the medium, and the manifesta- tion of surface energy in the medium is through the creation and/or expansion/dilation of voids. Two-phase theory is, therefore, devel- oped in order to track both the rock and void phases. Since voids are suggested to represent damage or weakness in the material, tracking the voids essentially boils down to tracking the damage in the system. The theory was generalized to allow for deformational work to go into generating fineness (grain size reduction) in the rock phase (or essentially pulverizing the rock) in addition to void gen- eration, and pulverizing the rock acts to create more surface energy in the medium (Bercovici & Ricard 2005). This theory avoids the rather nebulous approach of assuming that damage exists as some state variable without discerning how the damage is manifested in the system. An important question to ask though is how these manifestations of damage are related to the observed microphysics of damage. Void generation or Mode I cracks are well observed in brittle behaviour (and possibly brittle–ductile behaviour) at low pressures (Kohlstedt et al. 1995). At greater depths (∼15–18 km) the overburden pressure acts to suppress rock dilation (Kohlstedt et al. 1995), so the relevance of void generating damage to mid to deep lithospheric depths is of questionable importance (unless these voids were somehow filled with fluid which seems difficult given the petrologically inferred lack of water in the lithosphere). The relevance of grain size (and specifically the reduction in grain size) to the rheological properties of rocks and shear localization is well demonstrated (Karato 1983; Jin et al. 1998). The cause of reduction in grain size is generally associated with dynamic recrystallization (Karato et al. 1980). 1 I N T RO D U C T I O N Plate tectonics is the unifying theory of geology, yet much remains to be understood about the development of plate tectonics on Earth as well as its absence on the other terrestrial planets (Bercovici et al. 2000). Plate tectonics on Earth has been determined to have been in operation for at least two billion years and may well have been in operation much earlier (Cawood et al. 2006). These observations place an important constraint on numerical models in that Earth- like simulations of plate motion must be non-sporadic and long- lasting. The collection of studies labelled as plate generation refer to the attempt to understand how plate tectonics (or the surface manifestation of mantle convection) arise self-consistently within planetary convection, as opposed to viewing plate tectonics and planetary convection as separate entities. One of the most prominent features of plate tectonics that plate generation studies attempt to address is the existence of broad, strong plate interiors with weak, rapidly deforming boundaries (Weinstein & Olson 1992; Moresi & Solomatov 1998). The rapidly deforming boundaries tend to be very narrow features and are synonymous with shear localization. Also, the existence of strike-slip boundaries and toroidal motion remain The features of plate tectonics discussed above imply that the plates do not deform according to a simple Newtonian rheology. Experimental work on upper mantle rocks has shown that a signif- icant portion of the upper mantle deforms according to dislocation creep with a power law of approximately three (e.g. Karato & Wu 1993). Incorporation of non-Newtonian rheologies into plate gen- eration studies has not shown a significant improvement in gener- ating plate-like behaviours, especially in producing toroidal motion (Christensen & Harder 1991; Weinstein & Olson 1992; Bercovici 1993, 1995). Implementation of more exotic rheologies (e.g. stick- slip rheologies) has had more success at generating plate-like be- haviour (Bercovici 1993; Tackley 1998, 2000b), but the relevance of such rheologies to the actual lithosphere is questionable (Bercovici 2003). The longevity of plate boundaries (and their ability to be reactivated) implies that the low viscosity zone that constitutes the boundary is a time-dependent quantity that only decays after very long times (Gurnis et al. 2000). Therefore, tracking the time 1065 C⃝2008 The Authors Journal compilation C⃝2008 RAS W. Landuyt, D. Bercovici and Y. 1 I N T RO D U C T I O N Ricard 1066 to add another level of sophistication to previous work, but is still obviously a simplification of fully variable viscosity convection and hence convection in Earth. Our goal though is to ascertain the general plate behaviours (i.e. narrow boundary zones separated by rigid plate interiors) of two-phase damage theory in a convective system were we assume that the rheology is controlled by a single strong layer in the lithosphere. A simple but important addition to the evolution equation for fineness (inverse grain size) is added to the theory by the incorporation of a healing term. Healing in the fineness equation is representative of coarsening or grain growth in a poly-crystalline rock and is a well studied phenomena in Earth materials and in general (Karato 1989). One expects that increas- ing the fraction of deformational work which goes into generating damage as well as increasing the sensitivity of viscosity to grain size will allow for greater shear localization. In contrast, we expect that increasing the healing parameter will result in moderating the amount of localization within the lithosphere but potentially allow for a strengthening of the plate interior with respect to the bound- aries. While we do find that increasing the sensitivity of viscosity to grain size results in greater shear localization, the effect of in- creasing the healing parameter and fraction of deformational work stored as damage exhibits some behaviours different than expected. We also develop a series of scaling analyses for how the convergent and divergent boundary widths depend upon the model parameters and what damage inputs control the boundary behaviour. Scaling analyses are useful guides for exploring parameter space and un- derstanding the different localization behaviours demonstrated by the numerical experiments. In this study, we will inspect the abil- ity of both void and fineness generating damage in a convectively driven system to produce Earth-like features and determine what implications this may have for plate tectonics on Earth. evolution of the viscosity (or its controlling parameter) is impera- tive for plate generation studies. Previous studies have attempted to incorporate a time-dependent damage parameter (e.g. void or defect creation) to induce severe weakening in the plate that would address some of the deficiencies of the previous models (Bercovici 1998; Tackley 2000b; Auth et al. 2003; Ogawa 2003). 1 I N T RO D U C T I O N The relation between deformational work par- titioning into grain size reduction in the two-phase damage theory and the microphysics of dynamic recrystallization is not currently understood and remains an important and essential consideration for understanding this type of damage (Bercovici & Ricard 2005). Two-phase damage theory was recently incorporated by Bercovici & Ricard (2005) into a simple source-sink flow to determine the efficacy of both void and fineness generating damage to produce plate like flow (solid body translation, toroidal motion) in a 2-D sheet. Their results showed that fineness generating damage was very successful at generating plate like flow, while void generating damage merely enhanced the dipolar source-sink flow field and was, therefore, highly unplate-like (Bercovici & Ricard 2005). In this study, we will extend the previous work done on two- C⃝2008 The Authors, GJI, 174, 1065–1080 Journal compilation C⃝2008 RAS 2 M O D E L F O R M U L AT I O N The goal of this paper is to determine the effectiveness of different damage formulations in producing plate-like flow in a 2-D convec- tively driven system; this will involve determining how convergent and divergent zones as well as slowly deforming ‘plate-like’ zones respond to the given damage parameters. To that end we will em- ploy a lithosphere–mantle coupling model whereby a Newtonian mantle is overlain by a two-phase damage rheology lithospheric layer (Fig. 1); this model is similar to the formulation of Weinstein & Olson (1992). The lithospheric layer is of constant thickness h and is much smaller than the thickness of the underlying mantle D, (h ≪D). Material which flows across the boundary between 0 L D D + h h/D << 1 x z Newtonian Mantle vz , σxz = 0 vz , σxz , σzz viscosity(z) μm Non-Linear Rheology , μL (x) μL μL > μm μm Figure 1. The thin sheet model employed in this study along with the vertical viscosity profile. In this study, we will extend the previous work done on two- phase damage theory (Bercovici et al. 2001b; Ricard et al. 2001; Ricard & Bercovici 2003; Bercovici & Ricard 2003, 2005) by in- corporating two-phase damage physics into a coupled plate-mantle model that is convectively driven. Our model formulation attempts Figure 1. The thin sheet model employed in this study along with the vertical viscosity profile. C⃝2008 The Authors, GJI, 174, 1065–1080 Journal compilation C⃝2008 RAS Plate generation and two-phase damage theory in a model of mantle convection 1067 the lithosphere and mantle undergoes an instantaneous rheologi- cal transformation to the rheological properties of the region into which it flows. The boundary between the two regions is a no-slip surface such that stresses transmitted from the Newtonian layer to the lithosphere drive the flow in the surface layer. In the context of the thin sheet approximation that will be described hereafter, the temperature at the surface of the Newtonian mantle is assumed to be uniform and the thin layer is isothermal. With the assumed thermal profile of the lithosphere it follows that the top layer has no buoyancy variations to drive flow in the model. 2.1 Mantle convection model where the subscript m denotes variables in the mantle, and Pm, vm, wm and θ are the mantle non-hydrostatic pressure, ve- locity, vertical velocity and temperature perturbation, respectively (Schubert et al. 2001). The constant Ra is the Rayleigh number for the system. The convection equations above have also been non-dimensionalized by x = Dx′, t = (D2/κ)t′, vm = (κ/D)v′ m and P m = (μm κ/D2)P′ m, and the primes have subsequently been dropped. The constants D, κ and μm are the depth of the convect- ing system, thermal diffusivity and mantle viscosity, respectively. The above equations with the applied boundary conditions (to be discussed later) determine the behaviour of the Newtonian mantle in our model. where Aref is a reference value for fineness and μo is reference viscosity of the lithosphere. The viscosity exponent m is a dimen- sionless positive constant, and here we consider a reasonable range m (i.e. 1 ≤m ≤3) assuming a grain size sensitive deformation mechanism (e.g. diffusion creep) is in operation. Given the above momentum equations (eq. 5) we find that the effective matrix vis- cosity is given by where Aref is a reference value for fineness and μo is reference viscosity of the lithosphere. The viscosity exponent m is a dimen- sionless positive constant, and here we consider a reasonable range m (i.e. 1 ≤m ≤3) assuming a grain size sensitive deformation mechanism (e.g. diffusion creep) is in operation. Given the above momentum equations (eq. 5) we find that the effective matrix vis- cosity is given by μeff = (1 −φ)μL = μo(1 −φ)  Aref A m , (9) (9) where the (1 −φ) factor in the viscosity essentially arises from the deviatoric stress term in (eq. 5). 2.1 Mantle convection model α = Aη(φ) , η(φ) = φa(1 −φ)b, (7) (7) In order to focus on the effect of the various forms of damage on convection we employ a rather simple model for mantle convection, that is isoviscous, Boussinesq, infinite Prandtl number, Rayleigh– Benard convection. The equations for mass, momentum and energy are where A is the inverse void or grain size (depending on the curvature of the interface), η(φ) is a dimensionless function of porosity and a, b are constants ≤1. The above equation satisfies the requirement that interfacial area goes to zero at the different limits of porosity, and simple micromechanical models have shown how A relates to inverse grain/void size (Bercovici et al. 2001a). Following the previ- ous formulations of the matrix rheology we assume the lithospheric viscosity is given by where A is the inverse void or grain size (depending on the curvature of the interface), η(φ) is a dimensionless function of porosity and a, b are constants ≤1. The above equation satisfies the requirement that interfacial area goes to zero at the different limits of porosity, and simple micromechanical models have shown how A relates to inverse grain/void size (Bercovici et al. 2001a). Following the previ- ous formulations of the matrix rheology we assume the lithospheric viscosity is given by ∇· vm = 0 (1) 0 = −∇Pm + ∇2vm + Raθ ˆz (2) ∂θ ∂t + vm · ∇θ −wm = ∇2θ, (3) ∇· vm = 0 (1) 0 = −∇Pm + ∇2vm + Raθ ˆz (2) ∂θ ∂t + vm · ∇θ −wm = ∇2θ, (3) μL = μo  Aref A m , (8) (3) (8) where the subscript m denotes variables in the mantle, and Pm, vm, wm and θ are the mantle non-hydrostatic pressure, ve- locity, vertical velocity and temperature perturbation, respectively (Schubert et al. 2001). The constant Ra is the Rayleigh number for the system. The convection equations above have also been non-dimensionalized by x = Dx′, t = (D2/κ)t′, vm = (κ/D)v′ m and P m = (μm κ/D2)P′ m, and the primes have subsequently been dropped. The constants D, κ and μm are the depth of the convect- ing system, thermal diffusivity and mantle viscosity, respectively. The above equations with the applied boundary conditions (to be discussed later) determine the behaviour of the Newtonian mantle in our model. 2.2.3 Energy The two-phase damage equations originate from a series of papers (Bercovici et al. 2001a,b; Ricard et al. 2001), with subsequent papers refining various aspects of the theory (Bercovici & Ricard 2003, 2005; Ricard & Bercovici 2003). The equations presented below are in the geologically applicable ‘void limit’ as discussed in Ricard & Bercovici (2003), whereby the void phase has zero density, pressure and viscosity. The energy equation is separated into two coupled equations which govern the evolution of thermal energy and the rate of work done on the interface by pressure, surface tension and deformational work (Bercovici & Ricard 2005). The evolution of thermal energy is (1 −φ)ρc DT Dt −T D Dt  α dσ dT  −T α dσ dT ∇· v = Q −∇· q + B Dφ Dt 2 + σηkA Ap + (1 −f ) , (10) (10) 2.2.2 Momentum The momentum equations for the matrix are The momentum equations for the matrix are 0 = −∇[(1 −φ)P −σα] + ∇· [(1 −φ)τ] −(1 −φ)ρgˆz, (5) (5) where P is the matrix pressure,σ is the surface tension, α is the interfacial area density, τ is the deviatoric matrix stress and ρ is the density of the matrix phase. The deviatoric stress is given by τ = μL  ∇v + [∇v]t −2 3(∇· v)I  , (6) (6) where I is the identity matrix and μL is the top layer viscosity. The interfacial area density has been suggested to go as 2 M O D E L F O R M U L AT I O N Contrary to simi- lar model formulations (Weinstein & Olson 1992; Weinstein 1996) the lithosphere is recycled back into the mantle by calculating the vertical velocity at the plate-mantle interface, this avoids the rather unphysical situation where lithospheric material would build up at a subduction zone and become depleted at a ridge. the same as the matrix velocity, hence we do not need to solve independent mass and momentum equations for the void phase. 2.2.2 Momentum C⃝2008 The Authors, GJI, 174, 1065–1080 Journal compilation C⃝2008 RAS (16) where f ∗is the maximum permissible f φ, γ controls the vari- ability of f φ and f φ is assumed to depend on an even power of Dφ/Dt since it must be positive definite. As previously discussed in Bercovici et al. (2001b) and Bercovici & Ricard (2003), the above equation for f φ precludes singular solutions of the porosity damage equation (eq. 14) in areas of zero void growth (i.e. Dφ/Dt = 0). 2.2.1 Mass where T is the temperature, c is the matrix heat capacity, −bσ/dT is the interfacial entropy per unit area, q is an energy flux vector (e.g. heat diffusion) and Q is an intrinsic heat source. The quantity B is positive, has units of viscosity, and is related to the bulk viscosity term in the two-phase theory of McKenzie (1984); the term proportional to B represents irreversible work done during The equation for mass conservation is The equation for mass conservation is Dφ Dt = ∂φ ∂t + v · ∇φ = (1 −φ)∇· v, (4) (4) where v is the matrix (or rock phase) velocity and φ is the porosity. In the void limit the velocity of the void (or secondary) phase is where v is the matrix (or rock phase) velocity and φ is the porosity. In the void limit the velocity of the void (or secondary) phase is C⃝2008 The Authors, GJI, 174, 1065–1080 Journal compilation C⃝2008 RAS C⃝2008 The Authors, GJI, 174, 1065–1080 Journal compilation C⃝2008 RAS 1068 W. Landuyt, D. Bercovici and Y. Ricard this paper) the healing term would be representative of coarsening or grain growth (Karato 1989). Whether A represents inverse grain or void size depends upon the values of a and b in (eq. 7) as discussed previously. Similar to Bercovici & Ricard (2005) we find that variations in these parameters do not significantly affect the outcome of our results, and making the values of a smaller (which puts the fineness parameter in the inverse grain size regime) are easily offset by increasing f A. We will, therefore, associate the fineness parameter with inverse grain size in this paper and attempt to use constraints from grain size evolution to determine parameters in our evolution equation. isotropic compression or dilation. Following previous formulations for B we arrive at B = Kμo φ(1 −φ), (11) B = Kμo φ(1 −φ), (11) where K is a dimensionless factor accounting for pore or grain ge- ometry and is typically O(1) (Bercovici et al. 2001a; Bercovici & Ricard 2005). The (1 −φ) in denominator of (eq. 11) may not be necessary in the void limit, but the range of porosities in our study make this term negligible anyways. The quantity k A represents the rate of grain growth (Karato 1989) and η(φ) is a dimensionless function of porosity (see eq. 2.2.1 Mass 7); the term proportional to k A rep- resents the contribution of irreversible loss of interfacial area (via grain growth) to the evolution of thermal energy. The exponent p in the term proportional to k A is related to surface tension driven grain growth which will be further elaborated on later in this section. The viscous deformational work is given by where K is a dimensionless factor accounting for pore or grain ge- ometry and is typically O(1) (Bercovici et al. 2001a; Bercovici & Ricard 2005). The (1 −φ) in denominator of (eq. 11) may not be necessary in the void limit, but the range of porosities in our study make this term negligible anyways. The quantity k A represents the rate of grain growth (Karato 1989) and η(φ) is a dimensionless function of porosity (see eq. 7); the term proportional to k A rep- resents the contribution of irreversible loss of interfacial area (via grain growth) to the evolution of thermal energy. The exponent p in the term proportional to k A is related to surface tension driven grain growth which will be further elaborated on later in this section. The viscous deformational work is given by Previous analyses that have incorporated a damage evolution equation similar to the fineness evolution equation suggested the association of this equation to inverse grain size as well (Tackley 2000b; Auth et al. 2003), and in both cases choose p = 1 in eq. (15). The fineness evolution equation (eq. 15) in the absence of damage satisfies DA Dt ≈−kA Ap ⇒ 1 Ap−1 ≈(p −1)kAt. (17) (12) = (1 −φ)∇v : τ, (12) (17) a fraction 1 −f of which is partitioned into dissipative heating. The evolution of energy associated with work done on the interface and interface generation is a fraction 1 −f of which is partitioned into dissipative heating. The evolution of energy associated with work done on the interface and interface generation is Since we want to associate A with inverse grain size, A = 1/g, and the evolution of grain size undergoing grain growth is often found to vary as (kAt) 1 2 , 1 3 (e.g. Karato 1989; Evans et al. 2001) we therefore, choose p = 3. 2.2.1 Mass The association of the term proportional to k A with surface tension driven grain growth (and hence the curvature of grains) implies a direct relationship to the size of the grains, hence we assume the k A healing term is only in the equation governing the evolution of grain size (eq. 15) and not in eq. (14). The term proportional to k A in (eq. 15) acts to decrease the fineness (or increase grain size), and decreasing fineness leads to an increase in viscosity and, therefore, heals the damaged zone (i.e. plate boundary). We will consider cases where both the void- and fineness-generating damage mechanisms operate by them self as well as in tandem with each other. The partitioning fraction of damage in the void-generating case is given by where f = f φ + f A must be less than one. The association of the term proportional to k A with surface tension driven grain growth (and hence the curvature of grains) implies a direct relationship to the size of the grains, hence we assume the k A healing term is only in the equation governing the evolution of grain size (eq. 15) and not in eq. (14). The term proportional to k A in (eq. 15) acts to decrease the fineness (or increase grain size), and decreasing fineness leads to an increase in viscosity and, therefore, heals the damaged zone (i.e. plate boundary). We will consider cases where both the void- and fineness-generating damage mechanisms operate by them self as well as in tandem with each other. The partitioning fraction of damage in the void-generating case is given by fφ = f ∗ (Dφ/Dt)2 γ + (Dφ/Dt)2 , (16) fφ = f ∗ (Dφ/Dt)2 γ + (Dφ/Dt)2 , (16) 2.2.1 Mass Therefore, the pre- factor k A plays the role of the growth rate, which is experimentally found to be a function of temperature (Karato 1989). As we will discuss in the next section, temperature variations in our model lithosphere are not included which implies that the growth rate k A in our model is constant. The lithosphere is likely strongest at intermediate depths (30–70 km at 100 Myr) and, therefore, choosing a grain growth healing rate associated with this depth would be most appropriate in the thin-sheet formulation. The first term in eq. (15) allows for the input of deformational work to go into the reduction of grain size and increase the surface energy in the system. Grain size reduction in Earth minerals is experimentally found to occur by dynamic recrystallization (Karato et al. 1980), but there exists no complete development for the relationship between grain size and these processes (Hall & Parmentier 2003). Our evolution equation takes a similar form as previous studies, but we caution that our grain size reduction term comes about from the partitioning of deformational work between reversible and irreversible processes and not from a model that is meant to specifically represent any one experimentally determined grain size reduction mechanism. Recent work by Austin & Evans (2007) has also suggested that grain size reduction is determined by the rate of deformational work, and their proposed evolution equation for grain size (which is very similar to eq. 15) reproduces experimental results quite well. σ Dα Dt = −P Dφ Dt −B Dφ Dt 2 −σηkA Ap + f . (13) (13) Since changes in α can be manifested as either changes in porosity or changes in inverse grain/void size we can decompose (eq. 13) into one damage evolution equation for porosity and one for grain size (Bercovici & Ricard 2005). The damage equation for porosity is is σ A dη dφ Dφ Dt  = −P Dφ Dt  −B Dφ Dt 2 + fφ (14) (14) and the evolution equation for fineness is DA Dt = f A ση −kA Ap, (15) (15) where f = f φ + f A must be less than one. 2.2.1 Mass Choosing p = 1 leads to a logarithmic integration where the grain growth rate increases with time, while choosing p = 3, 4 allows for grain growth to be driven by surface tension and curvature. This simple analysis will provide the guidance for our choice of the healing exponent in eq. (15). Therefore, the pre- factor k A plays the role of the growth rate, which is experimentally found to be a function of temperature (Karato 1989). As we will discuss in the next section, temperature variations in our model lithosphere are not included which implies that the growth rate k A in our model is constant. The lithosphere is likely strongest at intermediate depths (30–70 km at 100 Myr) and, therefore, choosing a grain growth healing rate associated with this depth would be most appropriate in the thin-sheet formulation. The first term in eq. (15) allows for the input of deformational work to go into the reduction of grain size and increase the surface energy in the system. Grain size reduction in Earth minerals is experimentally found to occur by dynamic recrystallization (Karato et al. 1980), but there exists no complete development for the relationship between grain size and these processes (Hall & Parmentier 2003). Our evolution equation takes a similar form as previous studies, but we caution that our grain size reduction term comes about from the partitioning of deformational work between reversible and irreversible processes and not from a model that is meant to specifically represent any one experimentally determined grain size reduction mechanism. Recent work by Austin & Evans (2007) has also suggested that grain size reduction is determined by the rate of deformational work, and their proposed evolution equation for grain size (which is very similar to eq. 15) reproduces experimental results quite well. Since we want to associate A with inverse grain size, A = 1/g, and the evolution of grain size undergoing grain growth is often found to vary as (kAt) 1 2 , 1 3 (e.g. Karato 1989; Evans et al. 2001) we therefore, choose p = 3. Choosing p = 1 leads to a logarithmic integration where the grain growth rate increases with time, while choosing p = 3, 4 allows for grain growth to be driven by surface tension and curvature. This simple analysis will provide the guidance for our choice of the healing exponent in eq. (15). C⃝2008 The Authors, GJI, 174, 1065–1080 Journal compilation C⃝2008 RAS 2.3 Lithosphere model formulation We will employ the same dimensional scales as used for the mantle, except that we will in- clude an additional length scale Aref (m−1) to non-dimensionalize the fineness (inverse grain size) in order to keep the macroscopic length scale (D) separate from the microstructural one since they differ by about nine orders of magnitude. Substituting the aforemen- tioned scales into our governing equations (with the bar designating a vertically averaged quantity subsequently dropped) results in the following non-dimensional equations where the subscript L designates that the stresses are in the thin layer and we have neglected the buoyancy force of the top layer. where the subscript L designates that the stresses are in the thin layer and we have neglected the buoyancy force of the top layer. In the ˆz eq. (19) we will neglect the shear stress term since it goes as O(h/L) (since ∂τ xz/∂x ∼huL/L, where uL is the horizontal velocity in the lithosphere), which implies σzzL is constant. Taking the vertical average of both equations (and assuming the lithospheric thickness h is constant) results in 0 = ∂ ∂x  σ ¯α + (1 −¯φ)  ¯τxxL −¯P  −1 h  (1 −¯φ)τxzm|z=D (20) ∂σzzL ∂z = 0 ⇒σzzL|D+h D = 0 ⇒  σ ¯α + (1 −¯φ)  ¯τzzL −¯P  = τzzm|z=D (21) 0 = ∂ ∂x  σ ¯α + (1 −¯φ)  ¯τxxL −¯P  −1 h  (1 −¯φ)τxzm|z=D (20) (20) am bm −bm −am ∂uL ∂x wL h |z=1 = 1 h x 0 (1 −φ)τxzm|z=Ddx′ + τxzo −ˆσ Aλ τzzm|z=1 −ˆσ Aλ + μR F F , (29) 1 h x 0 (1 −φ)τxzm|z=Ddx′ + τxzo −ˆσ Aλ τzzm|z=1 −ˆσ Aλ + μR F F , (29) (21) (29) using the continuity of vertical and shear stress at the boundary between the lithosphere and Newtonian layer. We define σzzL = σ ¯α + (1−¯φ)(¯τzzL−¯P) tobethetotal vertical stress inthelithosphere, and the subscript m refers to stresses in the Newtonian mantle. Eq. (21) results from our approximation that σzzL is constant in the lithosphere, hence the vertically averaged σzzL is equal to σzzL at z = D + h (and also at z = D). To close the system of equations we also make the approximation that the average of a product is the product of the averages (e.g. φτL = ¯φ ¯τL). 2.3 Lithosphere model formulation The thin layer that represents the lithosphere is assumed to have a free-slip, impermeable surface on top and a no-slip interface with the Newtonian layer. Since our lithosphere thickness is assumed small we will employ the thin-sheet approximation to solve for the dy- namics of this layer (England & McKenzie 1982; Wdowinski et al. 1989; Ribe 1992; Weinstein & Olson 1992; Lemery et al. 2000). In the thin-sheet approximation, vertical gradients in horizontal velocity are assumed negligible in comparison to the horizontal The fineness generating damage equation presented in this paper is different from its previous form (Bercovici & Ricard 2005) in that it now explicitly contains a healing term. In the case where fineness represents inverse grain size (the only case considered in C⃝2008 The Authors, GJI, 174, 1065–1080 Journal compilation C⃝2008 RAS Plate generation and two-phase damage theory in a model of mantle convection 1069 1069 Plate generation and two-phase damage theory in a model of mantle convection 1069 gradients. The thin-sheet formulation, therefore, involves determin- ing the lithosphere’s vertically averaged horizontal velocity and the vertical velocity at the interface with the Newtonian layer. We thus examine the horizontal and vertical components of the momentum equation in a vertically averaged sense. The x and z components of the momentum equation in the lithosphere are: where the constant τxzo is determined using periodic boundary con- ditions, and where the constant τxzo is determined using periodic boundary con- ditions, and am = (1 −¯φ) 4 3μL + Kμo ¯φ  bm = (1 −¯φ) 2 3μL −Kμo ¯φ  λ =  ¯η + (1 −¯φ) d¯η d ¯φ  . (28) 0 = ∂ ∂x [σα + (1 −φ)(τxxL −P)] + ∂ ∂z [(1 −φ)τxzL] (18) 0 = ∂ ∂z [σα + (1 −φ)(τzzL −P)] + ∂ ∂x [(1 −φ)τxzL], (19) (18) λ =  ¯η + (1 −¯φ) d¯η d ¯φ  . (28) (28) 0 = ∂ ∂z [σα + (1 −φ)(τzzL −P)] + ∂ ∂x [(1 −φ)τxzL], (19) (19) We next consider the non-dimensionalization of the equations gov- erning the behaviour of the lithosphere. 2.3 Lithosphere model formulation 4), while the fineness evolution equa- tion becomes (23) The vertically averaged evolution equations for damage are The vertically averaged evolution equations for damage are ¯P = −σ ¯A d¯η d ¯φ −Kμo ¯φ ∇· ¯v + F, (24) where ∇· ¯v = ∇H · ¯v −wL h z=D, ¯v = (uL, 0, wL) (25) and F = f ∗ (1 −¯φ)2∇· ¯v γ + (1 −¯φ)2(∇· ¯v)2 ¯ (26) ¯P = −σ ¯A d¯η d ¯φ −Kμo ¯φ ∇· ¯v + F, (24) DA Dt = f AμR η ˆσ A−m ∗−ˆkA Ap, (33) where ˆkA = kA Ap−1 ref D2 κ , (34) ∗= (1 −φ)  ∇v : [∇v + [∇v]t −2 3(∇· v)]  . (35) DA Dt = f AμR η ˆσ A−m ∗−ˆkA Ap, (33) (24) (24) (33) where where where ∇· ¯v = ∇H · ¯v −wL h z=D, ¯v = (uL, 0, wL) (25) where ˆkA = kA Ap−1 ref D2 κ , (34) ∗= (1 −φ)  ∇v : [∇v + [∇v]t −2 3(∇· v)]  . (35) (25) (25) ˆkA = kA Ap−1 ref D2 κ , (34) and F = f ∗ (1 −¯φ)2∇· ¯v γ + (1 −¯φ)2(∇· ¯v)2 ¯ (26) ∗= (1 −φ)  ∇v : [∇v + [∇v]t −2 3(∇· v)]  . (35) (26) (35) under the same assumptions that lead to eqs (20) and (21). Sub- stituting in the expressions for ¯τxxL, ¯τzzL and ¯P eqs (22–24) into the momentum eqs (20 and 21) we get the following system of equations for ∂uL ∂x and wL h |z=D: The general scheme for solving the model system proceeds along the following steps. (1) Solve for the velocity and stress in the New- tonian mantle (eqs 1–2) given the current density distribution sub- ject to free-slip bottom and no-slip top boundary conditions. (2) The flow calculated in (1) generates a stress at the lithosphere–mantle interface that is used to calculate the velocity in the lithosphere via (eq. 29). (3) The lithospheric velocity is then used to drive cavity flow in the Newtonian mantle, and the velocity and stress in the Newtonian mantle are the sum of solutions from (1) and (3). 2.3 Lithosphere model formulation The vertically averaged stress is given by integrating eq. (6), and making use of the assumption that the vertical velocity is zero at the top boundary; this results in using the continuity of vertical and shear stress at the boundary between the lithosphere and Newtonian layer. We define σzzL = σ ¯α + (1−¯φ)(¯τzzL−¯P) tobethetotal vertical stress inthelithosphere, and the subscript m refers to stresses in the Newtonian mantle. Eq. (21) results from our approximation that σzzL is constant in the lithosphere, hence the vertically averaged σzzL is equal to σzzL at z = D + h (and also at z = D). To close the system of equations we also make the approximation that the average of a product is the product of the averages (e.g. φτL = ¯φ ¯τL). The vertically averaged stress is given by integrating eq. (6), and making use of the assumption that the vertical velocity is zero at the top boundary; this results in where am = (1 −φ) 4 3 μR Am + KμR φ  , bm = (1 −φ) 2 3 μR Am −KμR φ  . (30) (30) We have defined the following non-dimensional quantities in the above equations as q ˆσ = σ Aref D2 μmκ , (31) μR = μo μm , (32) ˆσ = σ Aref D2 μmκ , (31) ˆσ = σ Aref D2 μmκ , (31) ¯τxxL = ¯μL 4 3 ∂¯uL ∂x + 2 3 wL h |z=D  (22) ¯τzzL = ¯μL  −4 3 wL h z=D −2 3 ∂¯uL ∂x  . (23) (22) μR = μo μm , (32) μR = μo μm , (32) where μo is the reference lithospheric viscosity. The non- dimensional version of the mass equation is exactly the same as the dimensional case (eq. 2.5 Scaling analysis for fineness magnitude Without including a healing term in the damage eq. (15) it is obvious that fineness would continue to increase (i.e. grain size continues to decrease) without bound which is an unphysical scenario. Given that grain growth acts to increase the average grain size, even in the presence of deformation (Karato 1983, 1989) it is important to consider its effects on steady state fineness. We first examine how the magnitude of fineness depends upon the damage parameters ( f A, ˆkA and m), as well as consider the stability of the fineness evolution eq. (15) to an infinitesimal perturbation. We seek the maximum value of fineness at the boundaries where dA/dx is zero, and advection does not affect the fineness magnitude. Expanding the fineness field around its steady state value (Ao) uL dA dx = f AμR ˆση A−m ˙ε2 −ˆkA Ap. (36) (36) We assume functions with the proper symmetries for the velocity and fineness in the vicinity of the plate boundary, and then substitute them into eq. (15) to solve for the boundary widths. We thus assume uL = ±umax tanh(x/δ) A = Ao exp(−x2/δ2), (37) = ±umax tanh(x/δ) A ( 2/δ2) (37) ( / ) A = Ao exp(−x2/δ2), (37) (37) A = Ao + ϵ A′(t), (41) (41) where the ± in the velocity equation distinguishes the relations be- tween convergent (−) and divergent (+) zones, uL is the surface velocity and Ao is the steady-state maximum amplitude of fineness. The constant δ is the characteristic width for each boundary (conver- gent and divergent) and is assumed to be equal for both the fineness and velocity field (i.e. δ A = δv). Multiplying eq. (36) through by Am and substituting the above functional forms (eq. 37) into eq. (36) gives where A′ is the perturbation fineness and ϵ ≪1, and substituting this into where A′ is the perturbation fineness and ϵ ≪1, and substituting this into dA dt = f A Am τ 2 ˆσημR −ˆkA Ap (42) (42) we determine how the stability of steady state fineness is governed by the damage parameters. To 0th order in ϵ [assuming that the stress (τ) is constant] the steady-state fineness goes as we determine how the stability of steady state fineness is governed by the damage parameters. 2.3 Lithosphere model formulation We focus our analysis on both boundary widths and strain-rates for each numerical experiment to characterize the degree and type of localization present. The scaling analysis for δ above depends upon the steady-state fineness amplitude (Ao), and Ao is clearly a function of the input damage parameters. It is clear that eq. (40) provides a scaling law for both boundary width (δ) and boundary strain-rate umax/δ. We focus our analysis on both boundary widths and strain-rates for each numerical experiment to characterize the degree and type of localization present. The scaling analysis for δ above depends upon the steady-state fineness amplitude (Ao), and Ao is clearly a function of the input damage parameters. 2.4 Scaling analysis for boundary width/strain-rate In the study of plate generation, an important goal is the formation of narrow zones of weakness (plate boundaries) which separate strong plate-like interiors, especially when the analysis is confined to two dimensions. Before proceeding with a large number of numerical experiments it is useful to develop a scaling analysis that can guide our exploration of parameter space as well as provide insight into the different behaviours our numerical experiments produce. We are interested in determining how the plate boundary widths depend upon the free parameters in our model at steady state; to that end, we look at the fineness evolution eq. (33) in the absence of time- dependence: 2.3 Lithosphere model formulation (4) We am bm −bm −am ∂uL ∂x wL h |z=D = 1 h x 0 (1 −¯φ)τxzm|z=Ddx′ + τxzo −σ ¯Aλ τzzm|z=D −σ ¯Aλ + F F , (27) (27) C⃝2008 The Authors, GJI, 174, 1065–1080 Journal compilation C⃝2008 RAS C⃝2008 The Authors, GJI, 174, 1065–1080 Journal compilation C⃝2008 RAS W. Landuyt, D. Bercovici and Y. Ricard 1070 iterate on (2) and (3) until the L2 norm of the difference between successive iterations reaches 10−6 or less, at which point the litho- sphere and mantle are assumed to be in mechanical equilibrium. (E) Finally we update the temperature field in the mantle (eq. 3) and the damage variables in the lithosphere (eqs 4 and 33) with the equilibrium velocities determined at the end of (4). Convection in the Newtonian mantle is solved numerically via a spectral method that employs a Propagator Matrix method for solving Stokes flow (Hager & O’Connell 1981) and finite difference for the temperature equation. The cavity flow velocity field is calculated using a Prop- agator Matrix method as well. The sensitivity of lithospheric and convective dynamics to variations in damage parameters we will consider in this study include varying f φ and f A, k A and m, which are the fraction of deformational work partitioned into generating voids and fineness, grain growth healing rate, and the viscosity exponent for grain size sensitivity, respectively. of terms yields of terms yields  π m + p ˆkA Am+p o + (∓) √π (m + 1)3/2 Am+1 o umax δ  −4 3μR f A ˆση umax δ 2 = 0, (39) (39) with which we can estimate δ (or umax/δ, the boundary strain-rate) in terms of our governing free parameters. The term proportional to (umax/δ)0 in eq. (39) is associated with healing, the term propor- tional to (umax/δ)1 is associated with advection [(−) is for divergent and (+) is for convergent], and the term proportional to (umax/δ)2 is associated with deformational work. Solving for the boundary widths for convergence (δc) and divergence (δd) gives δd,c = ±1 +  1 + 16 3√π (m+1)3 (m+p)1/2 Ap−m−2 o μR fA ˆση ˆkA 2 (m+1)3/2 (m+p)1/2 Ap−1 o ˆkA umax . (40) (40) It is clear that eq. (40) provides a scaling law for both boundary width (δ) and boundary strain-rate umax/δ. C⃝2008 The Authors, GJI, 174, 1065–1080 Journal compilation C⃝2008 RAS 2.6 A note on boundary widths and the influence of temperature Table 1. List of the values for the model parameters: f φ is the fraction of deformational work that goes into void generating damage, f A is the fraction of deformational work that goes into fineness generating damage, m controls the sensitivity of viscosity to fineness, ˆkA is the healing rate related to grain growth and γ modulates the amount of void generating damage. Some important points can be made regarding the results of the scaling analysis for plate boundary widths. Convergent boundaries are predicted to be smaller than divergent boundaries for a given plate velocity, and this difference arises predominately from the dif- ference in sign of the velocity field (see eq. 37). Consideration of data from global seismicity would imply the opposite of the above prediction, specifically that divergent boundaries are narrower than convergent boundaries (Dumoulin et al. 1998). The difference in boundary widths between divergent and convergent zones seen on Earth are likely to not arise solely due to the difference in sign of the divergence field, but it also points out the importance of temper- ature variations in the lithosphere. The characteristic temperature of convergent and divergent boundaries in the lithosphere is very different, and this necessarily plays an important role for the varia- tions in boundary widths between convergence and divergence. The effects of horizontal variations in temperature within the plate and their subsequent influence on viscosity are not explicitly included in this model due to the assumed isothermal structure of the plate. While we include the zeroth order influence of temperature depen- dent viscosity in that μo > μm the existence of horizontal variations in viscosity due to horizontal variations in temperature likely influ- ences the difference between our scaling law predictions and Earth. Inspection of the scaling law in eq. (40) suggests that allowing for temperature variations in the lithosphere would most readily affect the viscosity (μo) and the healing rate (k A); an increase (decrease) in temperature would decrease (increase) viscosity while increas- ing (decreasing) healing rate. The divergent boundary width with temperature variations would, therefore, decrease compared to the isothermal case and vice versa for convergent boundaries. Given that the scaling analysis is derived by considering local variations we could just as easily assume that μo and k A are different for convergent and divergent boundaries. 2.5 Scaling analysis for fineness magnitude To 0th order in ϵ [assuming that the stress (τ) is constant] the steady-state fineness goes as Ao =  τ 2 ˆσημR  1 p−m  f A ˆkA  1 p−m , (43) ±2umax δ2 Am+1 o x tanh(x/δ) exp[−(m + 1)x2/δ2] = f A ˆση μRu2 max δ2 cosh−4(x/δ) −ˆkA Am+p o exp[−(m + p)x2/δ2]. (38) (43) and to first order in ϵ (38) dA′ dt = (m −p)ˆkA Ap−1 o A′. (44) (44) We next integrate eq. (38) around the plate boundary over a domain much greater than the boundary width δ. We make the assumption that the integral of eq. (38) from [−δ/2 −L, + δ/2 + L] is approx- imately equal to the integral with the limits (−∞, +∞) to facilitate evaluation of the above integral (i.e. δ ≪L). We also approximate tanh (x/δ) by x/δ which doesn’t produce any significant error since the Gaussian in the relevant term decays much faster than the linear term grows. The resultant integration of eq. (38) and organization We next integrate eq. (38) around the plate boundary over a domain much greater than the boundary width δ. We make the assumption that the integral of eq. (38) from [−δ/2 −L, + δ/2 + L] is approx- imately equal to the integral with the limits (−∞, +∞) to facilitate evaluation of the above integral (i.e. δ ≪L). We also approximate tanh (x/δ) by x/δ which doesn’t produce any significant error since the Gaussian in the relevant term decays much faster than the linear term grows. The resultant integration of eq. (38) and organization Two interesting points can be made. First, the steady-state fineness is predicted to be a function of the ratio of f A and ˆkA, second, stability of the steady-state to perturbations is determined solely by the sign of m −p. The scaling above for the magnitude of the steady-state fineness also provides a diagnostic to determine whether or not fineness magnitudes are determined by the competition between deformational work and healing. C⃝2008 The Authors, GJI, 174, 1065–1080 Journal compilation C⃝2008 RAS Plate generation and two-phase damage theory in a model of mantle convection 1071 2.6 A note on boundary widths and the influence of temperature 2.6 A note on boundary widths and the influence of temperature Since it is difficult to glean information from numerical experiments that fail to con- verge to a stable solution our goal is to find a range of parameters that will produce numerically resolvable, steady-state behaviours, while still remaining in parameter regimes that produce Earth-like convective patterns. We will look at the following plate character- istics: divergent and convergent boundary width (we will measure this quantity by the full-width at half-max for the divergence field), as well as the concentration of deformation and rigidity of plate interiors as measured by the integrated diagnostic plateness [Pl = (π −Pl∗)/(π −2), where Pl∗= (1/|u|avg) |˙εxx|dx] as defined by Weinstein & Olson (1992). The definition of plateness results in a sinusoidal velocity field having a plateness of 0, and boxcar velocity field having a plateness of 1. Looking at the individual the damage rheology is initiated the plate is assumed to have both constant porosity and fineness profiles, with values of 1 per cent and 1, respectively. The exponents (a and b) in the interfacial area density function η(φ) (7) are chosen to be 0.5 throughout the results section, and test cases with variations in a and b showed little to no difference in the system behaviour from the cases with a = b = 0.5. We employ 128 gridpoints in the horizontal direction and 50 gridpoints in the vertical direction. The chosen numerical resolution faithfully reproduced the results of test runs at higher resolutions but at significantly reduced computational times. Since it is difficult to glean information from numerical experiments that fail to con- verge to a stable solution our goal is to find a range of parameters that will produce numerically resolvable, steady-state behaviours, while still remaining in parameter regimes that produce Earth-like convective patterns. We will look at the following plate character- istics: divergent and convergent boundary width (we will measure this quantity by the full-width at half-max for the divergence field), as well as the concentration of deformation and rigidity of plate interiors as measured by the integrated diagnostic plateness [Pl = (π −Pl∗)/(π −2), where Pl∗= (1/|u|avg) |˙εxx|dx] as defined by Weinstein & Olson (1992). The definition of plateness results in a sinusoidal velocity field having a plateness of 0, and boxcar velocity field having a plateness of 1. Looking at the individual C⃝2008 The Authors, GJI, 174, 1065–1080 Journal compilation C⃝2008 RAS 2.6 A note on boundary widths and the influence of temperature The scaling law, therefore, seems capable of predicting boundary width variations seen on Earth when the appropriate lithospheric temperature variations are taken into consideration. We will discuss more later about the influ- ence of variations in viscosity due to temperature and its inclusion in subsequent models. g f φ f A(×10−4) m ˆkA γ 0 0.05 1.2 1 0 0 0.25 1.2 0.75, 1, 2.5 0 0 0.5 1.2 2, 2.5, 5, 10, 25, 50 0 0 0.75 1.2 2.5, 3, 3.75, 5, 7.5, 15 0 0 1 1.2 4 0 0 0.05 1.4 5 0 0 0.25 1.4 5 0 0 0.5 1.4 10, 25, 50, 100 0 0 0.75 1.4 15 0 0 0.05 1.6 5 0 0 0.25 1.6 15, 37.5 0 0 0.5 1.6 25, 37.5, 50, 100 0 0 0.75 1.6 37.5 0 0 0.05 1.8 10 0 0 0.1 1.8 20 0 0 0.25 1.8 50 0 0 0.5 1.8 75, 80, 85, 100, 200 0 0 0.75 1.8 150 0 0 0.05 2 25 0 0 0.1 2 50 0 0 0.25 2 125 0 0 0.5 2 200, 250, 375 0 0 0.75 2 375 0 0 2.5 2 1250 0 0 25 2 12 500 0 0 50–1000 2 250 000 0 0 0.5 2.5 725, 750, 1000 0 0.5 0 0 0 102, 1, 10−2, 10−4 0.4 0.1 1.2, 2 2.5, 250 10−2 0.25 0.25 1.2, 2 2.5, 250 10−2, 1, 103 0.2 0.3 1.2, 2 2.5, 250 10−2 0.1 0.4 1.2, 2 2.5, 250 10−2 the damage rheology is initiated the plate is assumed to have both constant porosity and fineness profiles, with values of 1 per cent and 1, respectively. The exponents (a and b) in the interfacial area density function η(φ) (7) are chosen to be 0.5 throughout the results section, and test cases with variations in a and b showed little to no difference in the system behaviour from the cases with a = b = 0.5. We employ 128 gridpoints in the horizontal direction and 50 gridpoints in the vertical direction. The chosen numerical resolution faithfully reproduced the results of test runs at higher resolutions but at significantly reduced computational times. 3 N U M E R I C A L R E S U LT S We next explore numerical solutions of our model in a sampling of parameter space. All of our convection simulations have a sim- ple Rayleigh–Bernard heating mode, and the Rayleigh number is 106 with an aspect ratio of two for each case. We choose to use this simple bottom heating mode in order to take advantage of the symmetries and steady-state behaviour of this convective system. In all cases we initiated our damage rheology calculations with a Newtonian convection simulation that has reached steady-state. For each set of parameters investigated (see Table 1 for list of all parameters), we attempted to find steady-state solutions to find ro- bust measurements of the plate characteristics (boundary widths, concentration of deformation and rigidity of plate interiors) that we are most interested in understanding. We also choose the litho- spheric plate viscosity to be four orders of magnitude greater than the viscosity of the Newtonian mantle (i.e. μR = 104). With typ- ical values of mantle scales (μm = 1021 Pa s,σ = 1 N m−1, κ = 10−6 m2 s−1, D ∼106 m and Aref ∼103 m−1 implying a grain size of 1 mm) the non-dimensional surface tension, ˆσ is approximately 1, and we will assume that it is throughout the calculations. When W. Landuyt, D. Bercovici and Y. Ricard 1072 boundaries, but this variation produces no noticeable improvement on localization for void-generating damage. This result corroborates the work of (Bercovici & Ricard 2005) in that pure void-generating damage is insufficient to allow for plate-like flow. It should be noted that Bercovici & Ricard (2005) found that void-generating damage was specifically unable to produce significant toroidal flow which is not relevant to this study; however both their and our results suggest that voids in the current two-phase framework do not allow for the types of plate boundary behaviour seen on Earth. A possible way to improve the plate-inducing capabilities of void-generating damage would be to change the sensitivity of viscosity to porosity. This was done in Bercovici & Ricard (2005) by specifying that the viscos- ity undergoes a sharp drop when porosity goes above some critical value, and Katz et al. (2006) employed a viscosity exponentially dependent on porosity. 3.2 Fineness-generating damage The fineness generating cases are distinguished by the variations in the parameters m, f A, ˆkA (see eq. 17). The healing exponent is assumed constant at p = 3 throughout to reduce the number of variables under consideration in this study. In the fineness evolution eq. (33) the variables f A and μR multiply each other and can be treated as a single variable. Most of the results we present in the following sections are for 0 ≤f AμR ≤1, and since μR = 104 this implies 0 ≤f A ≤10−4. As we show in the following sections the steady state results are not highly dependent upon the absolute magnitude of f AμR. Therefore, we can map out the general plate generating behaviours of our model at smaller values of f AμR, but at reduced computational times compared to calculations at larger f AμR. Our results also suggest that f A is not required to be large in order for significant amounts of localization to be generated. -4.4 0.0 4.4 0.00 0.22 Surface Velocity Porosity t = 0.05 Pl ~ .1 (a) (b) (c) -4.4 0.0 4.4 0.00 0.22 Temperature Surface Velocity Porosity t = 0.05 Pl ~ .1 x 0 1 2 (a) (b) (c) Figure 2. Numerical results for case with only void generating damage. In (a) we plot the porosity profile in the lithosphere, (b) we plot the surface velocity profile in the lithosphere and (c) we plot the temperature field. The parameter values are f φ = 0.5 and γ = 10−2, and at t = 0.05 the results for surface velocity are in steady state. This example of void generating damage is representative of all such cases where the model results are similar to the case of a Newtonian rigid lid. The contour interval for the temperature field is 0.1 (and will be the same contour interval for all plots of the temperature field). The plateness (Pl) for the velocity field is 0.1. 3.1 Void-generating damage The void-generating damage cases are distinguished by the variation in the parameter γ (and subsequently f φ), which primarily controls thesensitivityof void-generatingdamagetothedeformational work. We examine cases for four different values of γ (102, 1, 10−2, 10−4) with constant f ∗= 0.5, and show the results for γ = 10−2 in Fig. 2. Solutions for different γ display similar trends: plateness is very low (∼1, hence deformation is spatially distributed) and similar to the case where there is no damage (i.e. f A = f φ = 0), and porosity is greater over ridges than in subduction zones (Fig. 2a). That voids are generated over ridges and closed over subduction zones is indicative of ridges and subduction zones undergoing tensional and compres- sional environments, respectively. In the current void-generating damage framework it seems that we are unable to generate narrow plate boundaries and rigid plate interiors (Fig. 2b). Previous results employing void-generating damage in simple-shear found that shear localization occurred upon increasing γ and deformational work in tandem (Bercovici & Ricard 2003), but in the current study the de- formational work is approximately constant since we don’t vary the Rayleigh number. Decreasing the magnitude of γ results in a slight increase in the rate at which voids are generated around divergent 3 N U M E R I C A L R E S U LT S While these different viscosity formulations had varying degrees of success for the given model, we will not explore variations on the porosity sensitivity to viscosity since it is unclear of the relevance of the previous formulations to our study and a more appropriate form is unknown at this time. boundary widths will provide insight into how each deformational zone responds to model parameters, while plateness will quantify the narrowness of the boundaries as well as the rigidity of the plate interiors. C⃝2008 The Authors, GJI, 174, 1065–1080 Journal compilation C⃝2008 RAS Plate generation and two-phase damage theory in a model of mantle convection Further increases in f A forces the system to an episodic regime followed by a region -5.6 -4.2 -2.8 -1.4 0.0 0.000 0.167 0.333 0.500 + + + + ++++ • • • • • • •• •• •• + + + + ++ ++ • • • • • • •• •• •• stable plate like episodic epi spl + • log ( fA ) δd δc , kA = 2.5E5 kA = 2.5E2 stagnant lid sl Figure 5. The above plot shows the behaviours in boundary widths for variations in f A with the healing rate ˆkA kept constant for viscosity exponent m = 2 and two different healing rates. The grey inset is for ˆkA = 250 and the other plot is for ˆkA = 2.5 × 105, but the axes are the same for both plots. For both healing rates we find that at small values of f A (where small depends upon healing rate) the system is in a stagnant lid regime. Further increases in f A forces the system to an episodic regime followed by a region characterized by stable plate behaviour. Increases in f A beyond the steady state regions results in numerically unresolvable phenomena. Figure 3. Variations in surface velocity and fineness for variations in f A with ˆkA = 2.5 and m = 1.2. erations (Auth et al. 2003; Bercovici & Ricard 2005). However, in Bercovici & Ricard (2005) they looked at variations in f A/σ (with the surface tension σ assumed small) and were possibly able to al- low for more significant localization, though they primarily looked at generation of toroidal motion without characterizing boundary width variations. This study suggests that variations in the amount of fineness generating damage available in a convecting system at steady state produces the same amount of localization. Fig. 5 dis- plays the results for variations in boundary widths for variations in f A at two different healing rates. For a given healing rate at small values of f A the system is in a stagnant lid regime, and increases in f A cause a transition to time dependent behaviour characterized by episodic plate-like behaviour. Upon further increasing f A the system enters a stable plate-like regime. For a given healing rate, there is little variation in localization in the stable plate regime. Plate generation and two-phase damage theory in a model of mantle convection 1073 0 25 50 0 1 2 -100 0 100 m = 1.2, kA = 2.5 fA = .05x10-4 fA = .25x10-4 fA = .5x10-4 fA = .75x10-4 Fineness Surface Velocity x Figure 3. Variations in surface velocity and fineness for variations in f A with ˆkA = 2.5 and m = 1.2. 0 25 50 0 1 2 -100 0 100 m = 1.2, kA = 2.5 fA = .05x10-4 fA = .25x10-4 fA = .5x10-4 fA = .75x10-4 Fineness Surface Velocity x gure 3. Variations in surface velocity and fineness for variations in f th ˆk 2 5 and m 1 2 -5.6 -4.2 -2.8 -1.4 0.0 0.000 0.167 0.333 0.500 + + + + ++++ • • • • • • •• •• •• + + + + ++ ++ • • • • • • •• •• •• stable plate like episodic epi spl + • log ( fA ) δd δc , kA = 2.5E5 kA = 2.5E2 stagnant lid sl Fi 5 Th b l t h th b h i i b d idth f -5.6 -4.2 -2.8 -1.4 0.0 0.000 0.167 0.333 0.500 + + + + ++++ • • • • • • •• •• •• + + + + ++ ++ • • • • • • •• •• •• stable plate like episodic epi spl + • log ( fA ) δd δc , kA = 2.5E5 kA = 2.5E2 stagnant lid sl Figure 5. The above plot shows the behaviours in boundary widths for variations in f A with the healing rate ˆkA kept constant for viscosity exponent m = 2 and two different healing rates. The grey inset is for ˆkA = 250 and the other plot is for ˆkA = 2.5 × 105, but the axes are the same for both plots. For both healing rates we find that at small values of f A (where small depends upon healing rate) the system is in a stagnant lid regime. Plate generation and two-phase damage theory in a model of mantle convection With that being said, increasing the fineness inducing damage char- acterized by f A does allow for a viscosity drop that increases plate mobility without decreasing boundary widths (and hence increasing localization). 0.00 0.33 0.66 1.00 0.20 0.26 0.33 0.40 + + + + + + + + + 0.00 0.33 0.66 1.00 0.05 0.11 0.18 0.25 + + + + + + + + + 0.00 0.33 0.66 1.00 0.25 0.50 0.75 1.00 + + + + + + + + + 0.00 0.33 0.66 1.00 0 33 66 100 + + + + + + + + + Plateness umax fA ( x10-4 ) (a) (b) (c) (d) (e) (f) m = 1.2 , kA = 2.5 m = 1.6 , kA = 37.5 m = 2.0 , kA = 250 fA ( x10-4 ) 0 33 267 400 + + + + + + + + + 0 200 400 600 + + + + + + + + + 0.00 0.33 0.66 1.00 0.00 0.33 0.66 1.00 0.00 0.33 0.66 1.00 0.25 0.50 0.75 1.00 + + + + + + + + + Plateness (a) ( ) 0.00 0.33 0.66 1.00 0 33 66 100 + + + + + + + + + umax (b) d (b) (d) 0.00 0.33 0.66 1.00 0.05 0.11 0.18 0.25 + + + + + + + + + (c) (d) f 0 200 400 600 + + + + + + + + + 0.00 0.33 0.66 1.00 (f) 0.00 0.33 0.66 1.00 0.20 0.26 0.33 0.40 + + + + + + + + + fA ( x10-4 ) (e) (f) fA ( x10-4 ) 0 33 267 400 + + + + + + + + + 0.00 0.33 0.66 1.00 3.2.1 Variations in f A C⃝2008 The Authors, GJI, 174, 1065–1080 Journal compilation C⃝2008 RAS Plate generation and two-phase damage theory in a model of mantle convection 3.2.2 Variations in healing rate ˆkA m = 1.2 , kA = 2.5 m = 1.6 , kA = 37.5 m = 2.0 , kA = 250 We now consider how variations in healing rate ˆkA affect the be- haviours of convergent and divergent boundary widths (Figs 6c and e). Our results show that increasing the healing rate (at constant damage fraction f A and viscosity exponent m) significantly re- duces plate velocity (Fig. 6b), has little effect on plateness (Fig. 6a), and has varied effects on convergent and divergent boundary width (Figs 6c and e). Increasing the healing rate leads to an increased plate viscosity which acts to decrease the plate velocity and bound- ary strain-rate. Convergent boundary widths show unsystematic behaviour to variations in healing rate except when ˆkA becomes small and δc generally decreases mildly. The trends of δd for all m values suggest that the effect of increasing ˆkA initially leads to a reduced boundary width, but when ˆkA reaches a critical value divergent boundary width begins to increase and eventually reach an asymptote. The initial decrease in δd is consistent with our pre- vious analysis that a decrease in the healing length scale umax/ˆkA (dominated by the decrease in umax) will lead to a reduction in δd. Therefore, we see that an increase in healing can actually play a role in localization of divergent boundaries by causing the reduced Figure 4. The range of plate-like behaviours observed (+, with connecting dashed lines) and predicted (solid lines) from our study for the case of variations in fineness inducing damage characterized by f A. (a) Plateness versus f A, (b) Plate velocity versus f A, (c) Convergent boundary width versus f A, (d) Convergent boundary strain-rate versus f A, (e) Divergent boundary width versus f A and (f) Divergent zone strain-rate versus f A. Figure 4. The range of plate-like behaviours observed (+, with connecting dashed lines) and predicted (solid lines) from our study for the case of variations in fineness inducing damage characterized by f A. (a) Plateness versus f A, (b) Plate velocity versus f A, (c) Convergent boundary width versus f A, (d) Convergent boundary strain-rate versus f A, (e) Divergent boundary width versus f A and (f) Divergent zone strain-rate versus f A. Figure 4. 3.2.1 Variations in f A We consider variations in f A (which represents the partition of work towards fineness inducing damage) at constant values of ˆkA (healing rate) and m (viscosity exponent for grain size sensitiv- ity). Increases in f A result in increases in the plate mobility (de- fined as the ratio of plate velocity to rms velocity of the mantle) and boundary strain-rate (uL/δd,c), while the boundary widths and plateness remain fairly constant and insensitive to changes in f A, which is somewhat unexpected. These results imply that the fraction of deformational work that goes into fineness-generating damage is important for providing the reduction in strength (or increase in strain-rate) necessary for plate boundary lubrication, but con- tributes little to the localization of deformation. In Fig. 3, we show the fineness and surface velocity fields for m = 1.2, ˆkA = 2.5 and f A = (0.05–0.75)10−4. At f A = 0.05 × 10−4 the fineness field shows a small perturbation above the baseline value (of 1) and the associated boundary width is relatively small (see Figs 4c and e). As we increase f A the maximum values of fineness increase as expected (Ao ∼( f A/ˆkA)1/(p−m)) and there is a slight broadening of δc (see Fig. 4c). The convergent boundary width δc calculated at f A = 0.05 × 10−4 is smaller than at larger f A in Fig. 3, and we can easily see that as f A is reduced the variations in the fine- ness field get smoothed out until they disappear at f A = 0. The (c) x 0 1 2 Figure 2. Numerical results for case with only void generating damage. In (a) we plot the porosity profile in the lithosphere, (b) we plot the surface velocity profile in the lithosphere and (c) we plot the temperature field. The parameter values are f φ = 0.5 and γ = 10−2, and at t = 0.05 the results for surface velocity are in steady state. This example of void generating damage is representative of all such cases where the model results are similar to the case of a Newtonian rigid lid. The contour interval for the temperature field is 0.1 (and will be the same contour interval for all plots of the temperature field). The plateness (Pl) for the velocity field is 0.1. C⃝2008 The Authors, GJI, 174, 1065–1080 Journal compilation C⃝2008 RAS 3.2.2 Variations in healing rate ˆkA The range of plate-like behaviours observed (+, with connecting dashed lines) and predicted (solid lines) from our study for the case of variations in fineness inducing damage characterized by f A. (a) Plateness versus f A, (b) Plate velocity versus f A, (c) Convergent boundary width versus f A, (d) Convergent boundary strain-rate versus f A, (e) Divergent boundary width versus f A and (f) Divergent zone strain-rate versus f A. divergent boundary widths in Fig. 4(e) display less systematic be- haviour as a function of f A than the convergent boundary widths, and in Fig. 3 appear to not change significantly. The increase in strain-rate as f A is increased, therefore, comes about due to the increase in uL at approximately constant δc,d (Figs 4d and f). Since increasing the fraction of fineness inducing damage does nothing to increase the rigidity of the plate (and δc,d ≈constant) the plateness remains approximately unchanged. Increasing the fraction of defor- mational work that goes into the generation of fineness damage at constant ˆkA and m, therefore, does not seem to improve the plate characteristics in any dramatic fashion contrary to previous consid- C⃝2008 The Authors, GJI, 174, 1065–1080 Journal compilation C⃝2008 RAS C⃝2008 The Authors, GJI, 174, 1065–1080 Journal compilation C⃝2008 RAS W. Landuyt, D. Bercovici and Y. Ricard 1074 0 133 267 400 0.20 0.26 0.33 0.40++++++ + +++ + +++ + + ++++ + + + + 0 133 267 400 0.05 0.11 0.18 0.25 ++++++ + +++ + +++ + + ++++ ++ + + 0 133 267 400 0.25 0.50 0.75 1.00 ++++++ + +++ + +++ + + ++++ ++ + + 0 133 267 400 0 25 50 75+ + + ++ + + + + + + + + + +++ + + + + + Plateness umax kA kA (a) (b) (c) (d) (e) (f) m = 1.2 m = 1.6 m = 1.4 m = 1.8 m = 2 0 33 267 400 0 200 400 600 ++ + + ++ + + + + + ++ + + ++++ + + + + + + + + + ++ + + + + + ++ + + +++ + + + + + + 0 133 267 400 0 133 267 400 Figure 6. 3.2.3 Variations in f A and ˆkA with their ratio constant It seems that the effect of increasing ˆkA acts to distribute deformation by increasing the plate strength at old lithosphere, while the value of f A does not contribute significantly to weakening of the plate at subduction zones. These results again highlight the conclusion that the combination of plate velocity and healing may allow for narrow divergent boundaries, but increases in fineness inducing damage characterized by f A still remain insuf- ficient for generating narrow convergent boundaries. In Fig. 9 we (f) (e) kA (f) 0 33 267 400 ++ + + ++ + + + + + ++ + + ++++ + + + + + 0 133 267 400 0 133 267 400 0.20 0.26 0.33 0.40++++++ + +++ + +++ + + ++++ + + + + kA (e) m = 1.2 m = 1.6 m = 1.4 m = 1.8 m = 2 Figure 6. The range of plate-like behaviours observed (+, with connect- ing dashed lines) and predicted (solid lines) from our study for the case of variations in healing rate characterized by ˆkA with f A = 0.5 × 10−4. (a) Plateness versus ˆkA, (b) Plate velocity versus ˆkA, (c) Convergent bound- ary width versus ˆkA, (d) Convergent boundary strain-rate versus ˆkA, (e) Divergent boundary width versus ˆkA and (f) Divergent zone strain-rate versus ˆkA. grain size at a ridge to substantially heal within a short distance from the ridge. When the healing rate reaches the critical value, the velocity (and boundary strain-rate) approach their small- magnitude asymptotes. At this point the lithosphere begins to ap- proach the properties of a stagnant lid, and hence the boundary widths increase (divergent) or remain the same (convergent). Our expectation that increasing healing rate would lead to a modera- tion of localization while allowing for an increase in plate strength seems to be partially born out. While increasing healing rate does strengthen the plate interiors and allows for steady-state convec- tion, it will eventually choke off surface motion and cause a rigid lid. 3.2.3 Variations in f A and ˆkA with their ratio constant (d) 0 133 267 400 0.05 0.11 0.18 0.25 ++++++ + +++ + +++ + + ++++ ++ + + (c) (d) 0 200 400 600+ + + + ++ + + + + + ++ + + +++ + + + + + + 0 133 267 400 We further consider the physics of advection in plate boundary development by maintaining fixed relative sizes of damage and healing. The previous sections showed that increasing f A resulted in an increase in plate velocity and boundary strain-rate, while in- creasing the healing rate (ˆkA) resulted in a decrease in plate velocity and boundary strain-rate. Our results for constant f A/ˆkA at various values of m (note that the ratio f A/ˆkA is different for each m value, and is chosen to be the largest ratio that is numerically resolvable (e.g. f A/ˆkA for m = 1.2 is 0.25 × 10−4 while f A/ˆkA for m = 2 is 0.002 × 10−4)) are shown in Figs 7 and 8. We see that plate ve- locity remains fairly unchanged with variations in f A and ˆkA while f A/ˆkA is kept constant (Fig. 8b), whereas we say umax grows with an increasing ratio of f A to ˆkA in Figs 4 and 6. Our results show that di- vergent boundary width undergoes a decline as f A and ˆkA increase before eventually asymptoting to its minimum value (Fig. 8e). This result again shows the phenomena discussed in the previous section where a decrease in the healing length scale (umax/ˆkA) may allow for localization of divergent boundaries. Contrary to the previous section though the plate velocity remains constant as ˆkA goes up and the model does not approach the stagnant lid case; therefore, increasing f A simultaneously with the healing rate allows for di- vergent boundaries to undergo further localization than when f A remains small. The asymptotic value for δd is eventually controlled by the competition between healing and deformational work when both fineness inducing damage and healing rate dwarf the advective component. We have already seen that convergent boundaries re- spond somewhat negligibly to variations in f A, but here we see that δc actually grows for increasing healing rate and fineness inducing damage fraction (Fig. 8c). 3.2.2 Variations in healing rate ˆkA The range of plate-like behaviours observed (+, with connect- ing dashed lines) and predicted (solid lines) from our study for the case of variations in healing rate characterized by ˆkA with f A = 0.5 × 10−4. (a) Plateness versus ˆkA, (b) Plate velocity versus ˆkA, (c) Convergent bound- ary width versus ˆkA, (d) Convergent boundary strain-rate versus ˆkA, (e) Divergent boundary width versus ˆkA and (f) Divergent zone strain-rate versus ˆkA. 0 133 267 400 0.25 0.50 0.75 1.00 ++++++ + +++ + +++ + + ++++ ++ + + Plateness (a) 0 133 267 400 0 25 50 75+ + + ++ + + + + + + + + + +++ + + + + + umax (b) (d) ture to Fig. 5, except that stagnant lid (stable plate-like) behaviour would be at large (small) k A instead of small (large) f A. An intrigu- ing and surprising result is that increasing healing rate (in a specific range) may allow for localization at divergent plate boundaries via a reduction in the healing length scale. Plate generation and two-phase damage theory in a model of mantle convection The range of plate-like behaviours observed (+, with connecting dashed lines) and predicted (solid lines) from our study for the case of variations in viscosity exponent m with f A = 0.5 × 10−4. (a) Plateness versus m, (b) Plate velocity versus m, (c) Convergent boundary width versus m, (d) Convergent boundary strain-rate versus m, (e) Divergent boundary width versus m, and (f) Divergent zone strain-rate versus m. Figure 8. The range of plate-like behaviours observed (+, with connect- ing dashed lines) and predicted (solid lines) from our study for the case of simultaneous variations in f A and ˆkA. (a) Plateness versus f A, (b) Plate velocity versus f A, (c) Convergent boundary width versus f A, (d) Conver- gent boundary strain-rate versus f A, (e) Divergent boundary width versus f A and (f) Divergent zone strain-rate versus f A. m (Fig. 10a), irrespective of the ratio f A to ˆkA. Increases in the viscosity exponent, therefore, also increase the rigidity of the plate while also decreasing the boundary widths. As m is increased, the boundary zone strain-rate increases dramatically (Figs 10d and f), primarily due to the decrease in boundary width, but strain-rate is also influenced by the corresponding increase in plate velocity. Allowing for greater viscosity sensitivity to grain size by increasing m, therefore, allows for significantly greater plate-like behaviours by increasing plate velocity and plateness, while narrowing both divergent and convergent plate boundaries. -5 -3 -1 0.000 0.133 0.267 0.400 + + + ++ + + + + + + ++ + + + log ( fA ) fA μR / kA = .002 δd δc , Figure 9. The variation in plate boundary width as a function of damage fraction for 5 × 10−5 ≤f A ≤0.1. This case is for m = 2 and constant ratio of f A to ˆkA equal to 0.002 × 10−4. We can see that the variations in plate boundary width saturate well before f A approaches 0.1. -5 -3 -1 0.000 0.133 0.267 0.400 + + + ++ + + + + + + ++ + + + log ( fA ) fA μR / kA = .002 δd δc , In Fig. 11, we show the relevant plate characteristics for a case where m = 2.5, f A = 0.5 × 10−4, and ˆkA = 750 at steady state along with its time series. Plate generation and two-phase damage theory in a model of mantle convection (a) Plateness versus f A, (b) Plate velocity versus f A, (c) Convergent boundary width versus f A, (d) Conver- gent boundary strain-rate versus f A, (e) Divergent boundary width versus f A and (f) Divergent zone strain-rate versus f A. 1.0 1.4 1.8 2.2 0.20 0.26 0.33 0.40 + + + + + + + + + + + + + + 1.0 1.4 1.8 2.2 0.05 0.11 0.18 0.25 + + + + + + + + + + + + + + 1.0 1.4 1.8 2.2 0.25 0.50 0.75 1.00 + + + + + + + + + + + + + + 1.0 1.4 1.8 2.2 0 33 66 100 + + + + + + + + + + + + + + Plateness umax m m (a) (b) (c) (d) (e) (f) fA = 0.5 x10-4 , kA = 25, 50, 100, 200, 250 + + + + + + + + + + + + + + + + + + + + + + + + + + + + 1.0 1.4 1.8 2.2 1.0 1.4 1.8 2.2 0 33 267 400 0 200 400 600 Figure 10. The range of plate-like behaviours observed (+, with connectin dashed lines) and predicted (solid lines) from our study for the case o variations in viscosity exponent m with f A = 0.5 × 10−4. (a) Platenes versus m, (b) Plate velocity versus m, (c) Convergent boundary width versu m, (d) Convergent boundary strain-rate versus m, (e) Divergent boundar width versus m, and (f) Divergent zone strain-rate versus m. 3.2.4 Variations in m We finally consider how variations in viscosity exponent m affect the plate-like behaviour of our model at constant values of f A and ˆkA. The results shown in Fig. 10 are for various combinations of f A and ˆkA, but all show that an increase in m results in a decrease in boundary width for both δd and δc (Figs 10c and e), which is expected from our scaling analysis and previous observations (Auth et al. 2003; Bercovici & Ricard 2005). Our calculations also show that the plateness of the flow increases approximately linearly with Plate generation and two-phase damage theory in a model of mantle convection The time series values of plateness, Nus- selt number and maximum fineness show the transition from our initial state, through the period of transient oscillations to an even- tual steady state. The velocity field is very plate-like, with narrowly deforming boundaries and a rigid plate interior. The value of fine- ness at the subduction zone is approximately five, while the mini- mum value of fineness in the plate interior is about 0.05 for a range of fineness of about two orders of magnitude. Given our reference grain size of 1 mm, this would correspond to a range of grain sizes of about 200 μ m to 20 mm. While this range of values for grain size may be possible within the Earth, we would expect the grain size in a shear zone and plate interior to be somewhat reduced compared to our calculated values (Evans et al. 2001). In our efforts to find damage parameters that are able to produce plate like flows though it is encouraging that successful results don’t require unreasonable values of material parameters, such as grain size. Figure 9. The variation in plate boundary width as a function of damage fraction for 5 × 10−5 ≤f A ≤0.1. This case is for m = 2 and constant ratio of f A to ˆkA equal to 0.002 × 10−4. We can see that the variations in plate boundary width saturate well before f A approaches 0.1. see the effect of increasing f A to significantly larger values (and correspondingly ˆkA) demonstrates that boundary widths still remain saturated. Therefore, our analysis of behaviours at smaller f A dis- plays the variations brought about maintaining constant advection by fixing the ratio of damage to healing. 3.2.3 Variations in f A and ˆkA with their ratio constant The transition in convective behaviours would be similar in na- m = 2 0 10 -30 0 30 0 1 2 fA = .05x10-4 , kA = 25 fA = .25x10-4 , kA = 125 fA = .75x10-4 , kA = 375 0 50 -100 0 100 0 1 2 fA = .15x10-4 , kA = 0.6 fA = .5x10-4 , kA = 2 fA = .75x10-4 , kA = 3 m = 1.2 Fineness Surface Velocity x x Figure 7. Variations in velocity and fineness for different m, f A and ˆkA values, but keeping f A/ˆkA = constant. C⃝2008 The Authors, GJI, 174, 1065–1080 m = 2 0 10 -30 0 30 0 1 2 fA = .05x10-4 , kA = 25 fA = .25x10-4 , kA = 125 fA = .75x10-4 , kA = 375 x 0 50 -100 0 100 0 1 2 fA = .15x10-4 , kA = 0.6 fA = .5x10-4 , kA = 2 fA = .75x10-4 , kA = 3 m = 1.2 Fineness Surface Velocity x Figure 7. Variations in velocity and fineness for different m, f A and ˆkA values, but keeping f A/ˆkA = constant. C⃝2008 The Authors, GJI, 174, 1065–1080 Journal compilation C⃝2008 RAS C⃝2008 The Authors, GJI, 174, 1065–1080 Journal compilation C⃝2008 RAS Plate generation and two-phase damage theory in a model of mantle convection 1.0 1.4 1.8 2.2 0.20 0.26 0.33 0.40 + + + + + + + + + + + + + + 1.0 1.4 1.8 2.2 0.05 0.11 0.18 0.25 + + + + + + + + + + + + + + 1.0 1.4 1.8 2.2 0.25 0.50 0.75 1.00 + + + + + + + + + + + + + + 1.0 1.4 1.8 2.2 0 33 66 100 + + + + + + + + + + + + + + Plateness umax m m (a) (b) (c) (d) (e) (f) fA = 0.5 x10-4 , kA = 25, 50, 100, 200, 250 + + + + + + + + + + + + + + + + + + + + + + + + + + + + 1.0 1.4 1.8 2.2 1.0 1.4 1.8 2.2 0 33 267 400 0 200 400 600 0.00 0.33 0.66 1.00 0.05 0.11 0.18 0.25 + + + + + + + + + + + + + + + + + + + 0.00 0.33 0.66 1.00 0.25 0.50 0.75 1.00 + + + + + + + + + ++ + + + ++ + + + Plateness (a) (c) ( ) 0.00 0.33 0.66 1.00 0 33 66 100 + + + + + + + + + ++ + + + ++ + + + umax (b) (d) 1.0 1.4 1.8 2.2 0 33 66 100 + + + + + + + + + + + + + + umax (b) (d) 600 0.00 0.33 0.66 1.00 0.05 0.11 0.18 0.25 + + + + + + + + + + + + + + + + + + + 0.00 0.33 0.66 1.00 0.25 (c) ( ) (d) (d) m (d) (f) kA = 25 50 100 200 250 + + + + + + + + + + + + + + + + + + + + + + + + + + + + 1.0 1.4 1.8 2.2 1.0 1.4 1.8 2.2 0 33 267 400 0 200 400 600 (d) + + + + + + + + + + + + + + 1.0 1.4 1.8 2.2 0 200 400 600 (f) (f) (e) 1.0 1.4 1.8 2.2 0.20 0.26 0.33 0.40 + + + + + + + + + + + + + + m (e) 0.00 0.33 0.66 1.00 0.20 0.26 0.33 0.40 + + + + + + + + + + + + + + + + + + + (e) fA ( x10-4 ) m fA = 0.5 x10-4 , kA = 25, 50, 100, 200, 250 Figure 10. Plate generation and two-phase damage theory in a model of mantle convection 1075 0.00 0.33 0.66 1.00 0.20 0.26 0.33 0.40 + + + + + + + + + + + + + + + + + + + 0.00 0.33 0.66 1.00 0.05 0.11 0.18 0.25 + + + + + + + + + + + + + + + + + + + 0.00 0.33 0.66 1.00 0.25 0.50 0.75 1.00 + + + + + + + + + ++ + + + ++ + + + 0.00 0.33 0.66 1.00 0 33 66 100 + + + + + + + + + ++ + + + ++ + + + Plateness umax (a) (b) (c) (d) (e) (f) fA ( x10-4 ) fA ( x10-4 ) + + + + + + + + + ++ + + + ++ + + + + + + + + + + + + + + + + + + + 0 33 267 400 0 200 400 600 0.00 0.33 0.66 1.00 0.00 0.33 0.66 1.00 0.00 0.33 0.66 1.00 0.20 0.26 0.33 0.40 + + + + + + + + + + + + + + + + + + + 0.00 0.33 0.66 1.00 0.05 0.11 0.18 0.25 + + + + + + + + + + + + + + + + + + + 0.00 0.33 0.66 1.00 0.25 0.50 0.75 1.00 + + + + + + + + + ++ + + + ++ + + + 0.00 0.33 0.66 1.00 0 33 66 100 + + + + + + + + + ++ + + + ++ + + + Plateness umax (a) (b) (c) (d) (e) (f) fA ( x10-4 ) fA ( x10-4 ) + + + + + + + + + ++ + + + ++ + + + + + + + + + + + + + + + + + + + 0 33 267 400 0 200 400 600 0.00 0.33 0.66 1.00 0.00 0.33 0.66 1.00 Figure 8. The range of plate-like behaviours observed (+, with connect- ng dashed lines) and predicted (solid lines) from our study for the case of simultaneous variations in f A and ˆkA. 3.3 Combined void- and fineness-generating damage In this final section we consider the combined effects of both void- and fineness-generating damage mechanisms on plate-like C⃝2008 The Authors, GJI, 174, 1065–1080 Journal compilation C⃝2008 RAS 1076 W. Landuyt, D. Bercovici and Y. Ricard Temperature 0.0 0.5 1.0 0.0000 0.0257 0.0514 0.0771 time Pl -20 0 20 Surface Velocity 10 12 Nu 0.0 9.2 Fineness 0 15 Max A 0.000 0.035 t = 0.0514 Porosity 0.000 0.026 Max φ Figure 11. Results for parameter set m = 2.5, f A = 0.5 × 10−4 and ˆkA = 750. -4.2 0.0 4.2 0 2 0.000 0.026 Temperatur e Surface Velocity Finenes s Porosity t = 0.0025 fA = 0 fφ = 0.5 -12 0 12 0 12 0.000 0.037 t = 0.00353 -81 0 81 0 16 0.000 0.023 t = 0.0131 fA = 0.25 fφ = 0.25 fA = 0.5 fφ = 0 Figure 12. Results for case when both void and fineness generating damage are generated by deformational work. In these plots ˆkA = 2.5 × 106, m = 2 and γ = 10−2. behaviour in the model We have so far found that void generat- subsequent to frames shown The reason for the ultra localization 1076 W. Landuyt, D. Bercovici and Y. Ricard 0.0 0.5 1.0 0.0000 0.0257 0.0514 0.0771 time Pl 10 12 Nu 0 15 Max A 0.000 0.026 Max φ Temperature 0.0 0.5 1.0 0.0000 0.0257 0.0514 0.0771 time Pl -20 0 20 Surface Velocity 10 12 Nu 0.0 9.2 Fineness 0 15 Max A 0.000 0.035 t = 0.0514 Porosity 0.000 0.026 Max φ Figure 11. Results for parameter set m = 2.5, f A = 0.5 × 10−4 and ˆkA = 750. Figure 11. Results for parameter set m = 2.5, f A = 0.5 × 10−4 and ˆkA = 750. -12 0 12 0 12 0.000 0.037 t = 0.00353 fA = 0.25 fφ = 0.25 -4.2 0.0 4.2 0 2 0.000 0.026 Temperatur e Surface Velocity Finenes s Porosity t = 0.0025 fA = 0 fφ = 0.5 -12 0 12 0 12 0.000 0.037 t = 0.00353 -81 0 81 0 16 0.000 0.023 t = 0.0131 fA = 0.25 fφ = 0.25 fA = 0.5 fφ = 0 Figure 12. Results for case when both void and fineness generating damage are generated by deformational work. 3.3 Combined void- and fineness-generating damage In these plots ˆkA = 2.5 × 106, m = 2 and γ = 10−2. Temperatur e Surface Velocity Figure 12. Results for case when both void and fineness generating damage are generated by deformational work. In these plots ˆkA = 2.5 × 106, m = 2 and γ = 10−2. Figure 12. Results for case when both void and fineness generating damage are generated by deformational work. In these p γ = 10−2. behaviour in the model. We have so far found that void generat- ing is insufficient by itself to produce plate like flow in the cur- rent two-phase framework, which is in line with previous work (Bercovici & Ricard 2005). On the other hand, fineness generat- ing damage has shown to be successful at producing plate like flow under certain circumstances in this study as well as previously (Bercovici & Ricard 2005). Fig. 12 shows results for a case with both void and fineness generating damage, the void- and fineness- generating end member cases are presented alongside for com- parison. Previously we found that employing a damage rheology with only void generating damage and γ = 10−2 produced very unplate-like flows (see Fig. 12). However, when these same pa- rameters are employed with moderate fineness generating damage ( fφ = f A = 0.25, ˆkA = 2.5 × 106, m = 2) the result is local- ized plate boundaries and significantly more plate-like flow. The combined case in fact becomes too localized to numerically resolve subsequent to frames shown. The reason for the ultra localization in this case is due to the f A/η(φ) term in eq. (15). The void- generating damage allows for the porosity field to undergo larger variations than when f φ = 0, and hence η(φ) is reduced in the convergent and divergent zones. The reduction in η(φ) results in an increase in the effective amount of fineness generating dam- age, f A/η(φ). Physically, the reduction in η(φ) (or interfacial area density) allows for the continued input of damage to have a more significant impact than if the system was already heavily damaged, since inputting deformational work into a material with little dam- age results in a greater viscosity drop than if the material was already significantly deformed. While our results for boundary width vari- ations in f A (with f φ = 0 at constant ˆkA and m) (Fig. C⃝2008 The Authors, GJI, 174, 1065–1080 Journal compilation C⃝2008 RAS 4.1 Earth-like parameters and our model we eventually force the system to be similar to the case when f φ = 0. φ The results of allowing combined void- and fineness-generating damage in our convective model is summarized in Fig. 13. For some cases with combined forms of damage the system became numerically unresolvable (i.e. solution became too localized before reaching steady state), hence we choose to measure the maximum strain-rate when the convergent boundary width was less than three gridpoints. For each choice of m, k A and γ the plate velocity is generally the same, so increasing strain-rate [(u/δ)max] is indica- tive of a reduction in boundary width and hence localization. The results show that an increase in plate-like behaviour, specifically localization, is achieved by allowing for both forms of damage when the fineness generating parameters by themselves are associ- ated with stable plate-like behaviour (ˆkA = 2.5 × 105, f = 0.05 and ˆkA = 2.5 × 106, f = 0.5 in Fig. 13). In contrast, the com- bination of void and fineness generating damage, when the fine- ness parameters are associated with episodic or stagnant lid be- haviour (ˆkA = 2.5 × 106, f = 0.05 in Fig. 13), does not result in an increase in plate localization or mobilization. Combining the two types of damage mechanisms can result in more significant localization, but this seems to occur only when the fineness pa- rameters permit stable plate-like solutions. Addition of void gen- erating damage does not enhance plate like behaviour unless the fineness parameters allow for it. As previously discussed, increas- ing the parameter γ acts to force the system back to the case when f φ = 0 and decreasing γ causes the convective system to local- ize at a faster rate. These results suggest that an interesting inter- play between void and fineness generating damage may exist that could facilitate shear localization within the Earth. From a mineral physics perspective microcracks distributed in a poly-crystalline material may act to pin grain boundaries, therefore, inhibiting grain growth. If grain growth is stunted due to the presence of pores (by f φ ̸= 0) then grain size will remain smaller and viscosity will cor- respondingly remain small. Results from looking at natural samples has shown that grain size reduction can lead to the formation of brittle faults and shear localization (Jin et al. 1998). 4.1 Earth-like parameters and our model Plate generation necessitates breaking the strongest part of the litho- sphere; we would therefore, like to know if the range of parameters in our thin-sheet model is associated with the strongest portion of the lithosphere [i.e. brittle–ductile transition (Kohlstedt et al. 1995)]. One way of estimating this is through the grain growth rate k A in eq. (15). From mineral physics we know that the grain growth rate in olivine and other minerals is highly temperature dependent (Karato 1989). The growth rate has an Arrhenius type functional form that can be solved for temperature such that Figure 13. The effect of combined void and fineness generating damage on plate-like behaviour for different values of total damage and fineness healing. For this plot m = 2, f = 0.05, 0.5, ˆkA = 2.5 × 105, 2.5 × 106 and γ = 10−2. The quantity f is defined to be the sum of f A and f φ. kA = ko exp −H RT  ⇒T = H R ln  ko kA , (45) (45) where ko is the reference grain growth rate, H is the activation enthalpy, and R is the gas constant. Given the values of ˆkA we deter- mined to be capable of producing plate-like flows we can then get an estimate of the lithospheric temperature controlling the rheology in our model. Using values of k o = 10−8 m2 s−1 and H = 2 × 105 J (mol K)−1 (Karato 1989) and ˆkA = 250 –1000(2.3–9.0 × 10−21m2 s−1 in dimensional form) for m = 2 and f AμR = 0.5 from our calculations we arrive at a temperature range of T = 800–900 K. This range of values for temperature will put us in the brittle–ductile regime for most of the oceanic lithosphere. Our thin-sheet damage rheology is, therefore, associated with the strongest region of the lithosphere, which is what is desired in a thin-sheet model. The kinetics of grain growth at such low temperatures might be significantly different than eq. (45) predicts, but experimental data on single-phase grain growth likely predict much faster grain growth than actually occurs in the mantle due to the inhibition of grain growth by secondary phases (Evans et al. 2001). Therefore, our temperature estimate might be a lower limit of the temperature or depth represented by our vertically averaged rheology. 4.1 Earth-like parameters and our model Brittle fail- ure may lead to significant grain size reduction as well, though the reduction process may be more related to generation of fault gouge than grain size reduction via dynamic recrystallization or similar processes. The interaction of brittle and ductile deformation mechanisms remains a poorly understood rheological phenomena from both an experimental and theoretical perspective (Kohlstedt et al 1995) 4 D I S C U S S I O N A N D C O N C LU S I O N 0.0 0.5 1.0 0 1000 2000 fA / f | . max | kA = 2.5E6 kA = 2.5E5 f = 0.5 f = 0.05 f = 0.05 3.3 Combined void- and fineness-generating damage 4) didn’t show any significant localization it seems that a larger effective f A (with f φ > 0) is capable of generating much greater plate-like flow. When γ is increased to 103 (with the same fineness parameters) C⃝2008 The Authors, GJI, 174, 1065–1080 Journal compilation C⃝2008 RAS 1077 Plate generation and two-phase damage theory in a model of mantle convection 0.0 0.5 1.0 0 1000 2000 fA / f | . max | kA = 2.5E6 kA = 2.5E5 f = 0.5 f = 0.05 f = 0.05 Figure 13. The effect of combined void and fineness generating damage on plate-like behaviour for different values of total damage and fineness healing. For this plot m = 2, f = 0.05, 0.5, ˆkA = 2.5 × 105, 2.5 × 106 and γ = 10−2. The quantity f is defined to be the sum of f A and f φ. C⃝2008 The Authors, GJI, 174, 1065–1080 Journal compilation C⃝2008 RAS R E F E R E N C E S Austin, N. & Evans, B., 2007. Paleowattmeters: a scaling relation for dy- namically recrystallized grain size, Geology, 35(4), 343–346. Auth, C., Bercovici, D. & Christensen, U., 2003. Two-dimensional convec- tion with a self-lubricating, simple-damage rheology, Geophys. J. Int., 154, 783–800. Bercovici, D., 1993. A simple model of plate generation from mantle flow, Geophys. J. Int., 114, 635–650. Bercovici, D., 1995. A source-sink model of the generation of plate tectonics from non-Newtonian mantle flow, J. geophys. Res., 100, 2013–2030. Bercovici, D., 1998. Generation of plate tectonics from lithosphere–mantle flow and void-volatile self-lubrication, Earth planet. Sci. Lett., 154, 139– 151. Bercovici, D., 2003. The generation of plate tectonics from mantle convec- tion, Earth planet. Sci. Lett., 205, 107–121. Bercovici, D. & Ricard, Y., 2003. Energetics of a two-phase model of litho- spheric damage, shear localization and plate-boundary formation, Geo- phys. J. Int., 152, 581–596. Bercovici, D. & Ricard, Y., 2005. Tectonic plate generation and two-phase damage: void growth versus grain size reduction, J. geophys. Res., 110, 1–18. While our model does include the zeroth order effect of temper- ature on the viscosity of the lithosphere (i.e. lithospheric viscosity is much greater than Newtonian mantle viscosity), the effect of a laterally varying temperature on the lithospheric dynamics is an im- portant consideration to look into for the future. A laterally variable temperature in the plate would likely allow for the formation of even narrower divergent plate boundaries. The area of ridge formation is significantly warmer than assumed in the model, and including this effect would lead to a lower viscosity as well as increase the amount of healing in the grain size evolution equation. The viscosity of the region of old lithosphere would be similar to the zeroth order viscosity assumed in our model and, therefore, would likely be little affected by allowing for lateral temperature variations. Bercovici, D., Ricard, Y. & Richards, M., 2000. The relation between mantle dynamics and plate tectonics: a primer, in History and Dynamics of Global Plate Motions, Geophys. Monogr. Ser., Vol. 121, pp. 5–46, eds Richards, M.A., Gordon, R. & van der Hilst, R., Am. Geophys. Union, Washington, DC. Bercovici, D., Ricard, Y. & Schubert, G., 2001a. A two-phase model of compaction and damage, 1. General theory, J. geophys. Res., 106(B5), 8887–8906. Bercovici, D., Ricard, Y. & Schubert, G., 2001b. A two-phase model of compaction and damage, 3. Applications to shear localization and plate boundary formation, J. 4.2 Final thoughts This behaviour may also be important for the forma- tion of narrow rifts in that plate thinning under tension may allow for significant healing outside of the failure region thereby forming a localized rift zone. Increases in the viscosity exponent m lead to the formation of narrow plate boundaries (both convergent and di- vergent) as well as increases in the rigidity of the plate interior, and the values of m explored fall within the range of values expected for mantle rheologies (Karato & Wu 1993). These results suggest that the mechanism of localization at convergent and divergent bound- aries need not originate from the same process. Our model does not incorporate the generation of melts which will strongly influence the strength of ridges as well (Tackley 2000a,b), and melt forma- tion obviously leads to different localization mechanism than what affects subduction zones. While grain size sensitive creep (coupled with crack formation) may be needed to form narrow convergent boundaries, the generation of narrow divergent boundaries may only need some small damage input coupled with strong healing near the ridge. Allowing for both forms of damage causes a feed- back mechanism which allows for the most significant localization of plate boundaries in our study. The feedback mechanism begins with void-generation allowing for greater variability in porosity, the range of η(φ) (or interfacial area density) increases, which leads to greater damage input into increasing fineness (hence reduction in boundary viscosity), which focuses the variations in the poros- ity field, hence completing the cycle that facilitates the formation of narrow plate boundaries. While many studies have considered the effect of cracking and grain size reduction on plate generation or convection separately (Hall & Parmentier 2003; Bercovici & Ricard 2005), our study suggests that it is the interaction of these two forms of damage which allows for the formation of plate tec- tonics on Earth. Further investigation into the coupling between void- and fineness-generating damage in a depth-dependent prob- lem, hence allowing us to follow the plate boundary formation process in a vertical section of the lithosphere, is a necessary next step. dependent rheologies would enable us to explore the possibilities of fault formation and progression in a more complete manner. For ex- ample, localization may be initiated by grain size reduction at mid to deep lithospheric depths leading to failure at shallower depths via void generation completing the formation of a lithospheric fault. AC K N OW L E D G M E N T S The authors benefited from discussions with Shun-ichiro Karato and Garrett Leahy, as well as the feedback in the Yale mantle/core semi- nar. The suggestions from two anonymous reviewers also helped to clarify many different aspects of this paper. Support was provided by the National Science Foundation (NSF, grant EAR-0105269) and the Centre National de la Recherche Scientifique (CNRS). 4.2 Final thoughts These considerations are an important step for future analyses, but the current thin-sheet model does allow for a fairly comprehensive understanding of how two-phase damage theory could facilitate the formation of tectonic plates on Earth. 4.2 Final thoughts Increases in the viscosity exponent m lead to the formation of narrow plate boundaries (both convergent and di- vergent) as well as increases in the rigidity of the plate interior, and the values of m explored fall within the range of values expected for mantle rheologies (Karato & Wu 1993). These results suggest that the mechanism of localization at convergent and divergent bound- aries need not originate from the same process. Our model does not incorporate the generation of melts which will strongly influence the strength of ridges as well (Tackley 2000a,b), and melt forma- tion obviously leads to different localization mechanism than what affects subduction zones. While grain size sensitive creep (coupled with crack formation) may be needed to form narrow convergent boundaries, the generation of narrow divergent boundaries may only need some small damage input coupled with strong healing near the ridge. Allowing for both forms of damage causes a feed- back mechanism which allows for the most significant localization of plate boundaries in our study. The feedback mechanism begins with void-generation allowing for greater variability in porosity, the range of η(φ) (or interfacial area density) increases, which leads to greater damage input into increasing fineness (hence reduction in boundary viscosity), which focuses the variations in the poros- ity field, hence completing the cycle that facilitates the formation of narrow plate boundaries. While many studies have considered the effect of cracking and grain size reduction on plate generation or convection separately (Hall & Parmentier 2003; Bercovici & Ricard 2005), our study suggests that it is the interaction of these two forms of damage which allows for the formation of plate tec- tonics on Earth. Further investigation into the coupling between void- and fineness-generating damage in a depth-dependent prob- lem, hence allowing us to follow the plate boundary formation process in a vertical section of the lithosphere, is a necessary next step. characteristics we find. We have found that increasing fineness in- ducing damage f A (for fixed healing rate k A and viscosity expo- nent m) does not do much to improve the plate characteristics in our model, though allowing for void-generating damage in tandem with fineness-generating damage dramatically changes this result. Increases in the healing term k A can lead to an important reduction in divergent boundary width due to the reduction in the healing length scale. 4.2 Final thoughts The goal of this study is to test our hypothesis that two-phase dam- age theory will allow for shear localization leading to the develop- ment of plate tectonics on Earth. We have employed a 2-D model of mantle convection in order to test the plate generating capa- bilities of two-phase damage theory. The primary characteristics of plate tectonics on Earth that we have attempted to address in this study include the formation of narrow boundaries at both con- vergent and divergent zones, rigid plate interiors, and temporally persistent plate-like velocity profiles. We have attempted to under- stand how the different factors (i.e. advection, deformational work, and healing) affect the rheological variables (fineness and porosity) to produce the aforementioned plate-like features. We have explored how each form of damage, void and fineness generating (or grain size reducing), by itself and in tandem, can facilitate plate-like behaviour in a convectively driven system. Our results for void and fineness generating damage by themselves have shown some similar consequences to separate problems as previ- ous work (Bercovici & Ricard 2005), namely that void generating damage was unsuccessful while fineness generating damage was quite successful at inducing plateness in this study and toroidal motion in Bercovici & Ricard (2005). An important part of this study has been to understand how the range of different param- eters that govern grain size (fineness) evolution affect the plate C⃝2008 The Authors, GJI, 174, 1065–1080 Journal compilation C⃝2008 RAS C⃝2008 The Authors, GJI, 174, 1065–1080 Journal compilation C⃝2008 RAS W. Landuyt, D. Bercovici and Y. Ricard 1078 characteristics we find. We have found that increasing fineness in- ducing damage f A (for fixed healing rate k A and viscosity expo- nent m) does not do much to improve the plate characteristics in our model, though allowing for void-generating damage in tandem with fineness-generating damage dramatically changes this result. Increases in the healing term k A can lead to an important reduction in divergent boundary width due to the reduction in the healing length scale. This behaviour may also be important for the forma- tion of narrow rifts in that plate thinning under tension may allow for significant healing outside of the failure region thereby forming a localized rift zone. A P P E N D I X : S C A L I N G A NA LY S I S : P R E D I C T I O N S V E R S U S R E S U LT S Karato, S., 1983. Grain-size distribution and rheology of the upper mantle, Tectonophysics, 104, 155–176. Given the extensive numerical data generated, we test how well the scaling analyses developed in the previous sections fit the data. In Fig. A1, we show the results of plotting Ao (the maximum steady state value of fineness, see eq. 37) at both ridges and subduction zones versus eq. (43) for each numerical experiment. The fit of the data to the prediction is quite good, and indicates that our assumption that stress (deformational work) is constant in a given environment (i.e. divergent vs. convergent) is reasonably accurate. The slopes of the predicted fineness magnitudes are actually different for ridges and subduction zones owing to the fact that the second invariant of stress between these two different regions is different. These results suggest that the steady-state amplitude of fineness at subduction zones and ridges is governed by the competition between deforma- tional work and healing, and advection plays no role in determining the amplitude. Karato, S., 1989. Grain growth kinetics in olivine aggregates, Tectono- physics, 168, 255–273. Karato, S. & Wu, P., 1993. Rheology of the upper mantle: a synthesis, Science, 260, 771–778. Karato, S., Toriumi, M. & Fujii, T., 1980. Dynamic recrystallization of olivine single crystals during high temperature creep, Geophys. Res. Lett., 7, 649–652. Katz, R., Spiegelman, M. & Holtzman, B., 2006. The dynamics of melt and shear localization in partially molten aggregates, Nature, 442, 676– 679. King, S., 2001. Subduction zones: observations and geodynamic models, Phys. Earth planet. Int., 127, 9–24. Kohlstedt, D., Evans, B. & Mackwell, S., 1995. Strength of the lithosphere: Constraints imposed by laboratory experiments, J. geophys. Res., 100, 17 587–17 602. Lemery, C., Ricard, Y. & Sommeria, J., 2000. A model for the emergence of thermal plumes in Rayleigh-Benard convection at infinite Prandtl number, J. Fluid Mech., 414, 225–250. The results of comparing our numerical data for δc/umax and δd/umax with our scaling analysis is presented in Fig. A2(a). In Fig. A2(b) we plot ratio of the numerical result for δc,d/umax to the predicted value for each point in parameter space, since we expect McKenzie, D., 1984. The generation and compaction of partially molten rock, J. Petrol., 25, 713–765. Moresi, L. & Solomatov, V., 1998. Mantle convection with a brittle litho- sphere: thoughts on the global tectonic style of the Earth and Venus, Geophys. J. Int., 133, 669–682. A P P E N D I X : S C A L I N G A NA LY S I S : P R E D I C T I O N S V E R S U S R E S U LT S 0.0 15.8 31.5 47.3 0.0 16.5 33.1 49.6 + + + + + + + + + + + + + + + + + + + + +++ + + + + + + + + + + + + + +++++++ ++ + ++++ + ++ + + + + + + + + + + + + + + + + + + + + +++ + + + + ++ + + + + + + + +++++++ +++ +++++++ Ridge Subduction Zone Calculated Fineness Predicted Fineness Figure 14. The values of fineness plotted on the ordinate come from the numerical experiments, while the values of fineness plotted on the abscissa are from the scaling analyses eq. (43). The results from the numerical exper- iments and the scaling analyses are for the maximum values of steady-state fineness (Ao) in both subduction zones and ridges. 0.0 15.8 31.5 47.3 0.0 16.5 33.1 49.6 + + + + + + + + + + + + + + + + + + + + +++ + + + + + + + + + + + + + +++++++ ++ + ++++ + ++ + + + + + + + + + + + + + + + + + + + + +++ + + + + ++ + + + + + + + +++++++ +++ +++++++ Ridge Subduction Zone Calculated Fineness Predicted Fineness Ogawa, M., 2003. Plate-like regime of a numerically modeled thermal convection in a fluid with temperature-, pressure-, and stress-history-dependent viscosity, J. geophys. Res., 108, 2067, doi:10.1029/2000JB000069. Ribe, N., 1992. The dynamics of thin shells with variable viscosity and the origin of toroidal flow in the mantle, Geophys. J. Int., 110, 537– 552. Ricard, Y. & Bercovici, D., 2003. Two-phase damage theory and crustal rock failure: the theoretical ‘void’ limit, and the prediction of experimental data, Geophys. J. Int., 155, 1057–1064. Figure 14. The values of fineness plotted on the ordinate come from the numerical experiments, while the values of fineness plotted on the abscissa are from the scaling analyses eq. (43). The results from the numerical exper- iments and the scaling analyses are for the maximum values of steady-state fineness (Ao) in both subduction zones and ridges. Ricard, Y. & Froidevaux, C., 1986. Stretching instabilities and lithospheric boudinage, J. geophys. Res., 91, 8314–8324. Ricard, Y., Bercovici, D. & Schubert, G., 2001. Plate generation and two-phase damage theory in a model of mantle convection Plate generation and two-phase damage theory in a model of mantle convection 1079 Plate generation and two-phase damage theory in a model of mantle convection Gurnis, M., Zhong, S. & Toth, J., 2000. On the competing roles of fault reactivation and brittle failure in generating plate tectonics from mantle convection, in History and Dynamics of Global Plate Motions, Geophys. Monogr. Ser., Vol. 121, pp. 73–94, eds Richards, M.A., Gordon, R. & van der Hilst, R., Am. Geophys. Union, Washington, DC. weakening and asthenosphere, Geochem. Geophys. Geosystems (G3), 1, 2000GC000043. Wdowinski, S., O’Connell, R. & England, P., 1989. A continuum model of continental deformation above subduction zones: Application to the Andes and the Aegean, J. geophys. Res., 94, 10 331–10 346. Weinstein, S., 1996. Thermal convection in a cylindrical annulus with a non-Newtonian outer surface, PAGEOPH, 146, 551–572. Hager, B. & O’Connell, R., 1981. A simple global model of plate dynamics and mantle convection, J. geophys. Res., 86, 4843–4867. Hall, C. & Parmentier, E., 2003. Influence of grain size evolution on convec- tive stability, Geochem. Geophys. Geosystems (G3), 1, 2003GC000308. Weinstein, S. & Olson, P., 1992. Thermal convection with non-Newtonian plates, Geophys. J. Int., 111, 515–530. Jin, D., Karato, S. & Obata, M., 1998. Mechanisms of shear localization in the continental lithosphere: Inference from the deformation microstruc- tures of peridotites from the Ivrea zone, northwestern Italy, J. Struct. Geol., 20, 195–209. R E F E R E N C E S geophys. Res., 106(B5), 8925–8940. Cawood, P.A., Kroner, A. & Pisarevsky, S., 2006. Precambrian plate tecton- ics: criteria and evidence, GSA Today, 16(7), 4–11. Christensen, U. & Harder, H., 1991. Three-dimensional convection with variable viscosity, Geophys. J. Int., 104, 213–226. While a thin-sheet formulation for lithospheric dynamics cap- tures a significant component of lithospheric behaviour, allowing for depth-dependent rheologies might allow for a wider range of plate generating scenarios. As we noted previously the likelihood of void-generating damage manifesting itself at mid to deep litho- spheric depths is small, but it is entirely possible that localization in the lithosphere may vary as a function of depth. Allowing for depth- Dumoulin, C., Bercovici, D. & Wessel, P., 1998. A continuous plate-tectonic model using geophysical data to estimate plate margin widths, with a seismicity based example, Geophys. J. Int., 133, 379–389. England, P. & McKenzie, D., 1982. A thin viscous sheet model for conti- nental deformation, Geophys. J. R. astr. Soc., 70, 295–321. Evans, B., Renner, J. & Hirth, G., 2001. A few remarks on the kinetics of static grain growth in rocks, Int. J. Earth Sci., 90, 88–103. C⃝2008 The Authors, GJI, 174, 1065–1080 Journal compilation C⃝2008 RAS Plate generation and two-phase damage theory in a model of mantle convection A P P E N D I X : S C A L I N G A NA LY S I S : P R E D I C T I O N S V E R S U S R E S U LT S A two-phase model of compaction and damage, 2, Applications to compaction, deformation, and the role of interfacial surface tension, J. geophys, Res., 106(B5), 8907–8924. 0.00 0.08 0.16 0.25 0.00 0.02 0.04 0.06 + + ++++ ++++ + + ++++++ + + +++ + + + ++ + + +++ + + ++++ ++++ + ++++ + + + + + + +++ + +++ + + + +++++ + + + +++ + + + + + + + +++ + + +++ ++++ + + ++ + + + + + 0 18 35 53 0.000 0.333 0.667 1.000 +++++++++++++++++++++++++++++++++++++++++++++++++++++ +++++++++++++++++++++++++++++++++++++++++++++++++++++ Divergent Convergent numerical run # (a) (b) Figure 15. (a) Ordinate values are boundary widths over umax (or one over the strain-rate) that we determined from numerical experiments plotted against the values on the abscissa which are from the scaling analyses. (b) Ordinate values are the ratio of inverse strain-rate (δc,d/umax) for the scaling analysis to the numerical experiments plotted against the run number. This graph displays the constant scale that differs between the scaling predictions and numerical experiments. 0.00 0.08 0.16 0.25 0.00 0.02 0.04 0.06 + + ++++ ++++ + + ++++++ + + +++ + + + ++ + + +++ + + ++++ ++++ + ++++ + + + + + + +++ + +++ + + + +++++ + + + +++ + + + + + + + +++ + + +++ ++++ + + ++ + + + + + 0 18 35 53 0.000 0.333 0.667 1.000 +++++++++++++++++++++++++++++++++++++++++++++++++++++ +++++++++++++++++++++++++++++++++++++++++++++++++++++ Divergent Convergent numerical run # (a) (b) 0.00 0.08 0.16 0.25 0.00 0.02 0.04 0.06 + + ++++ ++++ + + ++++++ + + +++ + + + ++ + + +++ + + ++++ ++++ + ++++ + + + + + + +++ + +++ + + + +++++ + + + +++ + + + + + + + +++ + + +++ ++++ + + ++ + + + + + Divergent Convergent (a) Schubert, G., Turcotte, D. & Olson, P., 2001. Mantle Convection in the Earth and Planets, Cambridge Univ. Press, Cambridge, UK. Spiegelman, M., 1993a. Flow in deformable porous media, part 1, Simple analysis, J. Fluid Mech., 247, 17–38. Spiegelman, M., 1993b. Physics of melt extraction: theory, implications and applications, Phil. Trans. R. Soc. London, Ser. A, 342, 23–41. applications, Phil. Trans. R. Soc. C⃝2008 The Authors, GJI, 174, 1065–1080 Journal compilation C⃝2008 RAS A P P E N D I X : S C A L I N G A NA LY S I S : P R E D I C T I O N S V E R S U S R E S U LT S London, Ser. A, 342, 23–4 Tackley, P., 1998. Self-consistent generation of tectonic plates in three- dimensional mantle convection, Earth planet. Sci. Lett., 157, 9–22. Figure 15. (a) Ordinate values are boundary widths over umax (or one over the strain-rate) that we determined from numerical experiments plotted against the values on the abscissa which are from the scaling analyses. (b) Ordinate values are the ratio of inverse strain-rate (δc,d/umax) for the scaling analysis to the numerical experiments plotted against the run number. This graph displays the constant scale that differs between the scaling predictions and numerical experiments. Figure 15. (a) Ordinate values are boundary widths over umax (or one over the strain-rate) that we determined from numerical experiments plotted against the values on the abscissa which are from the scaling analyses. (b) Ordinate values are the ratio of inverse strain-rate (δc,d/umax) for the scaling analysis to the numerical experiments plotted against the run number. This graph displays the constant scale that differs between the scaling predictions and numerical experiments. Tackley, P., 2000a. Self-consistent generation of tectonic plates in time-dependent, three-dimensional mantle convection simulations, 1. Pseudoplastic yielding, Geochem. Geophys. Geosystems (G3), 1, 2000GC000036. Tackley, P., 2000b. Self-consistent generation of tectonic plates in time- dependent, three-dimensional mantle convection simulations, 2. Strain Tackley, P., 2000b. Self-consistent generation of tectonic plates in time- dependent, three-dimensional mantle convection simulations, 2. Strain Tackley, P., 2000b. Self-consistent generation of tectonic plates in time- dependent, three-dimensional mantle convection simulations, 2. Strain C⃝2008 The Authors, GJI, 174, 1065–1080 Journal compilation C⃝2008 RAS C⃝2008 The Authors, GJI, 174, 1065–1080 Journal compilation C⃝2008 RAS W. Landuyt, D. Bercovici and Y. Ricard 1080 between the numerical experiments and analytic prediction (where the analytic prediction has been multiplied by the constant deter- mined in Fig. A2b). The regression line fits all of the data points to within approximately 15 per cent which we will assume allows us to assign some validity to the analytic expression we derived. our scaling analysis to only fit the numerical data up to some multi- plicative constant (e.g. a geometrical factor of order 1 not included in a scaling analysis). The fit for convergent boundaries is especially good, but both boundaries exhibit the general features suggested by the scaling in eq. (40) as demonstrated by the linear relationship
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ARM Controller and EEG based Drowsiness Tracking and Controlling during Driving
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International Journal of Reconfigurable and Embedded Systems (IJRES) Vol. 6, No. 3, November 2017, pp. 127~132 ISSN: 2089-4864, DOI: 10.11591/ijres.v6.i3.pp127-132 International Journal of Reconfigurable and Embedded Systems (IJRES) Vol. 6, No. 3, November 2017, pp. 127~132 ISSN: 2089-4864, DOI: 10.11591/ijres.v6.i3.pp127-132  127  127 127 B. Naresh, Email: narib.naresh@gmail.com ARM Controller and EEG Based Drowsiness Tracking and Controlling during Driving B. Naresh, S. Rambabu, D. Khalandar Basha Department of Electronics and Communication Engineering, Institute of Aeronautical Engineering, India ABSTRACT This paper discussed about EEG-Based Drowsiness Tracking during Distracted Driving based on Brain computer interfaces (BCI). BCIs are systems that can bypass conventional channels of communication (i.e., muscles and thoughts) to provide direct communication and control between the human brain and physical devices by translating different patterns of brain activity commands through controller device in real time. With these signals from brain in mat lab signals spectrum analyzed and estimates driver concentration and meditation conditions. If there is any nearest vehicles to this vehicle a voice alert given to driver for alert. And driver going to sleep gives voice alert for driver using voice chip. And give the information about traffic signal indication using RFID. The patterns of interaction between these neurons are represented as thoughts and emotional states. According to the human feelings, this pattern will be changing which in turn produce different electrical waves. A muscle contraction will also generate a unique electrical signal. All these electrical waves will be sensed by the brain wave sensor and it will convert the data into packets and transmit through Bluetooth medium. Level analyzer unit (LAU) is used to receive the raw data from brain wave sensor and it is used to extract and process the signal using Mat lab platform. The nearest vehicles information is information is taken through ultrasonic sensors and gives voice alert. And traffic signals condition is detected through RF technology. Article history: Received July 05, 2017 Revised Sep 06, 2017 Accepted Sep 20, 2017 ARM controller Bluetooth Brain wave sensor DC motor Ultrasonic sensor Copyright © 2017 Institute of Advanced Engineering and Science. All rights reserved. Corresponding Author: B. Naresh, Department of Electronics and Communication Engineering, Institute of Aeronautical Engineering, Dundigal, Telangana, India. Email: narib.naresh@gmail.com B. Naresh, Department of Electronics and Communication Engineering, Institute of Aeronautical Engineering, Dundigal, Telangana, India. Email: narib.naresh@gmail.com B. Naresh, Department of Electronics and Communication Engineering, Institute of Aeronautical Engineering, Dundigal, Telangana, India. Email: narib.naresh@gmail.com B. Naresh, Department of Electronics and Communication Engineering, Institute of Aeronautical Engineering, Dundigal, Telangana, India. Email: narib.naresh@gmail.com 1. INTRODUCTION Driving is a complex task, requiring full concentration and a calm attitude. Stressed and strong emotions, whether they result from the driving task itself or unrelated matters, can affect a driver’s abilities. For example, research has shown that angry drivers are more likely to take risks such as speeding, rapidly switching lanes, tailgating and jumping red lights. EEG signal can be one of the features of BCI for detecting driver’s state of mind. The main objective of this paper is to study the different mental state recognition methods based on EEG signals. Later work can be done on analyzing different mental states of a driver in different traffic situation and based on the study, predicting whether driver is mentally fit or not. In recent past, it has been observed that drivers with mental fatigue lead to accident. In this proposol, we proposed a robust real-time embedded platform to monitor the loss of attention of the driver during day and night driving conditions. The alertness level can be measured by using different parameters, such as electro encephalogram (EEG) signals, visual features, blood samples, speech and others. The EEG- based method has been reported to be highly authentic for estimating the state of drowsiness. In a new method for multi fractal analysis of EEG signals Journal homepage: http://iaescore.com/journals/index.php/IJRES/index ISSN: 2089-4864 128  named generalized Higuchi fractal dimension spectrum (GHFDS) was proposed and applied in mental arithmetic task recognition from EEG signals. named generalized Higuchi fractal dimension spectrum (GHFDS) was proposed and applied in mental arithmetic task recognition from EEG signals. 2.1.2. Working Principle The principle of operation is quite simple. It consists of two dry sensors are detect and filters the EEG signals. The sensor tip detects electrical signals from the forehead of the brain. At the same time, the sensor picks up ambient noise generated by human muscle, computers, light bulbs, electrical sockets and other electrical devices. The sensor and ear clip picks up the information from grounds and reference, which allows think gear chip to filter out the electrical noise. The device measures the raw signal, power spectrum (alpha, beta, delta, gamma, theta), attention level, mediation level and blink detection. For every clock frequency of 512 Hz, it receives the raw data from EEG. Other measured values are made every second. Therefore, raw EEG data is a main source of information on EEG signals using Mind Wave MW001. 2. PROPOSED BLOCK DIAGRAM The above block diagram shows the control of vehicle through ARM Controller, it identifies the drowsiness while driving and also it detects the obstacles near to the vehicle. Figure 1 shows BCI- attention tracking. It consists of two sections, i.e. (a) design and implementation and (b) system hardware using ARM Processor. Figure 1. BCI- attention tracking (a) design and implementation and (b) system hardware using ARM processor Figure 1. BCI- attention tracking (a) design and implementation and (b) system hardware using ARM processor 2.1. Design and Implementation Electroencephalography (EEG) is the measurement of electrical activity in the living brain. In this project we used a brainwave sensor MW001 to analyze the EEG signals. This design discuss about processing and recording the raw EEG signal from the sensor MW001 in the MATLAB environment and through Serial transmission control commands will be passed to the voice chip. This MW001 sensor is not used for medical purpose, but it is used to measure neurons feedback. The Brain control interface is a direct communication pathway between the brain and an external device. 2.2. System Hardware LPC2148 Processor 2.2. System Hardware LPC2148 Processor In this paper it uses LPC2148 processor, is belongs to one of the family of ARM7. This processor employs the pipeline architecture, hence data processing and memory operations are executing parallel, it reduces the execution time. y In this paper it uses LPC2148 processor, is belongs to one of the family of ARM7. This processor employs the pipeline architecture, hence data processing and memory operations are executing parallel, it reduces the execution time. It consists of two instruction sets. It consists of two instruction sets.  Standard 32-bit ARMv5TE set  16-bit THUMB set 2.2.1. Thinkgear Asic Module  It is used to interface the dry electrode, One EEG channel with three contacts namely EEG, REF, and GND.  It is used to interface the dry electrode, One EEG channel with three contacts namely EEG, REF, and GND.  Improper fit detected through “Poor Signal Quality” warning from ASIC to reset if off the head for four consecutive seconds, or if it is receiving a poor signal for seven consecutive seconds.  Improper fit detected through “Poor Signal Quality” warning from ASIC to reset if off the head for four consecutive seconds, or if it is receiving a poor signal for seven consecutive seconds.  It is applicable for low power consumption devices.  Max power consumption 15mA @ 3.3 V  Raw EEG data output at 512 bits per second. 2.2.2. DC Motor A motor consists of a rotor and a permanent magnetic field stator. The magnetic field is maintained using either permanent magnets or electromagnetic windings. Motors are the devices that provide the actual speed and torque in a drive system. This family includes AC motor types (single and multiphase motors, universal, servo motors, induction, synchronous, and gear motor) and DC motors (brush less, servo motor, and gear motor) as well as linear, stepper and air motors, and motor contactors and starters. 3. SYSTEM SOFTWARE Using MATLAB’s GUIDE, the processes of laying out a GUI (i.e., its buttons, pop-up menus, etc.) and programming the operation of the GUI are divided conveniently into two easily managed and relatively independent tasks. 2.2.3. Ultrasonic Sensor Ultra Sonic Sensor Our ultrasonic range finder is capable of allowing the user to determine his or her distance from an object or wall. It is used to identify the obstacle, nearer to vehicle while driving. 2.1.4. The Communications Protocol 2.1.4. The Communications Protocol It consists of 7 basic steps, which are presented in following steps Load Think Gear library into MATLAB It consists of 7 basic steps, which are presented in following steps. Load Think Gear library into MATLAB Get a connection ID handle to Think Gear Attempt to connect the connection ID handle to serial port "COMx" Waiting to establish the connection Read packets from the connection In the first step are functions with parameters in the following order In the first step are functions with parameters in the following order  Libisloaded („Think gear‟)–returns true if the Think Gear library is loaded, and false otherwise.  Libisloaded („Think gear‟)–returns true if the Think Gear library  Load library ('Think gear.dll','think gear.h')–loads the functions defined in the header file and found library. Now, the function calllib () can call a function in the ThinkGear library.  Calllib ('Thinkgear'TG_GetDriverVersion')–returns the version of loaded library. In the next step, the function calllib ('Thinkgear', 'TG_GetNewConnectionId') gets a new connection ID handle to ThinkGear. The value -1 is returned if too many connections have been created. Select Connection ID, number of the serial port, Baud rate and type of data from the TG connect. The number of the serial port is given during the pairing of the device. The device can connect on modes 1200, 2400, 4800, 9600, 57600 and 115200 bits per second (bps). Here, we use the 9600 bps rate and stream 5V RAW mode, because these parameters have the minimum of transmission errors. IJRES ISSN: 2089-4864 2.1.3. Matlab Based Design Theory The MATLAB allows including thinkgear.dll. This environment has broad support in toolbox, which makes it ideal for a scientific research. This paper presents how recording and processing the raw EEG signal in MATLAB environment using Mind Wave sensor. a system of digital rules for message exchange between MATLAB environment and Mind Wave MW001 device is processing through communications protocol. This section also presents the main parameters of think gear library. IJRES Vol. 6, No. 3, November 2017 : 127 – 132 129  IJRES 3.1. Role of Matlab in BCI The MATLAB allows including thinkgear.dll. This environment has broad support in toolbox, which makes it ideal for a scientific research. This paper presents how recording and processing the raw EEG signal in MATLAB environment using Mind Wave sensor. The Communication Protocol shows a system of ARM Controller and EEG Based Drowsiness Tracking and Controlling during… (B. Naresh)  130 ISSN: 2089-4864 digital rules for message exchange between MATLAB environment and Mind Wave MW001 device. This section also presents the main parameters of thinkgear library. digital rules for message exchange between MATLAB environment and Mind Wave MW001 device. This section also presents the main parameters of thinkgear library. The Communication Protocol, shows a system of digital rules for message exchange between MATLAB environment and Mind Wave MW001 device. This section also presents the main parameters of thinkgear library. The connection is established through command: calllib ('Thinkgear ','TG_ Connect', Id, ComPortName,TG_BAUD_115200,TG_ STR EAM _5VRAW). In the next step, we must attempt to read a Packet of data from the connection. We use the TG_Read Packets () function with ID parameter and number of packet to read. The command calllib ('Thinkgear','TG_ReadPackets', Id, 1). Figure 2 shows Brian signal representation in MATLAB. Figure 2. Brian signal representation in MATLAB Figure 2. Brian signal representation in MATLAB The above graph representation includes the attention value and blink strength. Based on this signals device control file will be opened and the device will get controlled based on the brain signals. IJRES Vol. 6, No. 3, November 2017 : 127 – 132 [1] Chin-Teng Lin, Fu-Chang Lin, Shi-An Chen, Shao-Wei Lu, Te-Chi Chen, Li-Wei Ko, “EEG-based Brain-computer Interface for Smart Living Environmental Auto-adjustment”, Journal of Medical and Biological Engineering, Year: 2010, Vol. 30, Page: 237-245. 5. CONCLUSION The main goal of this paper is to design and implement a human thought controlled electrical switching system using BCI technology for the physically challenged people. Non invasive BCI method is used for capturing the brain signals with the help of brain wave sensor. Different cognitive state of the user like attention level, meditation level, and eye-blink can be used to measure the human thought. The signals are classified according their respective frequencies ranging from 0.5 Hz to 30 Hz. By setting the threshold value for an event, the interrupt is generated. This Interrupt is sent to the microcontroller unit for operating the appliances. 4. ANALYSIS OF HARDWARE SECTION ARM Controller read the value of raw ARM Controller read the value of raw EEG signal with the maximum frequency of 512 Hz. Sampling frequency is set on 512 Hz, and we control time delays in sampling. The value of the signal and time are written to the array data.The data which are stored in array will be compared with the threshold points given by the user. In this Paper, the Matlab section waits for three consecutive blink in order to send the hardware activation signal. Then based on the blink level signal, the cursor will be places upon any section to get the device control. Further the person has to raise the attention to switch the device. Figure 3 shows Brain wave signal identification in Matlab. Figure 4 shows Drowsiness identification through Matlab IJRES Vol. 6, No. 3, November 2017 : 127 – 132  131 ISSN: 2089-4864 IJRES IJRES Figure 3. Brain wave signal identification in Matlab Figure 4. Drowsiness identification through Matlab Figure 3. Brain wave signal identification in Matlab Figure 4. Drowsiness identification through Matlab Figure 3. Brain wave signal identification in Matlab [2] Wolpaw, J.R.,”Brain-computer interface research comes of age: traditional assumptions meet emerging realities.” Journal of motor behavior, Year: 2010, Vol. 42, Page: 351-353. [4] T. Kameswara Rao, M. Rajyalakshmi, Dr. T. V. Prasad, “An Exploration on Brain Computer Interface and Its Recent Trends”, International Journal of Advanced Research in Artificial Intelligence, Year: 2012, Vol. 1, Page: 17-22. ARM Controller and EEG Based Drowsiness Tracking and Controlling during… (B. Naresh) ] Anupama.H.S, N.K.Cauvery, Lingaraju. G.M, Brain computer interface and its types - a study , Internat Journal of Advances in Engineering & Technology, Year: 2012, Vol. 8, Page: 117-119. f , , , g ] Anupama.H.S, N.K.Cauvery, Lingaraju. G.M, “Brain computer interface and its types - a study”, Internation IJRES Vol. 6, No. 3, November 2017 : 127 – 132 REFERENCES [1] Chin-Teng Lin, Fu-Chang Lin, Shi-An Chen, Shao-Wei Lu, Te-Chi Chen, Li-Wei Ko, “EEG-based Brain-computer Interface for Smart Living Environmental Auto-adjustment”, Journal of Medical and Biological Engineering, Year: 2010, Vol. 30, Page: 237-245. g [2] Wolpaw, J.R.,”Brain-computer interface research comes of age: traditional assumptions meet emerging realities.” Journal of motor behavior, Year: 2010, Vol. 42, Page: 351-353. f , , , g [3] Anupama.H.S, N.K.Cauvery, Lingaraju. G.M, “Brain computer interface and its type Journal of Advances in Engineering & Technology, Year: 2012, Vol. 8, Page: 117-119. [4] T. Kameswara Rao, M. Rajyalakshmi, Dr. T. V. Prasad, “An Exploration on Brain Computer Interface and Its Recent Trends”, International Journal of Advanced Research in Artificial Intelligence, Year: 2012, Vol. 1, Page: 17-22.  132 ISSN: 2089-4864 132 ISSN: 2089-4864  BIOGRAPHIES OF AUTHORS BIOGRAPHIES OF AUTHORS BIOGRAPHIES OF AUTHORS B.Naresh working as Associate professor in Institute of aeronautical engineering, Hyderabd, Telangana. Area of interest is Embedded Systems and VLSI System Design. S.Rambabu working as Assistant professor in Institute of aeronautical engineering, Hyderabd, Telangana.Area of interest is VLSI. D.Khalander Basha working as Associate professor in Institute of aeronautical engineering, Hyderabd, Telangana. Area of interest is Image processing. B.Naresh working as Associate professor in Institute of aeronautical engineering, Hyderabd, Telangana. Area of interest is Embedded Systems and VLSI System Design. S.Rambabu working as Assistant professor in Institute of aeronautical engineering, Hyderabd, Telangana.Area of interest is VLSI. S.Rambabu working as Assistant professor in Institute of aeronautical engineering, Hyderabd, Telangana.Area of interest is VLSI. D.Khalander Basha working as Associate professor in Institute of aeronautical engineering, Hyderabd, Telangana. Area of interest is Image processing. D.Khalander Basha working as Associate professor in Institute of aeronautical engineering, Hyderabd, Telangana. Area of interest is Image processing. IJRES Vol. 6, No. 3, November 2017 : 127 – 132
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https://www.nature.com/articles/srep44696.pdf
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Experimental investigation on the coupled effect of effective stress and gas slippage on the permeability of shale
Scientific reports
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Diansen Yang1, Wei Wang1,2, Weizhong Chen1,3, Shugang Wang3 & Xiaoqiong Wang4 Diansen Yang1, Wei Wang1,2, Weizhong Chen1,3, Shugang Wang3 & Xiaoqiong Wang4 Permeability is one of the most important parameters to evaluate gas production in shale reservoirs. Because shale permeability is extremely low, gas is often used in the laboratory to measure permeability. However, the measured apparent gas permeability is higher than the intrinsic permeability due to the gas slippage effect, which could be even more dominant for materials with nanopores. Increasing gas pressure during tests reduces gas slippage effect, but it also decreases the effective stress which in turn influences the permeability. The coupled effect of gas slippage and effective stress on shale permeability remains unclear. Here we perform laboratory experiments on Longmaxi shale specimens to explore the coupled effect. We use the pressure transient method to measure permeability under different stress and pressure conditions. Our results reveal that the apparent measured permeability is controlled by these two competing effects. With increasing gas pressure, there exists a pressure threshold at which the dominant effect on permeability switches from gas slippage to effective stress. Based on the Klinkenberg model, we propose a new conceptual model that incorporates both competing effects. Combining microstructure analysis, we further discuss the roles of stress, gas pressure and water contents on gas permeability of shale. With recent development of novel technologies, unconventional gas extraction has made great success in the North America. Shale gas has been considered as an attractive low-carbon solution for the transition period to a future power by renewable energy1. As an important supplement to conventional energy resources, shale gas has obtained an increasing attention in the past years. Due to its extremely low permeability, shale gas is still difficult to be recovered, particularly in deep formations. For example, the average burial depth of shale gas in the US is about 800–2000 m, while the average depth in China is over 3500 m where the stress is significantly high (>​50 MPa)2. In order to better evaluate the production of shale gas, an accurate permeability of shale under in situ stress condi- tions is required. As the mean pore size of shale is often at the nanoscale, the permeability of shale is substantially low, typically less than 10−19 m2, which is difficult to be measured in the laboratory. Inert gases such as helium, azote, nitrogen, are often used to measure the low permeability. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports received: 16 August 2016 accepted: 13 February 2017 Published: 17 March 2017 Experimental investigation on the coupled effect of effective stress and gas slippage on the permeability of shale received: 16 August 2016 accepted: 13 February 2017 Published: 17 March 2017 Methods Pressure transient method and experimental setup. Pressure transient method or known as pressure pulse decay13,20,21 is often used to effectively measure low permeability of tight rocks (<​100 nD (10−19 m2)), as it is less time consuming. Recently, pressure transient measurements on ultra-tight rocks have been interpreted by accounting for the effects of gas-slippage22, transitional flow, and Knudsen diffusion flow23. Its principle was first proposed by Brace et al13. It consists of a sample connected with two reservoirs that have an equilibrated initial pressure P0 in the whole system. An increment pressure Δ​P0 is suddenly imposed into one of the two reservoirs and the pressure evolution in two reservoirs over time is recorded as demonstrated in Fig. 1a. Analyzing the var- iation of gas pressures, permeability can be then determined either using the method developed by Brace13,21 or back analysis method20.i y Based on the principle of pressure transient method, a specific setup of gas permeability measurements has been designed and developed in the Institute of Rock and Soil Mechanics of Wuhan, CAS. In this system, a triax- ial core holder, capable of accepting membrane-sheathed cylindrical samples (2.5 cm diameter) and of applying independent loading in the radial and axial directions, is connected to two gas reservoirs. In order to shorten the testing time, small reservoirs are designed and the volume of upstream reservoir is about 8.2 cm3 and the volume of downstream reservoir is about 6.6 cm3. Axial and confining (radial) stresses up to 56 MPa are independently applied using two ISCO 260D pumps with control up to ±​ 0.007 MPa. Gas pressure is controlled by a ISCO 500D pump. Different transducers (temperature, pressure, and displacement transducers) are connected to a computer to automatically record the experimental data. All parts of this apparatus are located in a temperature controlled cabinet where the temperature can be maintained constant with a deviation of +​/−​ 0.1 °C. The whole system is shown in Fig. 1b. Tests on aluminum samples have been carried out to calibrate the system and check possible leakage and the gas permeability accuracy of the system is up to 10−23 m2. Sample description. In this study, shale samples were recovered from the Lower Silurian Longmaxi (LM) shale formation located in Qianjiang, southeast of Chongqing. Longmaxi shale formation is comprised of dark gray to black graptolite shale, carbonaceous shale, siliceous shale, silty shale, and argillaceous siltstone24. Diansen Yang1, Wei Wang1,2, Weizhong Chen1,3, Shugang Wang3 & Xiaoqiong Wang4 Gas can shorten the testing time and can also avoid the hydration reaction of shale with high content of clay minerals that are sensitive to water. However, gas slippage effect could be very large in nanopores and thus could affect gas permeability measurement. Numerous investigations have been performed to study gas slippage phenomenon which was initially noted in the 1940s3–8. Due to gas slippage, the steady-state flow rate through small capillaries is higher, which results in an overestima- tion of permeability. To estimate the gas slippage effect on the permeability, Klinkenberg proposed an empirical equation written as = + K K b P (1 / ) (1) a int m = + K K b P (1 / ) a int m (1) where Ka is the apparent permeability, Kint is the intrinsic permeability, Pm is the average gas pressure in pores, and b is the slope of Klinkenberg straight line related to the mean free path of gas molecules. Many researchers have where Ka is the apparent permeability, Kint is the intrinsic permeability, Pm is the average gas pressure in pores, and b is the slope of Klinkenberg straight line related to the mean free path of gas molecules. Many researchers have 1State Key Laboratory of Geomechanics and Geotechnical Engineering, Institute of Rock and Soil Mechanics, Chinese Academy of Sciences, Wuhan, Hubei 430071, China. 2University of Chinese Academy of Science, Beijing, 100049, China. 3Geotechnical and Structural Engineering Research Center, Shandong University, Jinan, Shandong, China. 4Unconventional Natural Gas Institute, China University of Petroleum, Beijing 102249, China. Correspondence and requests for materials should be addressed to D.S.Y. (email: dsyang@whrsm.ac.cn) or S.G.W. (email: sdgeowsg@gmail.com) SCIEntIfIC REportS | 7:44696 | DOI: 10.1038/srep44696 1 www.nature.com/scientificreports/ Figure 1. Illustration of the pressure transient method for gas permeability measurement (a) principle of the method; (b) the experimental setup). Figure 1. Illustration of the pressure transient method for gas permeability measurement (a) principle of the method; (b) the experimental setup). investigated this phenomenon from the flow dynamics of gases in micro-channels with high Knudsen numbers (Kn), defined as the ratio of the molecular mean free path to the characteristic length of the flow path9,10. Methods such as the direct simulation Monte Carlo approach and the lattice Boltzmann method have been used to estimate the gas slippage effect3. A few new expanded models have been proposed to correct Klinkenberg’s equation4,6. Diansen Yang1, Wei Wang1,2, Weizhong Chen1,3, Shugang Wang3 & Xiaoqiong Wang4 In the experimental tests, Klinkenberg effect is often characterized using different gas pressures to measure perme- ability11. High gas pressure is often applied to reduce the gas slippage effect because high gas pressure can lead to a smaller mean free path of gas molecules and thus minimize Klinkenberg effect. However, high gas pressure also reduces the effective stress and thus increases permeability. The dependence of permeability on the stress has been well observed in the laboratory. Dong et al. in 2010 found that the permeability of shale was more sensitive to the effective confining stress than sandstone12. Based on numerous experimental data, stress dependent permeability could be described by either an exponential relationship13,14 or a power law15–18. Katsuki et al. in 2013 discussed poroelastic effects on the stress-dependent permeability of a stiff shale and found that pore pressure changed gas permeability of shale significantly19. However, there have been only a few studies to date aiming at understanding the coupled effect of gas slippage and effective stress principle on the permeability of shale. This paper will present an experimental investigation on this topic and discuss various factors influencing the gas permeability of shale. SCIEntIfIC REportS | 7:44696 | DOI: 10.1038/srep44696 Methods The organic content of shale is larger than 0.5%, with a mean TOC of 2.54%. LM shales are mainly composed of clay, quartz and calcite, while in contrast pyrite and feldspar are minor. The grain density of shale sample is about 2.72 g/cm3 and the total porosity is 0.25–3.25% and the initial water content is less than 4%. Two cylindrical samples with a diameter of 25.0 mm and a length of 40.4 mm were drilled from the same LM shale block and they were undamaged and the measured longitudinal wave velocity (Vp) at the initial state was nearly the same (2267 m/s for the sample No.1 and 2254 m/s for the sample No.2). The dry density of the sample is 2.32 g/cm3 and the connected porosity measured by Autopore IV9510 is 2.50%. The pore size distribution of LM shale has been characterized by the mercury intrusion porosimetry (MIP) method (Fig. 2a) and by the gas adsorption method SCIEntIfIC REportS | 7:44696 | DOI: 10.1038/srep44696 2 www.nature.com/scientificreports/ Figure 2. Pore size distribution of Longmaxi shale using (a) the MIP method and (b) the gas adsorption method. Figure 2. Pore size distribution of Longmaxi shale using (a) the MIP method and (b) the gas adsorption method. (Fig. 2b). The results show that the pore size of LM shale is complex and it covers a range from several nanometers to hundred nanometers and the average pore size is 3.0 nm. Experimental procedure. In order to evaluate the coupled effect of effective stress and gas slippage on gas permeability of shale, a series of gas permeability tests have been performed on the shale samples subjected to dif- ferent confining stresses and gas pressures. Sample No.1 was at unsaturated state and the water content was 1.24%, which was measured at the end of testing by drying it at 105 °C for 24 hours. Before testing, sample No.2 with an initial water content of 1.50% was dried. To estimate the effect of effective stress on gas permeability, a cycle of loading and unloading is performed on the sample No.1 with a constant confining stress at 15 MPa. The path of applied axial stress follows: 15 MPa →​ 30 MPa →​ 45 MPa →​ 50 MPa →​ 55 MPa →​ 45 MPa →​ 30 MPa →​ 15 MPa. The maximum deviatoric stress is about 40 MPa, under which little damage could occur in the shale samples. Methods At different stress levels, the initial gas pressure was maintained at 3 MPa, and the increment gas pressure for the pressure pulse varied between 0.2–0.4 MPa to measure gas permeability. At the end of the mechanical unloading, a hydrostatic stress of 16 MPa was applied on the sample No.1 and a series of gas permeability measurements were carried out with different gas pressures (1 MPa, 2 MPa, 3 MPa, 4 MPa) to study the gas slippage effect. Three levels of hydrostatic stress (12 MPa, 14 MPa, 16 MPa) were applied on the dry sample No.2 and under these stress conditions, different gas pressures were selected to measure the gas permeability of the sample. The histories of mechanical loading and gas pressure performed on the samples (No.1 and No.2) are illustrated in Fig. 3a and b, respectively. Finally, 14 pulse tests have been conducted for sample No.1 and 8 pulse tests for sample No.2 and they are shown in Fig. 3. The duration of the tests has lasted about 60 days. In this study, nitrogen was chosen to measure gas permeability and the temperature of the whole system was kept at 30 °C. SCIEntIfIC REportS | 7:44696 | DOI: 10.1038/srep44696 Results i Results Determination of the apparent gas permeability. The apparent gas permeability Ka was firstly evalu- ated using Brace’s equation13,25, Determination of the apparent gas permeability. The apparent gas permeability Ka was firstly evalu- ated using Brace’s equation13,25, αηβ α =    +    = −    ∆ ∆    k L S V V P t P 1 1/ 1/ , ln ( ) /t (2) a up down 0 (2) where Vup and Vdown (m3) are the volumes in the upstream reservoir and downstream reservoir; Pup is the gas pressure in the upstream reservoir (Pa) and Pf is the final equilibrium pressure of the system (Pa); η​ (Pa.s) is the SCIEntIfIC REportS | 7:44696 | DOI: 10.1038/srep44696 3 www.nature.com/scientificreports/ Figure 3. Mechanical loading path and gas pressure evolution during gas permeability tests (a) wet sample No.1, (b) dry sample No.2). Figure 3. Mechanical loading path and gas pressure evolution during gas permeability tests (a) wet sample No.1, (b) dry sample No.2). Nitrogen viscosity at the temperature of 30 °C and the mean pore pressure; β​ is the isothermal compressibility coefficient of the pore fluid (Pa−1) and it is gas pressure dependent; α​ is the decay exponent; L and S are the length and cross-sectional area of the sample, and t is the testing time (s).hl Nitrogen viscosity at the temperature of 30 °C and the mean pore pressure; β​ is the isothermal compressibility coefficient of the pore fluid (Pa−1) and it is gas pressure dependent; α​ is the decay exponent; L and S are the length and cross-sectional area of the sample, and t is the testing time (s).hl p g Brace’s formula was derived assuming that Darcy law is valid and gas is ideal gas. The fluid volume in the pores of the rock sample is ignored. However, this will not induce a large error for tight rock with very low porosity (e.g., granite, shale). To estimate the error, a series of permeabilities, chosen by changing the value determined from the formula (2) with an increment of several 10−22 m2, were used to calculate the theoretical pressure in the upstream and downstream reservoirs following Brace’s method as the following. ∆ ∆ ∆ = + . + . = − . + . = + . Results i + α α − − P V V V e P V V V e V V V P P , P P , P P P (3) t down t up f 0 up up down f 0 down up down f 0 0 up up down ∆ ∆ ∆ = + . + . = − . + . = + . + α α − − P V V V e P V V V e V V V P P , P P , P P P (3) t down t up f 0 up up down f 0 down up down f 0 0 up up down (3) Numerical results were then compared with experimental results (e.g., Fig. 4a and b). These two figures illus- trate the evolution of the gas pressure in the upstream and downstream reservoirs during the pulse tests of the sample No.1 (3rd stage) and No.2 (4th stage), respectively. The results show that the numerical results matches well against the experimental results with an appropriate Ka.h The obtained apparent gas permeability Ka and the gas permeability determined directly from Brace’s formula Ka B of the shale samples at different stages are listed in the Table 1 and 2. Table 1 presents the gas permeability evolution Ka of sample No.1 at different stages, and Table 2 lists permeabilities measured for sample No.2. The results show that the difference between the permeability determined from the formula (a) and the appropriate obtained during the numerical comparison is less than 5%. As the shale samples have a very low porosity (<​2%), we believe that the Brace method is reliable in determining gas permeability in this work. Permeability versus deviatoric stress with the same gas pressure. As shown in Table 1, the appar- ent gas permeability decreases during the loading process and increases during the unloading process, but the SCIEntIfIC REportS | 7:44696 | DOI: 10.1038/srep44696 4 www.nature.com/scientificreports/ Figure 4. Experimental and numerical data of gas pressure evolutions in upstream and downstream reservoirs during pressure transient tests for determination of gas permeability (a) 2nd stage of the wet sample, Ka =​ 4.3 ×​ 10−21m2, (b) 8th of the dry sample, Ka =​ 39.5 ×​ 10−21m2). Figure 4. Results i Experimental and numerical data of gas pressure evolutions in upstream and downstream reservoirs during pressure transient tests for determination of gas permeability (a) 2nd stage of the wet Ka =​ 4.3 ×​ 10−21m2, (b) 8th of the dry sample, Ka =​ 39.5 ×​ 10−21m2). Stage in Fig. 3a 1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th 11th 12th 13th 14th Confining stress (MPa) 15 15 15 15 15 15 15 15 15 16 16 16 16 16 Axial stress (MPa) 15 30 45 50 55 45 30 15 15 16 16 16 16 16 Gas pressure (MPa) 3 3 3 3 3 3 3 3 4 4 2 3 4 1 Ka (x10−21 m2) 17.6 4.3 3.9 3.7 3 3.1 3.5 3.7 3.6 3.6 4.1 3.7 3.6 4.8 Ka B (x10−21 m2) 17.1 4.4 3.9 3.6 3.1 3.2 3.5 3.6 3.5 3.5 4.1 3.6 3.5 4.7 Table 1. Apparent permeability of shale sample No.1 at different stages. Stage in Fig. 3a 1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th 11th 12th 13th 14th Confining stress (MPa) 15 15 15 15 15 15 15 15 15 16 16 16 16 16 Axial stress (MPa) 15 30 45 50 55 45 30 15 15 16 16 16 16 16 Gas pressure (MPa) 3 3 3 3 3 3 3 3 4 4 2 3 4 1 Ka (x10−21 m2) 17.6 4.3 3.9 3.7 3 3.1 3.5 3.7 3.6 3.6 4.1 3.7 3.6 4.8 Ka B (x10−21 m2) 17.1 4.4 3.9 3.6 3.1 3.2 3.5 3.6 3.5 3.5 4.1 3.6 3.5 4.7 Table 1. Apparent permeability of shale sample No.1 at different stages. Table 1. Apparent permeability of shale sample No.1 at different stages. Stage in Fig. 3b 1st 2nd 3rd 4th 5th 6th 7th 8th Hydrostatic stress (MPa) 16 16 12 12 12 16 14 14 Gas pressure (MPa) 1 4 1.15 2 3 4 1.14 2 Ka (x10−21 m2) 57 37 51 44 43 37 49.5 39.5 Ka B (x10−21 m2) 56.8 37.2 50.9 44.2 43.1 36.9 49.8 39.7 Table 2. Apparent permeability of shale sample No.2 under the constant hydrostatic stress for different gas pressures. Stage in Fig. Results i 3b 1st 2nd 3rd 4th 5th 6th 7th 8th Hydrostatic stress (MPa) 16 16 12 12 12 16 14 14 Gas pressure (MPa) 1 4 1.15 2 3 4 1.14 2 Ka (x10−21 m2) 57 37 51 44 43 37 49.5 39.5 Ka B (x10−21 m2) 56.8 37.2 50.9 44.2 43.1 36.9 49.8 39.7 Table 2. Apparent permeability of shale sample No.2 under the constant hydrostatic stress for different gas pressures. Table 2. Apparent permeability of shale sample No.2 under the constant hydrostatic stress for different gas pressures. permeability is not fully recoverable. The reduction of permeability can be explained by the compression of the shale due to the increase of stress which will reduce the effective porosity. At the first stage of loading, during which the deviatoric stress increases to 15 MPa, permeability change (from 17.6 ×​ 10−21 m2 to 4.3 ×​ 10−21 m2) is much larger compared with other stages during which the variation is less than 1 ×​ 10−21 m2. This could be due to the fact that the first stage is the hydrostatic loading while the following stages are all deviatoric loading. Permeability change is consistent with the deformation evolution of the sample. The axial strain obtained during the hydrostatic loading is larger than that during the deviatoric loading for the same magnitude variation of SCIEntIfIC REportS | 7:44696 | DOI: 10.1038/srep44696 5 www.nature.com/scientificreports/ Figure 5. Permeability versus gas pressure at different isotropic stress (a) wet sample No.1, (b) dry sample No.2). Figure 5. Permeability versus gas pressure at different isotropic stress (a) wet sample No.1, (b) dry sample No.2). stresses. Permeability decline is consistent with the compression of the sample during mechanical loading. The irreversible permeability loss is believed to be related to the irreversible strain, which is 0.18% in the axial direc- tion at the end of the cycle of loading and unloading. Permeability versus gas pressure under constant hydrostatic stress. The results in Table 1 and 2 demonstrate that measured permeability of the samples (No.1 and No.2) decreases with the increase of gas pres- sure under different constant hydrostatic stresses (Fig. 5) below a certain pressure value. This permeability reduc- tion is often explained by the effect of gas slippage which is weakened when the gas pressure increases. Sample No.2 was subjected to an isostatic stress of 16 MPa, 12 MPa, 14 MPa, successively. Results i For the same level of gas pres- sure (about 1 MPa), permeability is smaller at the isostatic of 14 MPa than at 12 MPa. This should be related to the dependence of permeability on the stress as presented above. The higher stress induces a large compression of shale and thus reduces the effective porosity and results in a decrease in permeability. Permeability of sample No.2 is much larger than of sample No.1 under similar conditions, which can be explained by the role of water saturation on gas permeability. Scientific Reports | 7:44696 | DOI: 10.1038/srep44696 Discussions Our experimental results show that measured apparent permeability is influenced by both confining stress and gas slippage effect. The effective stress principle by Terzaghi is widely used to characterize the influence of pore pressure on the material. Biot’s effective stress σ′ can be defined by σ σ ′ = − ′b p, where σ′ is the Cauchy stress tensor, p is pore pressure and b’ is Biot’s coefficient. In linear elastic poromechanics26,27, it is calculated by b’ =​ 1 − K/Ks, where Ks is the bulk modulus of solid grain, and K is the apparent elastic bulk modulus under drained conditions. For soft material such as sand and soils, K«Ks, and then the Terzaghi effective stress is recov- ered since b’ =​ 1. For hard material, b’ is often less than 1. Bemer et al.28 identified Cox shale Biot’s coefficient and obtained an average value equal to 0.52. Homand et al. found that Biot’s coefficient decreased from 0.95 to 0.55 when the axial stress increased from 8 to 24 MPa20. Cariou et al.29 characterized Biot’s coefficient of partially sat- urated Cox shale and identified the apparent Biot’s coefficient as 1. In this study, Biot’s coefficient is evaluated by analyzing the difference in axial strain induced by confining stress and by gas pressure. For the wet sample No.1, the axial strain is increased to 0.49% when the hydrostatic stress increases to 15 MPa. It is about −​0.017% when the gas pressure increases to 2.9 MPa. For the dry sample No.2, the axial strain variation is about 0.009%/MPa during gas pressure increasing period and 0.024%/MPa during hydrostatic stress loading period. The different contribution of hydrostatic stress and gas pressure on the axial strain shows that Biot’s coefficient of LM shale is Scientific Reports | 7:44696 | DOI: 10.1038/srep44696 6 www.nature.com/scientificreports/ less than 1.These displacement measurements confirm that different gas pressures will induce a non-negligible strain. The augment of effective stress, which is controlled by confining stress and gas pressure, will compact the pore volume and result in a decrease in permeability. Permeability decreases from 44×10−21 m2 to 39.5×10−21 m2 when the hydrostatic stress increases from 12 MPa to 14 MPa. Under the same hydrostatic stress, increasing gas pressure will expand the pores and result in an increase in permeability, which is the opposite effect compared with the influence of gas slippage. The relationship between permeability and gas pressure is nonlinear (Fig. Discussions 5) and the Klinkenberg empirical formula cannot be directly used to characterize the intrinsic permeability of the Longmaxi shale used in this work. We posit this would hold for other material sensible to the pore pressure change as well. Therefore, the influence of the effective stress on permeability cannot be ignored, and it is sug- gested to be considered to expand the Klinkenberg’s model.f p g Increasing gas pressure reduces effective stress, and also shortens the mean free path of gas molecules and thus mits the effect of the gas slippage phenomenon. The mean free path of the gas is defined as πδ λ = K T p 2 (4) B 2 πδ λ = K T p 2 B 2 (4) where KB is the Boltzmann constant (KB =​ 1.3805 × 10−23J/K), δ​ is the collision diameter of the gas molecule (for nitrogen, δ = . × −m 3 5 10 10 ), T is temperature (K), and P is the pressure (Pa). When gas pressure is 1 MPa, λ . is 7 69 nm and it becomes 1.92 nm when gas pressure increases to 4 MPa. The mean free path of the gas is com- parable to the average pore size of Longmaxi shale. As presented in the sample description section, the average pore size of LM shale is about 3.0 nm. The ratio of mean free path of the gas (λ​) to the average pore diameter of material (d) is named as the Knudsen number λ = K d / n . Different Knudsen number means different gas flow regimes in material3: continuum flow for Kn <​ 0.001, slip flow (0.001 <​ Kn <​ 0.1); transition flow (0.1 <​ Kn <​ 10) and free-molecule flow (Kn >​ 10). As shown in the MIP curve (Fig. 2), the pore size distribution of Longmaxi shale is wide thus a variety of flow types might occur in Longmaxi shale, similar to those observed by Dadmohammadi et al.22,23. As pores become smaller, gas slippage phenomenon will be more significant and Darcy’s law will be invalid. This phenomenon is often evaluated by Klinkenberg’s equation (Eq. 1). This equation establishes a linear relationship between the apparent permeability and the inverse of average pressure (1/pm). However, our experimental results (Fig. 5) cannot be explained by Klinkenberg’s linear equation. Discussions With increasing gas pressure, we also observe a pressure threshold at which the dominant effect on the net permeability changes from gas slippage to effective stress.i g pp gf Based on Klinkenberg’s work, Ashrafi et al. proposed a new expanded equation4, =    + −    K K b P a P 1 (5) a int m m 2 (5) This model describes a quadratic dependency between apparent gas permeability and mean pressure. The constants, a and b are dependent on the fluid properties and pore geometry, b is similar to the Klinkenberg con- stant and a is the slip effect with a non-constant velocity distribution and could be a representation of a dynamic or secondary slippage factor. This new equation can describe the nonlinear relationship between the apparent permeability and 1/p. However, this equation cannot completely explain the apparent permeability evolution. The obtained data in this work were used to fitting Klinkenberg and Ashrafi’s models and the results were shown in Fig. 5 which shows permeability change is different from the theoretical predictions from Klinkenberg and Ashrafi’s model. Specifically, when gas pressure increases to a certain threshold, net permeability increases again, and this is dominated by the effective stress principle. Moreover, both Klinkenberg’s equation and Ashrafi’s model consider that the pore size is constant while gas pressure increases. This is certainly not the case for geomaterial subjected to mechanical loading. Therefore, one needs to consider the coupled effect of effective stress and gas slippage in order to obtain an accurate estimation of permeability.htf pp g p y The exponential relationship30 is often used to express the dependence of the permeability on the effective stress and it is adopted as follows γ σ = − ∆′ K K exp( ( )) (6) 0 (6) where K is the permeability of the material subjected to the effective stress change (m2), K0 is the initial permea- bility of the material (m2), γ is a material property (Pa−1), σ ∆′ = ∆σ − ′b p ( ) is the effective stress change. This model can represent the increase of permeability due to the reduction in effective stress. Discussions To expand the Klinkenberg’s model to incorporate both gas slippage and effective stress effects, we propose a new conceptual model expressed as, where K is the permeability of the material subjected to the effective stress change (m2), K0 is the initial permea- bility of the material (m2), γ is a material property (Pa−1), σ ∆′ = ∆σ − ′b p ( ) is the effective stress change. This model can represent the increase of permeability due to the reduction in effective stress. To expand the Klinkenberg’s model to incorporate both gas slippage and effective stress effects, we propose a new conceptual model expressed as, γ σ =    +   ⋅ − ∆′ K K b P exp 1 ( ( )) (7) a int m (7) This model is capable of replicating the coupled and competing effect of both gas slippage effect and effective stress effect. It allows the dominant effect on the net permeability to switch from gas slippage to effective stress when increasing gas pressure. More tests will be performed in our future research activities to identify these parameters and better constraint this conceptual model.h This model is capable of replicating the coupled and competing effect of both gas slippage effect and effective stress effect. It allows the dominant effect on the net permeability to switch from gas slippage to effective stress when increasing gas pressure. More tests will be performed in our future research activities to identify these parameters and better constraint this conceptual model.h p p The measured gas permeability of the wet sample No.1 is lower than that of the dry sample No.2. Given that the porosity of LM shale is 2.5% and the water content is 1.46%, the degree of water saturation of sample No.1 is Scientific Reports | 7:44696 | DOI: 10.1038/srep44696 7 www.nature.com/scientificreports/ estimated to be 83%. The dependence of gas permeability on water content has been observed in the past on Cox shale25. The experimental results of Yang et al.25 show that there exists a quasi-linear relationship between log(Ka) and the saturation. The obtained results in this work confirm that permeability of shale strongly depends on water content and it should be considered during gas production design, because gas and water often co-exist in shale formations. References bl F., Walsh, J. B. & Frangos, W. T. Permeability of grani 14. David, C., Wong, T. F., Zhu, W. & Zhang, J. Laboratory measurement of compaction induced permeability change in porous rocks: implication for the generation and maintenance of pore pressure excess in the crust Pure Apply Geophys 143 425 456 (1994) 14. David, C., Wong, T. F., Zhu, W. & Zhang, J. Laboratory measurement of compaction induced permeability change in porous r implication for the generation and maintenance of pore pressure excess in the crust. Pure Apply Geophys. 143, 425–456 (1994) 5. Kwon, O., Kronenberg, A. & Gangi, A. Permeability of Wilcox shale and its effective pressure law, J. Geophys. Res. 106(B9) 19339–19353 (2001). 16. Shi, T. & Wang, C. Y. Pore pressure generation in sedimentary basins: overloading versus aquathermal. J. Geophys. Res. 91 2153–2162 (1986). ( ) 17. Wang, S., Elsworth, D. & Liu, J. Permeability evolution in fractured coal: the roles of fracture geometry and water-content. Int. J. Coal Geol. 87(1), 13–25 (2011). 8. Wang, S., Elsworth, D. & Liu, J. A mechanistic model for permeability evolution in fractured sorbing media. J. Geophys. Res 117(B06) (2012).ff 9. Katsuki, D. & Gutierrez, M. Poroelastic effects on the stress-dependent permeability of a stiff shale. Poromechanics V.1705–1714. doi 10.1061/9780784412992.202 (2013). 0. Homand, F., Shao, J., Giraud, A., Auvray, C. & Hoxha, D. Pétrofabrique et propriétés mécaniques des argilites. Comptes Rendu Géosciences. 338(12–13), 882–891 (2006).f 21. Rathnaweera, T. D., Ranjith, R. G. & Perera, S. A. Experimental investigation of geochemical and mineralogical effects o sequestration on flow characteristics of reservoir rock in deep saline aquifers. Sci. Rep. doi: 10.1038/srep19362 (2016). ql p q p p ( ) 22. Dadmohammadi, Y., Misra, S., Sondergeld, C. H. & Rai, C. S. Improved Petrophysical Interpretation of Laboratory Pressure-Step-Decay Measurements on Ultra-Tight Rock Samples. In Unconventional Resources Technology Conference. San Antonio, Texas. (2016a).f ql p q p p 22. Dadmohammadi, Y., Misra, S., Sondergeld, C. H. & Rai, C. S. Improved Petrophysical Interpretation of Laboratory Pressure-Step-De M t Ult Ti ht R k S l I U ti l R T h l C f S A t i T (2016 ) ql p q p p 22. Dadmohammadi, Y., Misra, S., Sondergeld, C. H. & Rai, C. S. Improved Petrophysical Interpretation of Laboratory Pressure-Step-Decay g p gy f 23. Dadmohammadi, Y., Misra, S., Sondergeld, C. H. & Rai, C. S. Discussions It is necessary to further characterize the gas permeability of unsaturated shale in the future, although it would be challenging in the laboratory to control the saturation of the sample to as the porosity of Longmaxi shale (2.50%) is much less than that of Cox shale (18%). ( ) ( ) In this study, nitrogen is used to measure gas permeability and it is a slightly sorptive gas which could induce swelling of shale. Nuttall et al. studied the adsorption of Devonian black shales and found CO2 have an adsorption capacity approximately 5 times greater than that of CH4 31. Battistuta et al.32 and Wang et al.17 studied the sorp- tive effect of different gas (CO2, CH4 and N2) on dry coal and found that CO2 sorption on coal induces a bigger swelling effect on the coal matrix than CH4 and N2. Wu and Zhang33 found that the swelling of matrix decreases gas permeability. In this study, the adsorption effect on permeability was checked by repeated gas permeability tests under the same condition at different stages as shown in Fig. 3a,b and Tables 1, 2 and the results show that permeability does not change even though the gas tests have lasted two weeks. The reason could be due to the fact that N2 has a relatively weak adsorption capacity. Thus, the swelling of shale due to gas adsorption can be ignored to characterize gas permeability in this study. References bl Simultaneous Estimation of Intrinsic Permeability, Effective Porosity, Pore Volume Compressibility, and Klinkenberg-Slip Factor of Ultra-Tight Rock Samples Based on Laboratory Pressure-Step-Decay Method. SPE-180266-MS, SPE Low Perm Symposium, Denver, Colorado, USA, Society of Petroleum Engineers (2016, May, 5–6). g gy f 23. Dadmohammadi, Y., Misra, S., Sondergeld, C. H. & Rai, C. S. Simultaneous Estimation of Intrinsic Permeability, Effective Por , , , , g , R , y,f y, Pore Volume Compressibility, and Klinkenberg-Slip Factor of Ultra-Tight Rock Samples Based on Laboratory Pressure-Step-Decay Method SPE-180266-MS, SPE Low Perm Symposium, Denver, Colorado, USA, Society of Petroleum Engineers (2016, May, 5–6) f Pore Volume Compressibility, and Klinkenberg-Slip Factor of Ultra-Tight Rock Samples Based on Laboratory Pressure-Step-Decay M h d SPE 180266 MS SPE L P S i D C l d USA S i f P l E i (2016 M 5 6) , y p y f g ( , y, ) 24. Cao, C. H., Lv, Z. G., Li, L. W. & Du, L. Geochemical characteristics and implications of shale gas from the Longmaxi Formation, Sichuan Basin, China. J. Nat Gas Geosci. doi: 10.1016/j.jnggs.2016.05.001 (2016). J j j gg 25. Yang, D. S., Billotte, J. & Su, K. Characterization of the hydrome permeability under deviatoric stress. Eng. Geol. 114, 116–122 (2010). j j gg 5. Yang, D. S., Billotte, J. & Su, K. Characterization of the hydromechanical behavior of argillaceous rocks with effective ga j j gg 25. Yang, D. S., Billotte, J. & Su, K. Characterization of the hydromechanical behavior of argillaceous ro permeability under deviatoric stress. Eng. Geol. 114, 116–122 (2010). D. S., Billotte, J. & Su, K. Characterization of the hydromechanical behavior of argillaceous rocks with effective gas ability under deviatoric stress. Eng. Geol. 114, 116–122 (2010). 5. Yang, D. S., Billotte, J. & Su, K. Characterization of the hydromechanical behavior of argillaceous rocks with effective ga permeability under deviatoric stress. Eng. Geol. 114, 116–122 (2010). g J y permeability under deviatoric stress. Eng. Geol. 114, 116–122 (2010). p y g ( ) 26. Biot, M. A. General theory of 3-dimensional consolidation. J. Appl. Phys. 12, 155–164 (1941). y g . A. General theory of 3-dimensional consolidation. J. Appl. Phys. 1 26. Biot, M. A. General theory of 3-dimensional consolidation. J. A y pp y 27. Coussy, O. Poromechanics. John Wiley and Sons, Chichester, England (2004). 28. Bemer, E., Longuemare, P. & Vincké, O. References bl 1. Gensterblum, Y. et al. Gas storage capacity and transport in shale gas reservoirs – A review. Part A: Transport processes. J. Uncon. Oil Gas Res. 12, 87–122 (2015).h 2. Li, Y. B., Li, Y., Wang, B. Q., Chen, Z. E. & Nie, D. The status quo review and suggested policies for shale gas development in China Renew. Sust. Energ. Rev. 59, 420–428 (2016). 3. Arabjamaloei, R. & Ruth, D. W. Lattice Boltzmann based simulation of gas flow regimes in low permeability porous media: Klinkenberg’s region and beyond. J. Nat. Gas Sci. Eng. 31, 405–416 (2016).i g g y g 4. Ashrafi-Moghadam, A. & Chalaturnyk, R. Expansion of the Klinkenberg’s slippage equation to low permeability porous media. Int. J. Coal Geol. 123, 2–9 (2014).l 5. Darabi, H., Ettehad, A., Javadpour, F. & Sepehrnoori, K. Gas flow in ultra-tight shale strata. J. Fluid Mech. 710, 641–658 (2012).l 5. Darabi, H., Ettehad, A., Javadpour, F. & Sepehrnoori, K. Gas flow in ultra tight shale strata. J. Fluid Mech. 710, 641 658 (2012). 6. Fathi, E., Tinni, A. & Yucel-Akkutlu, I. Correction to Klinkenberg slip theory for gas flow in nano-capillaries. Int. J. Coal Geol. 103 51–59 (2012).h 7. Klinkenberg, L. J. The permeability of porous media to liquids and gases. API Drilling and Production Practice. 200–213 (1941 h 8. Tanikawa, W. & Shimamoto, T. Comparison of Klinkenberg-corrected gas permeability and water permeability in sedimentary rocks. Int. J. Rock Mech. Min. Sci. Geomech. Abstr. 46, 229–238 (2009).l J ( ) 9. Harley, J. C., Huang, Y., Bau, H. H. & Zemel, J. N. Gas flow in micro-channels. J. Fluid Mech. 284, 257–274 (1995). y gl 10. Shan, X., Yuan, X. F. & Chen, H. Kinetic theory representation of hydrodynamics: a way beyond the Naiver Stokes equation. J. Fluid Mech. 550, 413–441 (2006).f 1. Ren, X. X., Li, A. F., Wang, Y. Z. & Chen, M. Q. Gas permeability experimental study of low permeability core considering effect o gas slippage, Nat. Gas Geosci. 26(4), 733–736 (2015). g pp g 12. Dong, J. J. et al. Stress-dependence of the permeability and porosity of sandstone and shale from TCDP Hole-A. Int. J. Rock. Mech. Min.Sci. 47(7), 1141–1157 (2010). 3. Brace, W. F., Walsh, J. B. & Frangos, W. T. Permeability of granite under high pressure, J. Geophys. Res. 73, 2225–2236 (1968). d h h b f d d b l h 13. Brace, W. Acknowledgementsh g This work is supported by the Chinese Fundamental Research (973) Program (2015CB057906), the National Natural Science Foundation of China (Grant Nos 51379200, 41572290, 51225902, 41672281), the Chinese government plan on the Recruitment of Global Young Talents and Youth Innovation Promotion Association CAS. These supports are gratefully acknowledged. Author Contributions D.S.Y. and S.G.W. designed the research and analyzed the data, all authors performed the research, D.S.Y. and S.G.W. wrote the paper, W.W. and X.Q.W. prepared the samples and carried out the tests, D.S.Y. and W.Z.C. analyzed the results. All authors discussed and approved the manuscript. The authors thank the reviewers for their constructive comments and suggestions to improve the paper. www.nature.com/scientificreports/ carbon dioxide on dry Selar Cornish coal. Int. J. Coal Geol. 34, 39 48 (2010). 3. Wu, T. H. & Zhang, D. X. Impact of Adsorption on Gas Transport in Nanopores. Sci. Rep. doi: 10.1038/srep23629 (2016). References bl Poroelastic parameters of Meuse/Haute Marne argillites: effect of loading and satur states. Appl. Clay Sci. 26, 359–366 (2004). 28. Bemer, E., Longuemare, P. & Vincké, O. Po states. Appl. Clay Sci. 26, 359–366 (2004). pp y 9. Cariou, S., Duan, Z., Davy, C., Skoczylas, F. & Dormieux, L. Poromechanics of partially saturated Cox argillite. Appl. Clay Sci. 56 36–47 (2012). 30. Seidle, J. P., Jeansonne, M. W. & Erickson, D. J. Application of Matchstick Geometry to Stress Dependent Permeability in Coals. SPE- 24361, SPE Rocky Mountain Regional Meeting, Casper, Wyoming, USA, Society of Petroleum Engineers (1992, May 18–21). 30. Seidle, J. P., Jeansonne, M. W. & Erickson, D. J. Application of Matchstick Geometry to Stress Dependent Permeability in Coals. SPE- 24361, SPE Rocky Mountain Regional Meeting, Casper, Wyoming, USA, Society of Petroleum Engineers (1992, May 18–21). 31. Nuttall, B. C., Eble, C. F., Drahovzal, J. A. & Bustin, R. M. Analysis of Devonian black shales in Kentucky for potential carbon dioxide sequestration and enhanced natural gas production. Report Kentucky Geological Survey/ University of Kentucky (DE-FC26- 02NT41442). http://www.osti.gov/scitech/servlets/purl/842849 (2005). 31. Nuttall, B. C., Eble, C. F., Drahovzal, J. A. & Bustin, R. M. Analysis of Devonian black shales in Kentucky for potential carbon dioxide sequestration and enhanced natural gas production. Report Kentucky Geological Survey/ University of Kentucky (DE-FC26- 02NT41442). http://www.osti.gov/scitech/servlets/purl/842849 (2005). Scientific Reports | 7:44696 | DOI: 10.1038/srep44696 8 www.nature.com/scientificreports/ Scientific Reports | 7:44696 | DOI: 10.1038/srep44696 Additional Informationh Competing Interests: The authors declare no competing financial interests. How to cite this article: Yang, D. et al. Experimental investigation on the coupled effect of effective stress an gas slippage on the permeability of shale. Sci. Rep. 7, 44696; doi: 10.1038/srep44696 (2017). Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ © The Author(s) 2017 Scientific Reports | 7:44696 | DOI: 10.1038/srep44696 9
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Novel recommendation for enhancing optical properties of CP-WLEDs by Ba2Si5N8Eu2+ phosphor
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International Journal of Electrical and Computer Engineering (IJECE) Vol. 11, No. 2, April 2021, pp. 1063~1067 ISSN: 2088-8708, DOI: 10.11591/ijece.v11i2.pp1063-1067  1063 Novel recommendation for enhancing optical properties of CP-WLEDs by Ba2Si5N8Eu2+ phosphor Van-Duc Phan1, Minh Tran2, Q. S. Vu3 1Faculty of Automobile Technology, Van Lang University, Ho Chi Minh City, Vietnam 2Optoelectronics Research Group, Faculty of Electrical and Electronics Engineering, Ton Duc Thang University, Ho Chi Minh City, Vietnam 3School of Engineering-Technology and Arts, Hong Bang International University, Ho Chi Minh City, Vietnam Article Info ABSTRACT Article history: In this paper, the Ba2Si5N8Eu2+ phosphor is proposed as the novel recommendation for enhancing the optical properties in terms of D-CCT, CRI, CQS, and LO of the CP-WLEDs. Firstly, we conducted the physical model of the CP-WLEDs in the LightTools software with the main parameters like the real LEDs. Furthermore, the scattering process in LEDs compound of the CP-WLEDs is simulated and investigated by the Matlab software. Then the influence of the Ba2Si5N8Eu2+ concentration on the D-CCT, CRI, CQS, and LO of the CP-WLEDs is investigated. Finally, the research results showed that the Ba2Si5N8Eu2+ concentration has a considerable effect on the D-CCT, CRI, CQS, and LO of the CP-WLEDs. From the results, we can state that the Ba2Si5N8Eu2+ phosphor can be considered as the novel recommendation for enhancing the optical properties of the CP-WLEDs. Received Jul 2, 2019 Revised Aug 4, 2020 Accepted Sep 8, 2020 Keywords: CP-WLEDs CQS CRI D-CCT LO Optical properties Red phosphor This is an open access article under the CC BY-SA license. Corresponding Author: Minh Tran Optoelectronics Research Group Faculty of Electrical and Electronics Engineering Ton Duc Thang University Ho Chi Minh City, Vietnam Email: tranhoangquangminh@tdtu.edu.vn 1. INTRODUCTION In the lighting revolution trend for improving civilian everyday life in over the world, the InGaNbased white-light-emitting diodes (LEDs) can be considered as the primary solution in comparison with the conventional lighting method based on the excellent advantages such as environment-friendly, energy efficiency, compactness, long lifetime, and designable features [1-3]. Nowadays, phosphor-converted LEDs (pcLED) combines a blue LEDs chip, and the yellow emitting phosphor is the novel solution for lighting in civil and industrial purposes [4, 5]. In the last few years, authors in many research focused on enhancing the lighting properties of the white LEDs. Authors in [6, 7] proposed and investigated the effect of the phosphor layer thickness and concentration on the optical properties of white LEDs and concluded that the lower phosphor concentration and higher phosphor thickness led to the higher luminous efficacy. Furthermore, the authors in [8] stated that the spatial color distribution (SPD) of white LEDs is significantly affected by the phosphor layer parameter such as thickness, concentration, and size. As studied in [9-12], the blue light and yellow light have a similar radiation pattern that can be lead to improving the SPD by varying the phosphor layer location of the white LEDs. Besides, the green and red phosphor by adding to the phosphor layer can be considered as a novel solution for enhancing the optical properties of the white LEDs as in [13-15]. Journal homepage: http://ijece.iaescore.com 1064  ISSN: 2088-8708 In this paper, the Ba2Si5N8Eu2+ is proposed as the novel recommendation for enhancing the optical properties in terms of D-CCT, CRI, CQS, and LO of the CP-WLEDs. Firstly, we conducted the physical model of the CP-WLEDs in the LightTools software with the main parameters like the real LEDs. Furthermore, the scattering process in the CP-WLEDs is simulated and investigated by the Matlab software. Then the influence of the Ba2Si5N8Eu2+ phosphor concentration on the D-CCT, CRI, CQS, and LO of the CP-WLEDs is investigated. Finally, the research results showed that the Ba2Si5N8Eu2+ concentration has a considerable effect on the D-CCT, CRI, CQS, and LO of the CP-WLEDs. From the results, we can state that the Ba2Si5N8Eu2+ phosphor can be considered as the novel recommendation for enhancing the optical properties of the CP-WLEDs. The rest of this paper can be formulated as follows. The physical model of CPW-LEDs, optical properties of the red phosphor is proposed in the second section. The third section presented the numerical results and some discussions. The last section concludes the research. 2. PHYSICAL CPW-LEDS MODEL AND OPTICAL PROPERTIES OF THE RED PHOSPHOR We use the LightTools software to simulate the real-world model of CPW-LEDs Figure 1(a) with the main parameters, as shown in Figure 1(b) [13-15]. Among these (oxy) nitride phosphors, Ba2Si5N8Eu2+ has high quantum efficiency and very low thermal quenching to improve the color rendering index Figure 2 [16-21]. (a) (b) Figure 1. (a) The white LEDs, (b) physical model Figure 2. Optical properties of the Ba2Si5N8Eu2+ phosphor Based on Mie theory [22-30], the scattering coefficients can be calculated as sca ( )   N (r )Csca ( , r )dr Int J Elec & Comp Eng, Vol. 11, No. 2, April 2021 : 1063 - 1067 (1) Int J Elec & Comp Eng ISSN: 2088-8708 abs ( )   N (r )Cabs ( , r )dr  1065 (2) 1 g ( )  2   p( ,  , r ) f (r ) cos  d cos  dr (3)  sca  sca (1  g ) (4) f (r )  f dif (r )  f phos (r ) (5) N (r )  N dif (r )  N phos (r )  K N .[ f dif ( r )  f phos ( r )] (6) c  K N  M (r )dr (7) 1 4 M (r )   r 3 [  dif f dif (r )   phos f phos (r )] 3 (8) 3. NEMERICAL RESULTS AND DISCUSSIONS Based on the Mie Theory, the scattering coefficients (SC) versus Ba2Si5N8Eu2+. concentration are shown in Figure 3 with the red, blue, and yellow lights. From Figure 3, we can see that the (SC) of the yellow light is better than others, and the SC of the red and blue lights are the same. The efficiency of the yellow light in the phosphor layer can cause the enhancing the optical properties of the CPW-LEDs. In another way, the scattering amplitude (SA) is investigated in Figure 4 with red, blue, and yellow lights. As shown in Figure 4, the SA of the blue light is better than others to verify the enhancing the optical properties when we added the red phosphor in the phosphor layer. Figure 3. SC Figure 4. The scattering amplitude The function of D-CCT of the phosphor concentration is illustrated in Figure 5. In this figure, we varied the phosphor concentration from 0 to 16% and investigated the D-CCT of the CPW-LEDs. As shown in Figure 5, it can be observed that the D-CCT increases massively from 1500 K to 8500 K while we vary the concentration of Ba2Si5N8Eu2+. Furthermore, the effect of the Ba2Si5N8Eu2+ phosphor concentration on the CRI and CQS of the CPW-LEDs is proposed in Figures 6 and 7, respectively. The CRI and CQS increase when the concentration of the Ba2Si5N8Eu2+ phosphor increases from 0% to 10% and then have a huge decrease with rising of the phosphor concentration from 10% to 16%. The maximum values of CRI and CQS are 85 and 79, which can be obtained with 10% concentration of the Ba2Si5N8Eu2+ phosphor. Finally, the LO versus the phosphor concentration is drawn in Figure 8. The LO has a massive fall with the rising of the Ba2Si5N8Eu2+ phosphor. From the results, we can state that the concentration of Ba2Si5N8Eu2+ has a considerable effect on the optical properties (D-CCT, CRI, CQS, and LO). Novel recommendation for enhancing optical properties of CP-WLEDs … (Van-Duc Phan) 1066  ISSN: 2088-8708 Figure 5. D-CCT Figure 6. CQS Figure 7. CRI Figure 8. The lumen output LO 4. CONCLUSION In this paper, Ba2Si5N8Eu2+ is proposed as the novel recommendation for enhancing the optical properties in terms of D-CCT, CRI, CQS, and LO of the CP-WLEDs. Firstly, we conducted the physical model of the CP-WLEDs in the LightTools software with the main parameters like the real LEDs. Furthermore, the scattering process in the CP-WLEDs is simulated and investigated by the Matlab software. Then the influence of Ba2Si5N8Eu2+ concentration on the D-CCT, CRI, CQS, and LO of the CP-WLEDs is investigated. Finally, the research results showed that Ba2Si5N8Eu2+ concentration has a considerable effect on the D-CCT, CRI, CQS, and LO of the CP-WLEDs. From the results, we can state that Ba2Si5N8Eu2+ can be considered as the novel recommendation for enhancing the optical properties of the CP-WLEDs. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] Sheng Liu, Xiaobing Luo, “Design of LED Packaging Applications,” LED Packaging for Lighting Applications, pp. 215-315, 2011. Gibney, Elizabeth, “Nobel for Blue LED That Revolutionized Lighting,” Nature, vol. 514, no. 7521, pp. 152-153, 2014. 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Validating the presence of Spanish Flag, Gonioplectrus hispanus (Cuvier, 1828) (Perciformes, Serranidae), from the south-western Gulf of Mexico
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Keywords Chernas, Epinephelinidae, grouper, range extension, species distribution Academic editor: Hudson Tercio Pinheiro  |  Received 2 April 2021  |  Accepted 28 August 2021  |  Published 21 September 2021 Citation: Del Moral-Flores LF, López-Segovia E, Escartín-Alpizar VR, Jiménez-Badillo ML (2021) Validating the presence of Spanish Flag, Gonioplectrus hispanus (Cuvier, 1828) (Perciformes, Serranidae), from the south-western Gulf of Mexico. Check List 17 (5): 1299–1303. https:// doi.org/10.15560/17.5.1299 Check List 17 (5): 1299–1303 https://doi.org/10.15560/17.5.1299 NOTES ON GEOGRAPHIC DISTRIBUTION Check List 17 (5): 1299–1303 https://doi.org/10.15560/17.5.1299 NOTES ON GEOGRAPHIC DISTRIBUTION Abstract We report the presence of Gonioplectrus hispanus (Cuvier, 1828) in Mexican waters of the Gulf of Mexico. Four specimens (198–224 mm standard length) were caught at two locations south of Veracruz. These records increases the number of species in the family Serranidae for Mexico to 113. We include morphometry and meristic data of our specimens. Check List 17 (5): 1299–1303 https://doi.org/10.15560/17.5.1299 Check List 17 (5): 1299–1303 https://doi.org/10.15560/17.5.1299 NOTES ON GEOGRAPHIC DISTRIBUTION Validating the presence of Spanish Flag, Gonioplectrus hispanus (Cuvier, 1828) (Perciformes, Serranidae), from the south-western Gulf of Mexico Luis Fernando Del Moral-Flores1, Eduardo López-Segovia1,2, Viridiana R. Escartín-Alpizar1, 2, María de Lourdes Jiménez-Badillo3 1 Laboratorio de Zoología, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Mexico City, Mexico • LFDMF: delmoralfer@gmail.com https://orcid.org/0000-0002-7804-2716 • VREA: viridianaescartin11d@gmail.com, https://orcid.org/ 0000-00018524-3850 2 Posgrado en Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México, Mexico City, Mexico • ELS: eduardosegovia@ comunidad.unam.mx https://orcid.org/0000-0001-8065-1521 3 Instituto de Ciencias Marinas y Pesquerías, Universidad Veracruzana, Veracruz, Mexico • MLJB: ljimenez@uv.mx https://orcid.org/0000- 0002-0324-433X * Corresponding author © The authors.  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. 2 Posgrado en Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México, Mexico City, Mexico • ELS: eduardosegovia@ comunidad.unam.mx https://orcid.org/0000-0001-8065-1521 1 Laboratorio de Zoología, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México, Mexico City, Mexico • LFDMF: delmoralfer@gmail.com https://orcid.org/0000-0002-7804-2716 • VREA: viridianaescartin11d@gmail.com, https://orcid.org/ 0000-00018524-3850 Introduction in coastal, tropical and subtropical ecosystems, includ­ ing mesophotic and rariphotic environments (Bullock and Smith 1991; Baldwin et al. 2018). One of the genera within the family is Gonioplectrus Gill, 1862, which is monotypic, with Gonioplectrus hispanus (Cuvier, 1828), Spanish Flag, restricted to Western Atlantic (Parenti and Randall 2020). The family Serranidae Swainson, 1839 is a monophy­ letic group and includes six subfamilies, 72 genera, and between 579 and 583 species (Fricke et al. 2020; Parenti and Randall 2020). This family has significant economic importance, as it supports commercial fisheries and sport fishing activities (Bullock and Smith 1991). In Mexico, it supports one of the main fisheries in the Western Atlan­ tic (Colás-Marrufo et al. 1998), and it is also important Gonioplectrus hispanus has a wide distribution from 1300 Check List 17 (5) North Carolina (USA) to south-eastern Brazil. How­ ever, the records of this species are scarce because it inhabits rocky areas and deep coral from 60 to 365 m (McEachran and Fechhelm 2005). The north-western distribution of this species includes the Gulf of Mex­ ico, Texas, and other parts of the United States (Briggs, 1958; Briggs et al. 1964; Moore 1975; Smith 1976; Bull­ ock and Smith 1991; Hoese and Moore 1998). There are no confirmed records based on deposited specimens in reference collections from Gulf of Mexico and the Mexi­ can Caribbean, although it is mentioned in the faunistic lists of Mexican Atlantic coasts (Lara-Domínguez et al. 1993; Baqueiro and Mendez 1994), including the coast at Los Tuxtlas (Schaldach et al. 1997) and as larvae on Campeche Bank (Flores-Coto et al. 2009). The only evi­ dence of its presence in Mexican Atlantic coasts is a pho­ tograph of a single specimen in the Instituto Nacional de Pesca (1976). The geographic coordinates of the collec­ tion site of this specimen are unknown. With the new records presented here, we confirm the presence of G. hispanus in the south-western Gulf of Mexico. determination was made with specialized keys by Bull­ ock and Smith (1991), McEachran and Fechhelman (2005), and Heemstra et al. (2002). Electronic calipers (0.01 mm) were used for morphological measurements, and the meristic data were counted according to Hubbs and Lagler (1958) with modifications for the group (Betancur-R. et al. 2001; Oliveira and Barbosa 2010; Sampaio et al. 2017). Gonioplectrus hispanus (Cuvier, 1828) Figure 2; Table 1 Common names. Spanish Flag; mero payaso, payasín (Spanish). New records. MEXICO – Veracruz • Gulf of Mexico; ca. 11.8 km from Punta Puntilla, municipality of Ángel R. Cabada; 18°46′56.7″N, 095°09′40.2″W; 280 m depth; 12.XI.2020; A. Campos-Pérez and E. Campos-Vicente leg.; fishing line; 2 specimens, CIFI-1715 (192 and 208 mm SL) • same locality, 300 m depth; 09.I.2021; A. Cam­ pos-Pérez leg.; fishing line; 1 specimen, CIFI-1801 (224 mm SL) • Gulf of Mexico; ca. 9 km from Playa Her­ mosa, municipality of Ángel R. Cabada; 18°44′28.8″N, 095°04′52.0″W; 180 m depth; 24.VI.2021; A. Campos- Pérez and E. Campos-Vicente leg.; fishing line; 1 speci­ men, CIFI-1820 (208 mm SL). Results Family Serranidae Swainson 1839 Genus Gonioplectrus Gill, 1862 Family Serranidae Swainson 1839 Genus Gonioplectrus Gill, 1862 Methods Four specimens of Gonioplectrus hispanus were caught accidentally by the artisanal fishery, using a bottom- longline made with nylon monofilament and 60 to 150 hooks (size 6) at depths of 180–300 m (Fig. 1). The speci­ mens were deposited in the Ichthyological Collection of the Facultad de Estudios Superiores Iztacala (CIFI), Uni­ versidad Nacional Autónoma de México. The taxonomic Figure 1. Sampling station of Gonioplectrus hispanus (black cross), ca. 11.8 km from Punta Puntilla, municipality of Ángel R. Cabada, Vera­ cruz, south-western Gulf of Mexico (18°46′56.7″N, 095°09′40.2″W). Previous records of G. hispanus in the western Atlantic (red dots). Figure 1 Sampling station of Gonioplectrus hispanus (black cross) ca 11 8 km from Punta Puntilla municipality of Ángel R Cabada Vera Figure 1. Sampling station of Gonioplectrus hispanus (black cross), ca. 11.8 km from Punta Puntilla, municipality of Ángel R. Cabada, Vera­ cruz, south-western Gulf of Mexico (18°46′56.7″N, 095°09′40.2″W). Previous records of G. hispanus in the western Atlantic (red dots). Del Moral-Flores et al. |  Gonioplectrus hispanus from the south-western Gulf of Mexico 1301 Figure 2. Gonioplectrus hispanus (CIFI-1715, 208 mm SL) collected in the south-western Gulf of Mexico, showing typical coloration for the species. The species was determined prior to the preservation of the specimen. Scale bar = 25 mm. Figure 2. Gonioplectrus hispanus (CIFI-1715, 208 mm SL) collected in the south-western Gulf of Mexico, showing typical coloration for the species. The species was determined prior to the preservation of the specimen. Scale bar = 25 mm. Table 1. Morphometric and meristic data of specimens of Gonio­ plectrus hispanus from the south-western Gulf of Mexico. Identification. Gonioplectrus hispanus is recognised by its a serrated preopercle with a large spine on the ventral edge directed forward. The opercle has three spines, and the second spine is most elongated. The dorsal fin has 8 spines and 11–13 rays, and the anal fin has 3 spines and 7 rays. The lateral line has 47–49 scales. The bases of the dorsal, anal, and pectoral fins are covered with scales. Gill rakers number 21–23. In addition, in vivo, G. hispanus shows a striking pink coloration, with seven bright yellow stripes on the sides from the head to the base of the tail fin. A diagonal yellow stripe runs from the snout through the eye and upper opercle to the back of the body. There are yellow patches in the interorbital region and cheek. Methods The pelvic fins are violet, and the anal fin has a crimson spot from the second anal spine as far as the first anal ray. There is a conspicuous, white, ven­ tral blotch, which is much larger than the eye-diameter. Morphometrics (mm) CIFI 1715 CIFI 1715 CIFI 1801 CIFI 1820 Total length 251 230 278 271 Standard length 208 192 224 222 Head length 88.66 82.21 96.06 93.82 Upper jaw length 39.47 37.06 42.25 43.97 Orbit diameter 15.78 15.35 16.03 16.24 Postorbital length 48.54 45.47 56.19 52.47 Interorbital width 14.70 12.62 16.13 15.84 Snout length 22.42 21.01 25.81 26.95 Suborbital length 9.53 8.55 11.20 11.48 Body depth 81.91 74.97 96.90 87.30 Caudal peduncule length 33.07 28.96 31.29 28.65 Caudal peduncule depth 32.58 30.70 33.78 34.92 Caudal fin base length 40.32 36.95 38.26 36.42 Dorsal fin base length 94.08 87.82 97.99 98.45 Anal fin base length 34.79 31.08 37.55 36.39 Predorsal length 84.15 81.70 93.45 96.12 Preanal length 144 134 156 15 Prepectoral length 84.11 74.2 87.96 85.13 Pectoral fin length 52.60 51.01 64.61 59.72 Prepelvic length 89.18 79.97 96.93 89.92 Pelvic fin length 42.61 41.66 46.10 46.51 Meristics Dorsal fin spines VIII VIII VIII VII Dorsal fin rays 11 13 13 13 Anal fin spines III III III III Anal fin rays 7 7 7 7 Pectoral fin rays 16 15 16 17 Caudal fin rays 16 17 17 17 Pored scales lateral line 49 48 48 50 Lateral line scales 85 81 79 83 Scales around the caudal peduncle 47 45 45 48 Upper gill rakers 6 7 6 5 Lower gill rakers 15 15 15 15 Authors’ Contributions Hoese HD, Moore RH (1998) Fishes of the Gulf of Mexico, Texas, Louisiana, and adjacent waters. Texas A&M University Press, College Station, USA, 416 pp. Conceptualization: LFDM. Data curation: VREA. For­ mal analysis: LFDM, ELS, MLJB, VREA. Visualiza­ tion: ELS, VREA. Writing – original draft: LFDM, ELS. Writing – review and editing: LFDM, MLJB, VREA. Hubbs CL, Lagler KF (1958) Fishes of the Great Lakes region. Univer­ sity of Michigan Press, Ann Arbor, USA, 213 pp. Instituto Nacional de Pesca (1976) Catálogo de peces marinos mexi­ S t í d i d t i i I tit t N i l d Instituto Nacional de Pesca (1976) Catálogo de peces marinos mexi­ canos. Secretaría de industria y comercio, Instituto Nacional de Pesca. Mexico City, Mexico, 462 pp.i Discussion https://doi.org/10.25268/bimc.invemar.2019.48.1.759 arque Nacional Natural Corales de Profundidad, usando buceo Colás-Marrufo T, Brulé T, Déniel C (1998) Análisis preliminar de las capturas de meros realizados a través de unidades de la flota mayor en el sureste del Golfo de México. Proceedings of the Gulf and Caribbean Fisheries Institute 50: 780–803. Its occurrence in Punta Puntilla is probably due to the dispersal of larvae, which influences the presence of adults on coral reefs. Kendall and Fahay (1979) captured larvae of G. hispanus in North Carolina, which represents the northernmost record. However, in Mexico, there is a gap in the knowledge of deep communities compared to other countries where this species occurs (Simon et al. 2016; Baldwin et al. 2018; Chasqui-Velasco and González- Corredor 2019) due to poor sampling efforts and the diffi­ culty sampling mesophotic and rariphotic environments. Our new data of G. hispanus are an important contri­ bution to understanding Mexico’s mesophotic and rari­ photic ichthyofauna, and confirm the presence of this species in the southwestern Gulf of Mexico. Flores–Coto C, Espinosa–Fuentes ML, Zavala García F, Sanvicente Añorve L (2009) Ictioplancton del sur del Golfo de México: un compendio. Hidrobiológica 19 (1): 49–76. Fricke R, Eschmeyer WN, Fong JD (2020) Species by family/sub­ family. Version 2020. http://researcharchive.calacademy.org/ research/ichthyology/catalog/SpeciesByFamily.asp. Accessed on: 2021-1-28. Garcia-Júnior J, Nóbrega MF, Lins-Oliveira JE (2015) Coastal fishes of Rio Grande do Norte, north-eastern Brazil, with new records. Check List 11 (3): 1659. https://doi.org/10.15560/11.3.1659 Check List 11 (3): 1659. https://doi.org/10.15560/11.3.1659 González-Gándara C (2020) Composition, distribution and relative abundance of fishes linked to fringing reefs of southern Veracruz, Gulf of Mexico. GSC Biological and Pharmaceutical Sciences 12 (2): 217–228. https://doi.org/10.30574/gscbps.2020.12.2.0268 (2): 217–228. https://doi.org/10.30574/gscbps.2020.12.2.0268 Guitart DJ (1977) Sinopsis de los peces marinos de Cuba. Tomo III. Academia de Ciencias de Cuba, Instituto de Oceanología, Ha­ vana, Cuba, 325–608. Heemstra PC, Anderson WD, Lobel PS (2002) Serranidae. Groupers (seabasses, creolefish, coney, hinds, hamlets, anthiines, and soap­ fishes). In: Carpenter KE (Ed.) The living marine resources of the Western Central Atlantic. Vol. 2: bony fishes, part 1 (Acipenseri­ dae to Grammatidae). FAO Species Identification Guide for Fish­ ery Purposes and American Society of Ichthyologist and Herpe­ tologists Special Publication, Rome, Italy, 601–1374. Acknowledgements We thank the local fishermen, A. Campos Pérez and E. Campos Vicente, who collected specimens for us, and the community of Salinas Roca Partida for its help dur­ ing with the fieldwork. We also acknowledge the finan­ cial support of SNI-CONACyT. This work was financed by the project PAPIIT-UNAM IA207820. Heemstra PC, Randall JE (1993) FAO species catalogue. Vol. 16. Groupers of the world (family Serranidae, subfamily Epinepheli­ nae). An annotated and illustrated catalogue of the grouper, rock­ cod, hind, coral grouper and lyretail species known to date. FAO Fisheries Synopsis 125 (16): 1–382. Discussion Gonioplectrus hispanus occurs in moderately deep waters down to depths of 460 m (Oliveira and Barbosa 2010; Baldwin et al. 2018) and co-exists with other ser­ ranids, usually at the periphery of tropical reefs (Bullock and Smith 1991). Specifically, it lives in coral reefs of the rariphotic zone (139–309 m; Baldwin et al. 2018). This species has no value as human food due to its small size, but it is often used as ornamental fish in the aquarium trade (Oliveira and Barbosa 2010). Its capture is difficult below 100 m, and barriers, such as thermoclines at these depths, function as biological boundaries (Heemstra and Randall 1993; Oliveira and Barbosa 2010). The species has been recorded sporadically along 1302 Check List 17 (5) the Caribbean coasts of Colombia and Venezuela, as well as in the Caribbean Islands (Cervigón 1971; Gui­ tart 1977; Thompson and Munro 1978; Betancur-R. et al. 2001; Baldwin et al. 2018). It has been recently recorded in Brazilian waters as a result of deep-sea fishing and mesophotic exploration (Oliveira and Barbosa 2010; Gar­ cia-Júnior et al. 2015; Sampaio et al. 2017). Bullock and Smith (1991) reported this species in the north-western Atlantic, and in Mexico, it has been detected in the south­ ern Gulf of Mexico, between Laguna de Términos and the Yucatán Peninsula, although the location was not specified (Lara-Domínguez et al. 1993). Despite recent ichthyofaunal surveys in reef areas in the south-eastern Gulf of Mexico and on the Yucatán continental shelf, G. hispanus has not been detected (Colás-Marrufo et al. 1998; Robertson et al. 2019; González-Gandara 2020). letin of the Florida State Museum, Biological Sciences Series 2 (8): 223–318. letin of the Florida State Museum, Biological Sciences Series 2 (8): 223–318. Briggs JC, Hoese HD, Hadley WF, Jones RS (1964) Twenty-two new fish records for the north–western Gulf of Mexico. Texas Journal of Science 16 (1): 113–116. Bullock LH, Smith GB (1991) Seabasses (Pisces: Serranidae). Mem­ oirs of the Hourglass Cruises 8 (2): 1–243. Cervigón F (1971) Los peces marinos de Venezuela. Complemento i Cervigón F (1971) Los peces marinos de Venezuela. Complemento II. Contribuciones Científicas Universidad de Oriente Porlamar 1: 5–36. Chasqui-Velasco LH, González-Corredor, JD (2019) Peces registrados en ambientes mesofóticos de Bajo Frijol, la porción más somera del Parque Nacional Natural Corales de Profundidad, usando buceo técnico CCR. Boletín de Investigaciones Marinas y Costeras 48 (1): 89–101. References Kendall AW Jr., Fahay MP (1979) Larva of the serranid fish Gonio­ plectrus hispanus with comments on its relationships. Bulletin of Marine Science 29 (1): 117–121. Baldwin CC, Tornabene L, Roberston DR (2018) Below the meso­ photic. 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Transcriptomic analysis of a-linolenic acid content and biosynthesis in Paeonia ostii fruits and seeds
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Results Gas chromatograph-mass spectrometry indicated that ALA content was highest in the kernel, moderate in the testa, and lowest in the pericarp. Therefore, we used RNA-sequencing to compare ALA synthesis among these three tissues. We identified 227,837 unigenes, with an average length of 755 bp. Of these, 1371 unigenes were associated with lipid metabolism. The fatty acid (FA) biosynthesis and metabolism pathways were significantly enriched during the early stages of oil accumulation in the kernel. ALA biosynthesis was significantly enriched in parallel with increasing ALA content in the testa, but these metabolic pathways were not significantly enriched during pericarp development. By comparing unigene transcription profiles with patterns of ALA accumulation, specific unigenes encoding crucial enzymes and transcription factors (TFs) involved in de novo FA biosynthesis and oil accumulation were identified. Specifically, the bell-shaped expression patterns of genes encoding SAD, FAD2, FAD3, PDCT, PDAT, OLE, CLE, and SLE in the kernel were similar to the patterns of ALA accumulation in this tissue. Genes encoding BCCP, BC, KAS I– III, and FATA were also upregulated during the early stages of oil accumulation in the kernel. In the testa, the upregulation of the genes encoding SAD, FAD2, and FAD3 was followed by a sharp increase in the concentrations of ALA. In contrast, these genes were minimally expressed (and ALA content was low) throughout pericarp development. Conclusions In this study, we used three tissues with high, moderate, and low ALA concentrations as an exemplar system in which to compare tissue-specific ALA accumulation mechanism in P. ostii. The genes and TFs identified herein might be useful targets for future studies of ALA accumulation in the tree peony. This study also provides a framework for future studies of FA biosynthesis in other oilseed plants. Background Paeonia ostii is a potentially important oilseed crop because its seed yield is high and the seeds are rich in α-linolenic acid (ALA). However, the molecular mechanisms underlying ALA biosynthesis during seed kernel, seed testa, and fruit pericarp development in this plant are unclear. We used transcriptome data to address this knowledge gap. Version of Record: A version of this preprint was published at BMC Genomics on April 23rd, 2021. See the published version at https://doi.org/10.1186/s12864-021-07594-2. Version of Record: A version of this preprint was published at BMC Genomics on April 23rd, 2021. See the published version at https://doi.org/10.1186/s12864-021-07594-2. Page 1/34 Background Edible oils for human consumption are mainly derived from plant seeds, which contain several types of fatty acids (FAs). Among the FAs, linoleic acid (LA; C18:2Δ9,12, a ω-6 FA) and α-linolenic acid (ALA; C18:3Δ9,12,15) are essential dietary nutrients for humans; these FAs cannot be synthesized independently by humans [1]. ω-6 FA and ALA not only exert a hypocholesterolemic effect against coronary heart Page 2/34 Page 2/34 disease and hypertension when used as human dietary supplements, but also are critical for fetal development and breastfeeding support [1, 2]. It has been suggested that a ratio of ω-6 to ω-3 FAs < 5 is optimal for human beings [3], and several sources have testaulated that ancient human diets had ω-6 to ω-3 FA ratios of ~ 1 [4]. Indeed, the high ratio of ω-6 to ω-3 FAs (~ 15:1) in the typical modern human diet is thought to be a major factor contributing to the high rates of cardiovascular disease in modern human societies [5]. One explanation for this imbalance is that the bulk of the oil seed crops consumed by modern humans, including soybean, peanut, maize, sunflower, and rape, have relatively low levels of ω-3 FAs, such as ALA [5]. However, tree peony seed oil, which is rich in ALA, has a ω-6 to ω-3 FA ratio of < 1.0 [6]. For this reason, tree peony seed oil has been recognized as a high-quality edible oil, and was identified as a new food resource by the Chinese Ministry of Health in 2011 [7]. The tree peony (family Paeoniaceae, genus Paeonia, section Moutan DC), which was historically known as "The King of Flowers", is a perennial shrub that is widely distributed in China [8]. There are nine wild species in section Moutan DC, which differ with respect to preferred habitat, but all rich in unsaturated FAs (>90%) and ALA (26.7–50%) [6, 9]. To date, transcriptomic, proteomic, and microRNA sequencing have been used to investigate the mechanisms underlying ALA synthesis in tree peony seeds [10–14]. Previous studies have focused on enzymes that are key to ALA biosynthesis in various tree peony species, including FAD3 in P. rockii, P. potaninii and P. lutea seed [15]; FAD2 and FAD8 in P. ostii [10]; and ACCase, FATA, LPCAT, FADs, and DGAT in the developing endosperm of P. ostii var. lishizhenii [11]. Wang et al. Background [14] identified 115 genes and 24 proteins associated with the ALA metabolism in P. ostii seeds. However, despite these studies of FA metabolism in the tree peony, additional functional genes require further exploration. In oil seed plants, stored lipids accumulate primarily as a result of FA synthesis in the plastids and triacylglycerol (TAG) assembly in the endoplasmic reticulum (ER). Acetyl-coenzyme A (CoA) is the unique building block used for FA production in plants [16]. In the plastid, de novo FA synthesis begins when acetyl-CoA is converted into malonyl-CoA by the acetyl-CoA carboxylase (ACC); malonyl-CoA is then either elongated to C16 and C18 by a series of enzymes or desaturated to C18:1-ACP by delta-9-stearoyl-ACP desaturase (SAD) [17, 18]. In most oilseed plants, more than 95% of the newly-synthesized FAs (primarily C18:1) are exported from plastids to the ER as CoA thioesters[19, 20]. The biosynthesis of polyunsaturated FAs (PUFAs) primarily relies on the further desaturation of C18:1 by omega-6 fatty acid desaturase (omega-6 FAD) and omega-3 FAD in the plastid or ER [16]. FAs, both those synthesized de novo or those have been modified through acyl editing, are assembled into glycerol-3-phosphate (G3P) to form TAG [21]. Generally, in plants and eukaryotic algae, TAG is mainly synthesized along the acyl-CoA- dependent Kennedy pathway and the acyl-CoA-independent pathway. Once TAGs have been synthesized, they are surrounded by a layer of phospholipids and amphipathic proteins, and then released to form oil bodies in the cytoplasm. The FA metabolic pathway is complex, and the details of this pathway differ substantially among plant species and tissue types. In castor beans and oil palms, transcriptome analyses showed that differences in FAs among seed tissues were associated with certain genes and transcription factors (TFs) [22, 23]. Page 3/34 Page 3/34 Interestingly, previous studies have showed that FA composition and relative abundance differ among wild P. ostii trees [6, 9], as well as among seed kernels and testas [6, 24]. This implies that FA synthesis and TAG assembly in developing tree peony seeds are differentially regulated among species. Zhang et al. [13, 15] compared FAD transcription (i.e., SAD, FAD2, and FAD3) among three species of tree peonies, and found that FAD transcription was greater in P. rockii seeds than in P. potaninii and P. lutea seeds. In addition, various transcriptomes of P. ostii seeds or endosperms are also available [10, 11, 14]. Background However, to date, no integrated analysis of the developing pericarp, seed coat, and endosperm in P. ostii has been performed. In this study, we chose P. ostii as an exemplar tree peony species primarily because, compared with other tree peonies, P. ostii has higher seed yields and ALA content. This species is also suitable for large-scale cultivation south of the Yangtze River, China. In this study, we aimed to use comparative transcriptomics to assess the expression levels of certain genes associated with ALA accumulation during the development of three P. ostii tissues: the seed kernel, the seed testa, and the fruit pericarp. The identification of candidate genes that may affect levels of ALA accumulation will provide molecular tools for future genetic modifications, aimed at increasing the diversity and yield of seed oils produced by the tree peony and other oilseed plants. In this study, we chose P. ostii as an exemplar tree peony species primarily because, compared with other tree peonies, P. ostii has higher seed yields and ALA content. This species is also suitable for large-scale cultivation south of the Yangtze River, China. In this study, we aimed to use comparative transcriptomics to assess the expression levels of certain genes associated with ALA accumulation during the development of three P. ostii tissues: the seed kernel, the seed testa, and the fruit pericarp. The identification of candidate genes that may affect levels of ALA accumulation will provide molecular tools for future genetic modifications, aimed at increasing the diversity and yield of seed oils produced by the tree peony and other oilseed plants. Morphological changes in developing P. ostii fruits and seeds At the study location (south of the Yangtze River), P. ostii fruit and seed development lasted about 120 days. P. ostii fruits and seeds grew rapidly between 7 and 49 days after fertilization (DAF), and at 49 DAF were almost as large as the mature forms (Fig. 1a–c). The fruit peel was green during the early stages of development, but began to turn yellow at about 70 DAF (Fig. 1a). Examination of the seeds in longitudinal section indicated that embryogenesis and seed-filling occurred from 0 to 70 DAF; cotyledon embryos were obvious at 70 DAF. Between 70 and 119 DAF, the seed coat became thinner and browner as compared to the period from 49 to 70 DAF (Fig. 1d). At 119 DAF, the seeds were mature (Fig. 1b, d). Dynamic changes in FA composition in the pericarp, kernel, and testa All five major FAs [ALA, LA, oleic acid (OA, C18:1Δ9), stearic acid (SA, C18:0) and palmitic acid (PA, C16:0)] were detected in the pericarp, testa, and kernel samples taken between 21 and 119 DAF (Fig. 2). The three most abundant UFAs during kernel development were OA, LA, and ALA. In the kernel, OA, LA, and ALA concentrations were 2.4±0.08 mg/g, 6.35±0.34 mg/g, and 2.5±0.24 mg/g, respectively, at 35 DAF, increasing to 58.79±2.21 mg/g, 92.56±4.98 mg/g, and 134.29±2.23 mg/g, respectively, at 77 DAF (Fig. 2a). After 77 DAF, OA, LA, and ALA concentrations decreased gradually, to 52±0.73 mg/g, 77.36±1.39 mg/g, and 131.26±1.68 mg/g, respectively, at 84 DAF, and to 41.96±1.21 mg/g, 58.56±1.94 mg/g, and 94.96±3.64 mg/g, respectively, at 119 DAF (Fig. 2a). Although the concentrations of all five FAs were similar throughout most of testa development (21–63 DAF and 77–119 DAF; Fig. 2b), Page 4/34 Page 4/34 ALA, LA, and OA peaked sharply at 70 DAF, with ALA concentration showing the greatest increase (to 20.27 ± 3.08 mg/g). FA concentrations fluctuated but remained low throughout pericarp development; PA and LA were the most abundant FAs (Fig. 2c). In all three tissues, SA concentrations remained consistently low. ALA, LA, and OA peaked sharply at 70 DAF, with ALA concentration showing the greatest increase (to 20.27 ± 3.08 mg/g). FA concentrations fluctuated but remained low throughout pericarp development; PA and LA were the most abundant FAs (Fig. 2c). In all three tissues, SA concentrations remained consistently low. Unigene Assembly, Analysis, And Quantification Of Gene Expression The transcriptomes of the kernel, testa, and pericarp samples taken at 35, 49, 63, 77, 91, and 119 DAF (T1–T6) from two separate trees (specimens CS0009 and CS0016) were sequenced. Across the 36 samples, we obtained approximately 49.69 M reads per sample (Table 1). A total 227,837 unigenes were detected, with an average length of 755 bp (Additional file 1: Table S1). The heatmap of the Pearson correlation coefficients of gene expression levels between pairs of genes showed that the gene expression levels were significantly correlated throughout pericarp development and during rapid oil accumulation in the kernel (Additional file 2: Figure S1). Across the six developmental stages, more genes were co-expressed in all three tissues than were expressed only in one tissue (Additional file 3: Figure S2). In addition, more genes were expressed specifically in the pericarp than in either the kernel or the testa. More genes were specifically expressed in the seed kernel at 49 DAF than at any other stage(Additional file 3: Figure S2). Page 5/34 Table 1 Sequencing statistics for the 36 samples taken during the development of Paeonia ostii seeds. Unigene Assembly, Analysis, And Quantification Of Gene Expression Sample Total Raw Reads(Mb) Total Clean Reads(Mb) Total Clean Bases(Gb) Clean Reads Q20(%) Clean Reads Q30(%) Clean Reads Ratio(%) CS0009_p_35 65.12 51.42 7.71 97.09 91.51 78.96 CS0009_p_49 53.48 41.89 6.28 97.08 91.53 78.32 CS0009_p_63 79.5 63.54 9.53 97.22 91.84 79.92 CS0009_p_77 85.27 63.95 9.59 96.95 91.22 74.99 CS0009_p_91 75.27 59.6 8.94 97.1 91.58 79.18 CS0009_p_119 79.37 58.11 8.72 97 91.2 73.22 CS0009_t_35 42.71 33.45 5.02 97.35 91.95 78.32 CS0009_t_49 68.7 51.46 7.72 96.62 90.23 74.9 CS0009_t_63 56.31 43.43 6.51 97.09 91.59 77.12 CS0009_t_77 56.61 41.72 6.26 96.93 91.16 73.69 CS0009_t_91 65.53 48.97 7.35 96.94 91.21 74.73 CS0009_t_119 49.76 41.65 6.25 98.13 94.01 83.71 CS0009_k_35 51.22 40.63 6.1 97.12 91.56 79.34 CS0009_k_49 55.96 44.77 6.72 97.22 91.84 80.01 CS0009_k_63 80.62 61.78 9.27 96.94 91.19 76.63 CS0009_k_77 56.87 42.97 6.45 96.99 91.24 75.55 CS0009_k_91 66.07 48.77 7.32 96.86 91.06 73.82 CS0009_k_119 58.84 43.22 6.48 96.76 90.8 73.45 CS0016_p_35 64.68 49.97 7.49 96.95 91.25 77.25 CS0016_p_49 70.79 55.9 8.38 97.05 91.41 78.95 CS0016_p_63 59.62 46.02 6.9 96.92 91.18 77.19 CS0016_p_77 72.86 56.46 8.47 96.99 91.3 77.48 CS0016_p_91 81.99 64.04 9.61 96.95 91.24 78.1 CS0016_p_119 42.01 31.77 4.77 96.89 90.75 75.62 CS0016_t_35 104.07 83.87 12.58 97.81 93.25 80.59 CS0016_t_49 66.34 50.45 7.57 97.06 91.51 76.04 Page 6/34 Page 6/34 Sample Total Raw Reads(Mb) Total Clean Reads(Mb) Total Clean Bases(Gb) Clean Reads Q20(%) Clean Reads Q30(%) Clean Reads Ratio(%) CS0016_t_63 61.83 48.98 7.35 97.06 91.39 79.22 CS0016_t_77 73.54 57.14 8.57 97.15 91.65 77.71 CS0016_t_91 61.93 47.81 7.17 97.19 91.81 77.2 CS0016_t_119 109.95 81.97 12.3 97.34 92.47 74.56 CS0016_k_35 52.81 41.03 6.15 97 91.31 77.69 CS0016_k_49 61.03 48.59 7.29 97.2 91.78 79.61 CS0016_k_63 52.57 39.83 5.97 96.97 91.28 75.76 CS0016_k_77 57.55 48.62 7.29 98.16 94.06 84.47 CS0016_k_91 50.84 37.42 5.61 96.84 90.95 73.6 CS0016_k_119 47.16 34.85 5.23 96.75 90.75 73.9 Ontology and KEGG pathway enrichment of the differentially expressed genes Ontology and KEGG pathway enrichment of the differentially expressed genes Ontology and KEGG pathway enrichment of the differentially expressed genes The up- and downregulated genes at each of the six time points in the kernel, testa, and pericarp are shown in Additional file 4: Table S2. We found that the proportion of genes up- and downregulated varied among tissues and time points. The heatmap of the hierarchical DEG clusters indicated that more genes were differently expressed during the first three stages of kernel development (Additional file 5: Figure S3). We then determined which gene ontology (GO) functions were enriched in the DEGs. GO functions are grouped in three categories: molecular function, cell component and biological process. At 49 DAF, twice as many genes in the kernel and testa were enriched in metabolic process as compared to the pericarp (Additional file 6: Figure S4). In the kernel, 49 DAF was a period of rapid oil accumulation (Fig. 2a). Consistent with this, the genes differentially expressed in the kernel at 49 DAF as compared to 35 DAF were primarily associated with the lipid metabolism (226 DEGs) and the carbohydrate metabolism (309 genes; Fig. 3a). Other metabolic pathways, such as fatty acid metabolism, fatty acid biosynthesis, linoleic acid metabolism, glycosphingolipid biosynthesis-ganglioseries, glycosphingolipid synthesis-globo and isoglobe series, starch and sucrose metabolism were also significantly enriched in these DEGs (Fig. 3b). Most of the metabolic pathways were overrepresented in the genes downregulated between 49 DAF and 35 DAF, including the linoleic acid metabolism pathway; linoleic acid is the substrate of ALA synthesis (Fig. 3c). However, the fatty acid metabolism, fatty acid biosynthesis, pyruvate metabolism, and plant hormone signal transduction pathways were overrepresented in the genes upregulated between 49 DAF and 35 DAF (Fig. 3c). With respect to the genes differently expressed between 63 DAF and 49 DAF, more pathways were significantly enriched in the downregulated genes than in the upregulated genes (Fig. 3d). Interestingly, the fatty acid metabolism and fatty acid biosynthesis Page 7/34 Page 7/34 pathways, which were significantly enriched at 49 DAF, were not significantly enriched at any other time point (Additional file 7: Figure S5). In the testa, 187 genes associated with the lipid metabolism were differentially expressed between 49 In the testa, 187 genes associated with the lipid metabolism were differentially expressed between 49 DAF and 35 DAF. Ontology and KEGG pathway enrichment of the differentially expressed genes Pathways related to the lipid metabolism (i.e., glycerolipid metabolism, glycerophospholipid metabolism, glycosphingolipid biosynthesis-ganglio series, and cutin/suberine and wax biosynthesis) and to the carbohydrate metabolism (i.e., starch/sucrose metabolism and galactose metabolism) were significantly enriched in these DEGs (Fig. 4a). The glycerolipid metabolism pathway was overrepresented in similar numbers of up- and downregulated DEGs at 49 DAF (Fig. 4b). Several other lipid-associated pathways (i.e., glycerophospholipid metabolism, glycosphingolipid biosynthesis- ganglio series, and steroid biosynthesis) were more enriched in the upregulated DEGs than in the downregulated DEGs at 49 DAF (Fig. 4b). Similarly, pathways related to starch accumulation and sugar synthesis (i.e., fructose/mannose metabolism and galactose metabolism) were also enriched in more upregulated than downregulated DEGs at 49 DAF (Fig. 4b). This might be due to the intensive membrane lipid synthesis, seed coat thickening, and dry matter accumulation that occurs during this stage in the testa. Compared with 35 DAF, 287 genes associated with the lipid metabolism were differently expressed at 63 DAF in the testa; the metabolic pathways significantly enriched in these DEGs included fatty acid elongation, glycerolipid metabolism, sphingolipid metabolism, glycosphingolipid biosynthesis-ganglio series, and cutin/suberine and wax biosynthesis (Fig. 4c). At 63 DAF, most of the DEGs were associated with the alpha linolenic acid pathway; in this pathway, the number of upregulated genes was similar to the number of downregulated genes (Fig. 4d). No lipid metabolism pathways were enriched at any other stage of testa development (Additional file 8: Figure S6). DAF and 35 DAF. Pathways related to the lipid metabolism (i.e., glycerolipid metabolism, In the pericarp, certain lipid metabolism pathways, including glycerolipid metabolism, fatty acid elongation, sphingolipid metabolism, and cutin/suberine and wax biosynthesis, were similarly enriched in all DEGs in different stages (Additional file 9: Figure S7 and Additional file 10: Figure S8). Expression Patterns Of Unigenes Associated With Oil Accumulation Based on the functional annotations of the DEGs, we identified 1373 unigenes associated with lipid metabolism (Additional file 11: Table S3). These unigenes were associated with 10 metabolic pathways: de novo plastid FA synthesis, elongation, desaturation and export (138 unigenes); triacylglycerol biosynthesis and eukaryotic phospholipid synthesis & editing (176 unigenes); prokaryotic and eukaryotic galactolipid, sulfolipid, and phospholipid synthesis (25 unigenes); sphingolipid biosynthesis (58 unigenes); fatty acid elongation, wax biosynthesis, cutin synthesis, and suberin synthesis and transport (251 unigenes); triacylglycerol and fatty acid degradation (99 unigenes); phospholipid signaling (108 unigenes); lipid trafficking (27 unigenes); oxylipin metabolism (39 unigenes); and mitochondrial fatty acid synthesis (43 unigenes) (Additional file 12: Table S4). Of these 1373 unigenes, 314 were associated with FA and TAG biosynthesis (Additional file 12: Table S4). Specifically, 13 unigenes were associated with the pyruvate dehydrogenase complex (PDHC), which catalyzes the oxidative decarboxylation of pyruvate to produce acetyl-CoA. The PDHC contains three Page 8/34 Page 8/34 subunits: pyruvate dehydrogenase (PDH, 10 unigenes), dihydrolipoyl acyltransferase (DHLAT, 2 unigenes), and dihydrolipoamide dehydrogenase (LPD, 1 unigene). These unigenes were highly expressed throughout the development of the pericarp, testa, and kernel. In addition, 25 unigenes encoded subunits of ACC, a multi-subunit enzyme that includes biotin carboxylase (BC), the biotin carboxyl carrier protein (BCCP), and carboxyltransferase (CT). Finally, seven unigenes were homologous to α-CT; one unigene was homologous to β-CT; 13 unigenes were homologous to BC; and four unigenes were homologous to BCCP (Additional file 12: Table S4). In particular, CL15202, homologous to α-CT, was highly expressed across all three tissues, but was most strongly upregulated in the kernel. Similarly, CL9207.Contig2, homologous to homomeric ACC (HmACC), was also highly expressed during the development of all three tissues, but was most highly expressed in the testa. CL16462 (homologous to α-CT) and unigene 719 (homologous to β-CT) were strongly upregulated in the pericarp, but expressed only at low levels in the kernel and the testa. In addition, homologs to BC (CL7345.contig8) and BCCP (CL18970.contig3 and unigene17464) were significantly upregulated in the kernel at 35 and 49 DAF, as compared to later developmental stages (63–119 DAF); these genes were expressed only at low levels throughout the development of the testa and pericarp (Additional file 13: Figure S9). Three unigenes were associated with the catalysis of the malonyl-ACP elongation cycle: CL8489.Contig2, associated with 3-ketoacyl-ACP synthase isoform I (KAS I); CL1611.Contig2, associated with KAS II; and unigene32463, associated with KAS III. Expression Patterns Of Unigenes Associated With Oil Accumulation In the kernel, unigene32372 expression increased from 35–49 DAF, and then decreased fairly steadily until the end of the experiment (Fig. 5b). Three unigenes encoding lysophosphatidylcholineacyltransferase (LPCAT) (unigene39796, CL18110.Contig2, and CL18110.Contig24) and two unigenes encoding lysophosphatidylethanolamineacyltransferase (LPEAT) (CL10703.Contig3 and CL20149.Contig2) were identified as DEGs. We also identified 19 unigenes encoding PLD. In the kernel, the expression profile of one of these unigenes (CL1337.Contig6) resembled a bell-shaped curve, peaking at 63 DAF (Fig. 5b). Other phospholipase D (PLD) genes were also identified as DEGs, but did not exhibit a smooth peak in expression. cholinephosphotransferase (PDCT) (unigene32372), was significantly differentially expressed among these tissues. In the kernel, unigene32372 expression increased from 35–49 DAF, and then decreased fairly steadily until the end of the experiment (Fig. 5b). Three unigenes encoding lysophosphatidylcholineacyltransferase (LPCAT) (unigene39796, CL18110.Contig2, and CL18110.Contig24) and two unigenes encoding lysophosphatidylethanolamineacyltransferase (LPEAT) (CL10703.Contig3 and CL20149.Contig2) were identified as DEGs. We also identified 19 unigenes encoding PLD. In the kernel, the expression profile of one of these unigenes (CL1337.Contig6) resembled a bell-shaped curve, peaking at 63 DAF (Fig. 5b). Other phospholipase D (PLD) genes were also identified as DEGs, but did not exhibit a smooth peak in expression. We identified 45 unigenes associated with TAG biosynthesis. Of these, 16 unigenes encoded glycerol-3- phosphate acyltransferase (GPAT), 10 unigenes encoded lysophosphatidic acid acyltransferase (LPAAT), 10 unigenes encoded phosphatidate phosphatase (PAP), and 4 unigenes encoded diacylglycerol acyltransferase (DGAT). Specifically, four unigenes were strongly upregulated in the kernel at 49 DAF as compared to 35 DAF: unigene42999 encoding GPAT; CL1842.Contig1 and CL1842.Contig2 encoding LPAAT; CL4004.Contig1 and CL18619.Contig1 encoding PAP; and CL5087.Contig1 encoding DGAT (CL5087.Contig1) (Additional file 13: Figure S9). This pattern was observed in neither the testa nor the pericarp. Five unigenes encoding phospholipid:diacylglycerol acyltransferase (PDAT) were identified as DEGs. One of these (CL7876.Contig4) was highly expressed throughout pericarp and testa development, but was downregulated at 49 DAF as compared to 35 DAF in the kernel. In the kernel, the expression of another PDAT-encoding unigene (unigene24505) increased dramatically from 35–49 DAF and then again from 77–91 DAF (Fig. 5b). This gene was highly expressed in neither the testa nor the pericarp (Additional file 13: Figure S9). We identified 29 unigenes associated with the formation of oil bodies in the cytoplasm that were also DEGs. Of these unigenes, six encoded oleosin (OLE), 19 encoded caleosin (CLE), and four encoded steroleosin (SLE) (Additional file 12: Table S4). Expression Patterns Of Unigenes Associated With Oil Accumulation These unigenes were strongly upregulated during pericarp and testa development, but were highly expressed in the kernel at 49 DAF only (Additional file 13: Figure S9). Eighteen unigenes encoding SAD were also identified as DEGs. Of these, four (CL2824.Contig13, CL2824.Contig14, CL2824.Contig25, and CL2824.Contig26) were more highly expressed in the kernel and the testa than in the pericarp (Additional file 13: Figure S9). In addition, in the kernel, the expression levels of CL2824.Contig25 steadily increased from 35–63 DAF, remained constant from 63–77 DAF, and decreased steadily from 77–119 DAF (Fig. 5b). One unigene was shown to encode fatty acyl-ACP thioesterase A (FATA) (unigene17393), and three were shown to encode FATB (unigene2022, unigene5608, and unigene6721). Unigene6721 was differently expressed among the different developmental stages of the pericarp, testa, and kernel, but unigene17393 was only highly expressed in the kernel (with an expression peak at 49 DAF; Additional file 13: Figure S9). We also identified 10 unigenes encoding long-chain acyl-CoA synthetase (LACS) (Additional file 12: Table S4). Ten unigenes were associated with PUFA biosynthesis: three unigenes encoded FAD2 (CL15521.Contig1, CL15521.Contig2, and CL15521.Contig3), and two unigenes encoded FAD6 (CL5945.Contig3 and CL5945.Contig8). In the kernel, the expression level of one of the FAD2-encoding genes (CL15521.Contig3) increased from 35–77 DAF, and decreased from 77–119 DAF (Fig. 5b). This unigene was also sharply upregulated at 77 DAF in the testa (Additional file 13: Figure S9). An additional four unigenes encoded FAD3 and FAD8 (unigene2598, unigene43483, CL1023.Contig1, and CL1023.Contig4), and one unigenes encoded FAD7 (unigene21286) (Additional file 11: Table S3). Based on our phylogenetic analysis of ω-3 FADs from Arabidopsis thaliana and P. ostii (Additional file 14: Figure S10), we identified four microsomal FAD3 genes and one plastid FAD7/8 gene in P. ostii. In the kernel, the expression level of one of these FAD3-encoding genes (CL1023.Contig4) increased steadily from 39–91 Page 9/34 Page 9/34 DAF, followed by an abrupt decrease (Fig. 5b). Similarly, CL1023.Contig4 was sharply upregulated at 77 DAF in the testa (Additional file 13: Figure S9). In contrast, CL1023.Contig4 was expressed only at low levels throughout pericarp development. Several unigenes encoding enzymes important for acyl editing and the Lands cycle were also identified. The three unigenes encoding CDP-choline:DAG choline phosphotransferase (CPT) (unigene7809, unigene16109, and unigene16131) were differentially expressed during the development of the kernel, pericarp, and testa. One unigene, encoding phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT) (unigene32372), was significantly differentially expressed among these tissues. Expression Patterns Of Unigenes Associated With Oil Accumulation All of these unigenes were stably expressed at very low levels during pericarp development, but were highly expressed throughout the development of the testa and very highly expressed throughout the development of the kernel (Additional file 13: Figure S9). In the kernel, bell-shaped expression profiles were observed for one CLE-encoding unigene (CL18131.Contig3), two OLE-encoding unigenes (unigene5123 and unigene17798), and one SLE- encoding unigene (CL19272.Contig1) (Fig. 5b). TFs associated with FA and TAG biosynthesis TFs associated with FA and TAG biosynthesis Page 10/34 We predicted the DEGs that might encode TFs, and classified these into TF families (Fig. 6a). Of these TF DEGs, 301 belonged to the MYB family, 164 to the AP2-EREBP family, 161 to the FAR1 family, 132 to the NAC family, 115 to the MADS family, 114 to the bHLH family, 70 to the WRKY family, and 14 to the bZIP family. Within the TF DEGs, we also identified the unigenes related to the FA metabolism: nine in the WRI family, 20 in the bZIP family, one in the LEAFY family, six in the FUSCA (FUS) family, five in the ABSCISIC ACID-INSENSITIVE family, and 82 in the MYB family (Additional file 15: Table S5). WRI1 (CL22311) was more strongly upregulated in the kernel than in the testa and the pericarp, especially during the early stages of FA accumulation (Fig. 6b). FUSCA (unigene25640) was also more strongly upregulated in the kernel than in the pericarp or the testa (Fig. 6b). The two unigenes encoding bZIP (unigeneCL3725 and unigeneCL15124) were constitutively expressed in all three tissues (Additional file 16: Fig. 11). The expression patters of the many TF DEGs in the MYB family differed among tissues. One unigenes (CL14921.Contig3) was highly expressed in the pericarp, but expressed only at low levels in the kernel and testa. Similarly, unigene25046 was also differentially expressed among tissues. In the kernel, unigene25046 expression increased from 35–49 DAF, and then decreased sharply until the end of the experiment (Additional file 16: Fig. 11). In the testa, unigene25046 was upregulated at 49 DAF, this high level of expression was maintained until the end of the experiment. In the pericarp, unigene25046 was expressed at low levels throughout the experiment. We also considered the unigenes encoding zinc finger CCCH domain-containing protein 54 (C3H54) and ethylene-responsive transcription factor (RA212). Unigene32844, encoding C3H54, and unigene43518, encodingRA212, were both upregulated during FA accumulation in the kernel, but were expressed only at low levels in the pericarp and testa (Fig. 6b). Quantitative real-time PCR (qRT-PCR) validation of key DEGs In general, the expression patterns of the 13 key genes chosen for validation were similar between the qRT-PCR and RNA-seq analyses (Fig. 5b and 7; Additional file Figure S12-15). For example, seven unigenes (encoding SAD, FAD2, FAD3, PDAT, OLE, CLE, and SLE), which exhibited bell-shaped patterns of expression during kernel development in the RNA-seq data, were observed to exhibit similar expression patterns in the qRT-PCR data. Importantly, both RNA-seq and qRT-PCR indicated that these seven unigenes were only expressed at low levels throughout the development of the testa and pericarp. However, the qRT-PCR data indicated that FAD3 expression in the testa increased sharply at 63 DAF (Fig. 7b). RNA-seq and qRT-PCR analyses showed that the remaining six unigenes were upregulated in the kernel, but expressed at low levels throughout the development of the pericarp and testa. Discussion The patterns of FA accumulation differed noticeably among the pericarp, testa, and kernel. Throughout development, FA content in the pericarp was generally low, as was FA content in the testa. In contrast, concentrations of UFAs, particularly ALA, increased rapidly in the testa beginning at 63 DAF, peaking at 70 DAF. At 70 DAF, ALA content in the testa was 10-fold that of ALA content at 56 DAF; this coincided with the maximum thickening of the testa (Fig. 1d). This is interesting because the concentrations of UFAs, Page 11/34 Page 11/34 especially ALA, exhibited an inverted V-shaped pattern of change during testa development. To our knowledge, this is the first demonstration of changes in ALA content during testa development. The kernel began to accumulate oil rapidly once the external shape and size of the seed had stabilized. The concentrations of UFAs, especially ALA, followed a bell-like curve as the kernel matured. ALA concentration peaked at 77 DAF; at this point, the cotyledon embryo had formed completely, the seed had reached maximum size, and the pericarp was yellow. In contrast, Li et al. [10] found that ALA content was greatest at the seed-browning stage. Our results showed ALA concentrations in the P. ostii were high in the endosperm, moderate in the testa, and low in the pericarp. As these three tissues have different ALA concentrations, they represent good research systems for comparative studies of the mechanisms of ALA synthesis and accumulation in P. ostii. Transcriptomes have previously been used to study the mechanisms underlying ALA synthesis in tree peony seeds [10–14]. Here, we identified genes associated with the lipid metabolism that were differentially expressed during the development of the seed kernel, testa, and pericarp. Although the FA metabolism is the primary metabolic process in plant cells, the pathways associated with FA biosynthesis and metabolism were not significantly enriched during pericarp and testa development. The metabolic pathways that were most significantly enriched in the pericarp and testa DEGs (e.g., fatty acid elongation, glycerolipid metabolism, cutin/suberine and wax biosynthesis, and sphingolipid metabolism) were primarily associated with the wax and cutin biosynthesis that is required for the development of the pericarp and the testa. However, at stage 63 DAF in the testa (and not at any other stages), ALA content increased sharply, and the genes encoding SAD, FAD2, and FAD3 were strongly upregulated (Fig. 7). Discussion In addition, the ALA biosynthesis and fatty acid biosynthesis pathways were significantly enriched at this stage in the testa. The inverted V-shape of the change in ALA content in the testa at 56–84 DAF was significantly correlated with the increase in ALA and FA biosynthesis. More upregulated genes than downregulated genes were identified in the kernel at 49 DAF, possibly because oil rapidly accumulates in the kernel during this period. Consistent with this, some of the genes upregulated in the kernel at 49 DAF were significantly enriched in lipid metabolism pathways, especially FA biosynthesis and metabolism. The genes significantly upregulated at 49 DAF in the kernel included those encoding α-PDH, BCCP, BC, FATA, KAS I–III, PLD, PDCT, and LPAAT. Most of these genes are known to play major roles in de novo FA biosynthesis. Genes associated with the pyruvate metabolism and with plant hormone signal transduction also tended to be upregulated, rather than downregulated, at 49 DAF in the kernel. These two pathways are also related to the FA metabolism: pyruvate metabolism is the precursor of acetyl CoA production, while plant hormone signaling pathways may activate a variety of TFs to regulate the FA metabolism [16]. Thus, the rapid synthesis and accumulation of FAs at 49 DAF in the kernel might be primarily due to the activation of the FA synthesis pathway and the upregulation of key genes. Two types of ACCs, heteromeric ACC (HeACC) and HmACC, were expressed during the development of the P. ostii tissues. HeACC forms a multisubunit enzyme with BC, BCCP, alpha-CT, and beta-CT [25]. The genes encoding these subunits were differentially expressed among the three tissues throughout development. The genes encoding HmACC were highly expressed during testa development, but expressed at low levels in the other two tissues. Specifically, ACC in plant plastids is heterogeneous, while ACC in plant cytoplasm Page 12/34 Page 12/34 is homogenous; ACC in the cytoplasm catalyzes malonyl monoacyl CoA to produce long-chain FAs, flavonoids, anthocyanins, and other secondary metabolites [26, 27]. Thus, we speculated that the step of de novo FA biosynthesis catalyzed by ACCs is specifically catalyzed by HmACC in the testa and by HeACC in the kernel. However, in the Gramineae, the ACC in the plastids is homogenous [28]. Discussion The genes encoding KAS I, KAS II, and KAS III, which are important catalysts of FA synthesis, were significantly upregulated at 49 DAF in the kernel (Additional file 13: Figure S9), while the genes encoding SAD (CL2824.Contig14 and CL2824.Contig25) exhibited a bell-shaped expression pattern. These genes were not similarly upregulated in the testa and the pericarp. The upregulation of the SAD and KAS genes during kernel development might be related to the rapid production of 18:1-ACP in this tissue. Consistent with this, the upregulation of the gene encoding FATA in the kernel, particularly at 49 DAF, might ensure that 18:1-ACP is produced rapidly via SAD catalysis and transported to the ER for downstream reactions. Thus, the rapid upregulation of KAS I, KAS III, KAS III, SAD, and FATA, as well as BCCP and BC, at 49 DAF might be required to ensure that sufficient C18:1-dominated fatty acyl flux are transported to the ER for acyl editing or TAG assembly. ω-3 FADs are located in the plastid or the ER, and are encoded by a multigene family [20]. ω-3 FADs act on C18:2 to produce C18:3 (or on C16:2 to produce C16:3). There are two types of ω-3 FADs: ω-3 FADs are located in the plastid or the ER, and are encoded by a multigene family [20]. ω-3 FADs act on C18:2 to produce C18:3 (or on C16:2 to produce C16:3). There are two types of ω-3 FADs: microsomatic FAD3, which is located in the ER and acts on PC or other phospholipids with cytochrome b as the electronic donor, and plastid FAD7/8, which is located in the plastid and acts on phosphatidylglycerol and galactose lipids with ferritin as the electronic donor [16, 20]. In A. thaliana, FAD7/8 were shown to be plastid isozymes of FAD3; FAD3, FAD7, and FAD8 were all present as single copies in A. thaliana [29–31]. Here, based on gene annotation, we identified five unigenes (unigene21286, unigene2598, unigene43483, CL1023.Contig1, and CL1023.Contig4) as encoding the temperature- sensitive ω-3 FAD7/8 (Additional file 11: Table S3). However, our phylogenetic analysis suggested that four microsomal FAD3 genes and one plastid FAD7/8 gene were expressed in P. ostii. One of the genes encoding a microsomal FAD3 (CL1023.Contig4) was most highly expressed at 77 DAF in the kernel (Fig. 7). One gene encoding a plastid FAD7/8 (Unigene21286) was expressed during kernel development at a low level. Discussion ALA content in the kernel also peaked at 77 DAF, suggesting that FAD3 might play a vital role in the formation of ALA. In the testa, CL1023.Contig4 was also highly expressed at 63 DAF, followed by in a sharp increase in ALA content at 70 DAF. In contrast, FAD3 was not highly expressed in the pericarp, and ALA content in the pericarp was very low. Although a previous study concluded that the high concentrations of ALA in P. ostii seeds were due to FAD8 activity and abundance [10], our results suggested that high concentrations of ALA in the kernel might be associated with the activity and abundance of FAD3, not FAD8. Other plants with high concentrations of ALA, such as flax, perilla, and sea-buckthorn, invariably express both FAD3 and FAD7/8 [32–35]. For example, the upregulation of FAD3 in flax was associated with high ALA content during seed development [32, 33]. In Perilla frutescens, FAD3 was more highly expressed than FAD7/8 during seed development, but FAD7/8 was more highly expressed than FAD3 in the leaves [34]. In the fruits of the sea-buckthorn (Hippophae rhamnoides) levels of FAD3 and FAD7/8 expression were similarly upregulated, leading to high levels of ALA [35]. However, Page 13/34 Page 13/34 even among species with high ALA content, the enzymes that play key roles in ALA synthesis differ, suggesting that ALA synthesis pathways also differ among plants, or even among tissues. De novo FA synthesis, and the subsequent acyl flux through the Kennedy pathway, produces oils containing saturated and monounsaturated FAs [16]. However, the production of more diverse TAG acyl compositions in plants requires the flux of acyl groups through the membrane lipids PC or PE [16, 21]. Therefore, the accumulation of TAG assemblies containing various PC/PE-modified FAs requires the coordinated flux of acyl groups into PC/PE for desaturation, and the subsequent flux of acyl groups out of the PC/PE and into TAG assemblies (Fig. 5a). In conjunction, acyl editing and the Lands cycle regulate acyl flux, to ensure that acyl groups are modified to increase the diversity of TAG acyl compositions [21]. In the three P. ostii tissues, we detected 28 unigenes associated with acyl editing and the Lands cycle, including three CPT-encoding unigenes, one PDCT-encoding unigene, three LPCAT-encoding unigenes, two LPEAT-encoding unigenes, and 19 PLD-encoding unigenes. Discussion In particular, one PDCT-encoding unigene (unigene32372) and one PLD-encoding unigene (CL1337.Contig6) showed bell-shaped expression patterns during kernel development, but were expressed minimally in the pericarp and testa (Additional file 13: Figure S9). This indicated that, compared with the testa and pericarp, the high expression of these genes in kernel was very important for overall carbon flux into TAG in P. ostii in the form of PUFAs. To our knowledge, unigene32372 and CL1337.Contig6 have not previously been identified as key genes in tree peony seed development. In A. thaliana, the reduced oleate desaturase 1 gene (rod1) was shown to encode a novel enzyme (PDCT); when rod1 expression was inhibited, the accumulation of PC-modified fatty acids in the TAG assembly decreased ~ 40%, although overall levels of TAG remained constant [36]. As PC/PE is the site for extra- plastidial FA modification, the flux of the de novo DAG moiety through PC can provide a PC-derived DAG pool with a different acyl composition than that of de novo DAG (Fig. 5a). However, the relative proportion of TAG synthesized from the Kennedy pathway using de novo DAG to TAG synthesized from PC-derived DAG is unclear in most plants. Indeed, the PC-derived DAG pool, rather than the de novo DAG pool, might be the primary source of DAG for TAG synthesis in various plants [36, 37]. The DGAT enzyme has also been implicated in the formation of TAG from de novo DAG and acyl-CoA (the final step of the Kennedy pathway; Fig. 5a, blue line). For example, DGAT1, DGAT2, and DGAT3 drive TAG production in Arabidopsis [38], the peanuts [39], and tung tree [40]. Here, although four DGAT-encoding unigenes were identified in P. ostii, only one unigene (CL5087.Contig1) was strongly upregulated at 49 DAF in the testa, weakly upregulated at same stage in the kernel, and expressed minimally throughout development in the pericarp (Additional file 19: Figure S14). TAG synthesis may also be catalyzed by PDAT, using FAs from the PC pool and PC-derived DAG as the substrate (Fig. 5a, green line). Here, the PDAT-encoding unigene (unigene24505) showed a bell-shaped expression pattern in the developing kernel, but was minimally expressed in the testa and the pericarp (Additional file 19: Figure S14). This indicated that in the P. ostii kernel, PDAT reflected the amount of PFUA-containing TAG more accurately than DGAT. Thus, our results indicated that the PDAT pathway (Fig. Discussion 5a, green line), rather than the Kennedy pathway (Fig. 5a, blue line), might be the primary mechanism of TAG generation in the P. ostii kernel. Zhang et al. [13] reported that PDAT levels were significantly associated with TAG accumulation in P. rokii. However, a previous study Page 14/34 Page 14/34 concluded that the PDAT pathway was probably not an important pathway for TAG generation in tree peony seeds, due to the low abundance of PDAT-encoding gene transcripts [10]. In mature seeds, TAGs can be stored as oil bodies, surrounded by a phospholipid monolayer and abundant amphipathic proteins (e.g., OLE, CLE, and SLE) [41–43]. Throughout development, the expression levels of the gene homologs encoding OLE, CLE, and SLE either increased or were consistently high in the kernel and the testa, ensuring efficient ALA accumulation, but remained low in the pericarp (Additional file 20: Figure S15). In all three tissues, the CLE-encoding genes were more strongly upregulated than the OLE- or SLE-encoding genes. However, during the development of Brassica napus seeds and Prunus sibirica kernels, OLE was much more highly expressed than CLE and SLE [41, 44]. This suggested that mechanisms of oil formation differ among plants. In mature seeds, TAGs can be stored as oil bodies, surrounded by a phospholipid monolayer and abundant amphipathic proteins (e.g., OLE, CLE, and SLE) [41–43]. Throughout development, the expression levels of the gene homologs encoding OLE, CLE, and SLE either increased or were consistently high in the kernel and the testa, ensuring efficient ALA accumulation, but remained low in the pericarp (Additional file 20: Figure S15). In all three tissues, the CLE-encoding genes were more strongly upregulated than the OLE- or SLE-encoding genes. However, during the development of Brassica napus seeds and Prunus sibirica kernels, OLE was much more highly expressed than CLE and SLE [41, 44]. This suggested that mechanisms of oil formation differ among plants. TFs, such as WRI1, FUS3, ABI3, bZIP, LEC1, LEC2, MYB, and GL2, have previously been shown to play key roles in seed oil synthesis and deposition [45]. Here, transcriptional profiling revealed that WRI1 and FUSCA participated in the positive regulation of genes associated with oil synthesis during kernel development (Fig. 7b). Notably, the AP2 TF WRI1 is upregulated by LEC1, LEC2, ABI3, and FUS3 in Arabidopsis [45]. During kernel development, both FUS3 and WRI1 were upregulated (Fig. Discussion 6b), suggesting that the gene encoding WRI1 might be a direct target of FUS3 in the developing kernel. Additionally, the co-upregulation of plastidial ACC (BCCP and BC), KAS I–III, SAD, and FATA with WRI1 during kernel development (Additional file 13: Figure S9) suggested that WRI1 might be involved in the transcriptional regulation of these target genes, as was shown in Arabidopsis seeds. Thus, WRI1 (CL22311) might play a critical role in the regulatory network controlling ALA accumulation during kernel development. An important regulator of gene transcription in the soybean is zinc finger CCCH domain containing protein (GmZF351). Transgenic experiments using Arabidopsis showed that GmZF351 directly regulated WRI1, BCCP, KAS III, OLE and other genes; in addition, GmZF351 overexpression in soybeans increased oil content [46]. Here, we found that the expression pattern of unigene32844, which encoded the TF zinc finger CCCH domain-containing protein 54 (C3H54), in the seed kernel were consistent with the changes in ALA content (Figs. 2 and 6b). Therefore, this TF might be a useful research target for future studies of the regulation of ALA biosynthesis. Plant materials Fruits and seeds of P. ostii were collected in 2016 and 2017 at Shanghai Chenshan Botanical Garden (31°4′52″N, 121°10′14″E), Shanghai, China. These P. ostii were introduced to the garden more than 10 years ago, and have been grown under the same environmental and cultivation conditions since that time. We observed the development of the fruits and seeds produced by these trees over two growing seasons (April–August, 2016 and 2017). Each year, the budded flowers of both selected plants (CS0016 and CS0009) were hand-pollinated with the pollen collected from a third P. ostii tree (CS0010). The pollination date was recorded as 0 DAF. Between 0 and 119 DAF, seeds were collected every seven days from both trees, for a total of 17 samples. After seed collection, the pericarp, testa, and kernel were separated manually, frozen in liquid nitrogen, and stored at -80°C. Separated samples were used for FA detection, transcriptome sequencing, and qRT-PCR. Conclusions Our results indicated that the optimal time for tree peony oil collection was 77 DAF, when the fruit peel was yellow and oil quantity and quality were greatest. That is, FA concentration increased dramatically during cotyledon embryogenesis, and continued to increase steadily to peak at 77 DAF. This rapid increase in seed oil content was associated with the differential expression of many genes associated with FA and TAG synthesis during embryogenesis. Many FA biosynthesis and metabolism pathways were significantly enriched in these DEGs in the kernel at the early stages of rapid oil accumulation. ALA content also increased sharply in the testa during development; many of the genes differentially expressed in the testa at this stage were significantly enriched in ALA and FA biosynthesis. In total, we identified 1373 unigenes related to lipid metabolism in P. ostii. Of these, unigenes with bell-shaped Page 15/34 Page 15/34 expression patterns (e.g., those encoding SAD, FAD2, FAD3, PDCT, PDAT, OLE, CLE, and SLE) and unigenes that were upregulated at 49 DAF (e.g., those encoding BCCP, BC, KAS I–III, and FATA) may be useful targets for future investigations of lipid metabolism in the tree peony. Similarly, many regulatory enzymes (e.g., PDH, ACC, KAS I–III, SAD, FATA, FAD2, FAD3 LPCAT/LPEAT, CPT, PDCT, DGAT, PDAT, OLE, CLE, and SLE) and TFs (WRI1 and FUS3), which are crucial for the biosynthesis of acetyl-CoA, FA, TAG, and the oil body, and which were significantly enriched during kernel development, deserve further investigation. In this study, we used three tissues with high, moderate, and low ALA concentrations as an exemplar system in which to compare tissue-specific ALA accumulation mechanism in P. ostii. We found that key genes related to ALA synthesis were differentially expressed among the three tissues. Thus, our results help to provide a framework for future studies of the tree peony, particularly those aimed at improving tree peony seed oil production through breeding, genetic diversification, and gene excavation. Measurement of FA accumulation To explore how FA composition changed in the pericarp, testa, and kernel throughout development, we measured the FA concentrations in the pericarp, testa, and kernel samples taken from both trees between 21 and 119 DAF (a total of 42 samples per tree per year). The samples used for FA analysis were dried at 60°C to a constant weight. Total lipids were extracted from the dried biomass as previously described [47], and FAs were methylated as previously described in spinach [48], with minor modifications. First, the dried, powdered sample was added to 3 mL of a 1:2 chloroform-methanol (v/v) mixture. Next, this solution was incubated in a water bath at 35°C for 1 h at 120 rpm for lipid extraction. After the full extraction, 1.0 mL supplementary chloroform was added to the solution, and the mixture was vortexed. Then, 1.8 mL ddH2O was added, to generate a solution with a final chloroform:methanol:ddH2O ratio of 1:1:0.9 (v/v). The solution was then centrifuged at 4000 g for 15 min. The chloroform layer was withdrawn, and then dried with sample concentrators under a nitrogen evaporator. The concentrated lipids were then re-dissolved in 2 mL of an H2SO4-methanol solution (2% H2SO4). After charging with Page 16/34 Page 16/34 nitrogen gas, the solution was vortexed for 1 min, and then incubated in a 90°C water bath for 1 h. After incubation, 1 mL ddH2O and 1 mL hexane were added to the solution. The solution was vortexed, and then centrifuged at 4000 g for 15 min. The supernatant was transferred to a new tube, concentrated using bubbling nitrogen, and stored at 4°C for gas chromatograph-mass spectrometry (GC-MS) analysis. We used 50 mg/mL nonadecanoic acid in hexane as the internal standard. The FA methyl esters were measured using a GC-MS (GC7890/MS5975, Agilent) on a HP-88 capillary column (60 m long x 0.25 mm internal diameter; 0.2 mm; Agilent). FA methylation was tested and analyzed following Yu et al.6 Unigene assembly and annotation After sequencing, the raw reads were preprocessed to remove clipped adapter sequences, low-quality reads (Q value £ 20 or containing ambiguous nucleotides), and contaminated sequences. The clean reads were de novo assembled using Trinity [49]. Redundant sequences were removed, and remaining sequences were clustered based on homology. Based on overlap, the fragments were merged or extended into longer transcripts to form a set of non-redundant unigenes. To annotate the obtained unigenes, we used BLASTX (e-value < 0.00001) to search several public databases in the following order: NCBI non- redundant (Nr), Swiss-Prot, KEGG, and COG/KOG. The Gene Ontology (GO) [50] annotation for unigenes was obtained using Blast2GO [51]. GO classification and enrichment analyses were performed using WEGO [52]. RNA extraction and cDNA library construction As preliminary results indicated that patterns of FA accumulation differed substantially among the three tissues, we sequenced the transcriptomes of the kernel, testa, and pericarp throughout development to explore the molecular mechanisms underlying FA accumulation in the three tissues. RNA was extracted from kernel, testa, and pericarp samples taken at 35, 49, 63, 77, 91, and 119 DAF from CS0016 in 2016 and 2017, and CS0009 in 2017. Total RNA was extracted from these 54 samples using RNA Exaction Kits (E.Z.N.A. HP Plant RNA Kit, Omega Bio-Tek), and then purified using the RNeasy Plant Mini Kit (Qiagen), following the manufacturer’s protocols. The concentration and quality of each RNA sample was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies). All samples had an OD260/OD280 ratio of 2.0–2.1, and an RNA integrity number >7.0. The extracted total RNA was treated with DNase I, and Oligo(dT) primers were used to isolate mRNA. Then, cDNA was synthesized using these mRNA fragments as templates. Short fragments were purified and resolved with EB buffer for end repair. Single A (adenine) nucleotides were then added. Next, the short fragments were connected with adapters, and suitable fragments were selected for PCR amplification. The Agilent 2100 Bioanalyzer and the ABI StepOnePlus Real-Time PCR System were used for the quality control of the sample library. The generated cDNA libraries were sequenced at BGI-Shenzhen (XX), using an Illumina HiSeq 4000 system. Identification of DEGs We mapped the clean reads to unigenes using Bowtie2 [53], and then calculated gene expression level with RSEM [54]. Unigene expression levels were calculated and normalized using the Fragments Per Page 17/34 Kilobase per Million mapped fragments (FPKM) method. We then used several methods to cluster the gene expression data; clustering genes based on expression patterns helps to identify genes with similar functions, which may be associated with the same biological functions. We performed principal components analysis (PCA) of all samples using the princomp function in R. We used Cluster [55] and the Euclidean matrix formula to cluster the expressed genes and sample schemes simultaneously. The resulting clusters were visualized using Java Treeview. We also used the heatmap function in R to draw a complete clustering graph. We used Mfuzz, which employs a loose clustering algorithm, to analyze the time series data. Kilobase per Million mapped fragments (FPKM) method. We then used several methods to cluster the gene expression data; clustering genes based on expression patterns helps to identify genes with similar functions, which may be associated with the same biological functions. We performed principal components analysis (PCA) of all samples using the princomp function in R. We used Cluster [55] and the Euclidean matrix formula to cluster the expressed genes and sample schemes simultaneously. The resulting clusters were visualized using Java Treeview. We also used the heatmap function in R to draw a complete clustering graph. We used Mfuzz, which employs a loose clustering algorithm, to analyze the time series data. We identified DEGs using DEseq2, EBseq, NOIseq, and PossionDis as required. DEseq2, which is based on the negative binomial distribution, was used as described by Michael et al. [56]. EBseq, which is based on the empirical Bayesian model, was used as described by Leng et al. [57]. NOIseq, which is based on the noisy distribution model, was used as described by Tarazona et al. [58]. PossionDis, which is based on the Poisson distribution, was used as described by Audic et al. [59]. We next classified the identified DEGs based on GO and KEGG annotations. We calculated GO functional enrichment and KEGG pathway enrichment using phyper in R. Analysis of unigene expression patterns and associated TFs All protein sequences were downloaded from Swiss-Prot. Based on the functional annotations of the DEGs, we selected all DEGs associated with the lipid metabolism. To better understand the molecular factors underlying ALA accumulation during kernel development, we focused on the DEGs associated with FA and TAG biosynthesis. The sequence of three ω-3 FADs from Arabidopsis thaliana (AtFAD3_AT2G29980, AtFAD7_AT3G11170, and AtFAD8_AT5G05580) were downloaded from the NCBI. We aligned the A. thaliana ω-3 FADs with those from P. ostii (unigene21286, unigene2598, unigene43483, CL1023.Contig1, and CL1023.Contig4) using clustalX [60]. We then constructed a phylogenetic tree based on this multiple sequence alignment using FastTree2 [61]. To investigate how TFs regulated tree peony oil biosynthesis, we analyzed all of the DEGs that were also TFs. First, we used Getorf  to detect the ORF of each DEG. These ORFs were then mapped to the transcription factor protein domain database (from PlntfDB) using Hmmsearch [62]. We predicted the TF coding ability of each DEG based on the characteristics of each TF family. Based on the Swiss-Prot annotations of the DEGs, we identified the TF DEGs related to FA metabolism. qRT-PCR validation of the expression patterns of key DEGs involved in oil accumulation Each 20 µL reaction mixture contained 10 μl of TB Green Premix Ex Taq II (Tli RNaseH Plus) (Takara), 0.8 μl of each primer (10 μM), 0.3 μl of cDNA template (1 μg), and 8.1 μl of RNase-free water. PCRs for each gene were performed in triplicate, with the following thermal cycling conditions: 95°C for 30 s; 40 cycles of 95°C for 5 s and 64°C for 30s; and 95°C for 15 s. Primer specificity was confirmed by melting curve analysis. The relative expression levels of the tested genes were calculated using the 2-ΔΔCt method [63], with the actin genes as internal controls. Abbreviations ACC: acetyl-CoA carboxylase; ACP: acyl carrier protein; ALA: alpha linolenic acid; BC: biotin carboxylase; BCCP: biotin carboxyl carrier protein; CT: carboxyltransferase; CLE: caleosins; CPT: CDP-choline:DAG choline phosphotransferase; DAG: diacylglycerol; DAF: days after fertilization; DEG: differentially expressed gene; DGAT: diacylglycerol acyltransferase; EPT: CDP-ethanolamine:DAG ethanolaminephosphotransferase; ER: endoplasmic reticulum; FA: fatty acid; FAS: fatty acid synthase; FATA/B: acyl-ACP thioesterase A/B; FAD2: oleate desaturase; FAD3: linoleate desaturase; FPKM: fragments per kilobase per million mapped fragments; G3P: glycerol-3-phosphate; GPAT: glycerol-3- phosphate acyltransferase; KAS I, II, III: ketoacyl-ACP synthase I, II, III; KEGG: kyoto encyclopedia of genes and genomes; LA: linoleic acid; LACS: long-chain acyl-CoA synthetase; LEC1: leafy cotyledon1; LPA: lysophosphatidic acid; LPAAT: lysophosphatidyl acyltransferase; LPC: lysophosphatidylcholine; LPE: lysophosphatidylethanolamine; LPCAT: lysophosphatidylcholineacyltransferase; LPEAT: lysophosphatidylethanolamineacyltransferase; MCMT: malonyl-CoA ACP transacylase; OA: oleic acid; OLE: oleosin; PA: phosphatidic acid; PAP: phosphatidic acid phosphohydrolase; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PDAT: phospholipid: diacylglycerol acyltransferase; PDCT: phosphatidylcholine: diacylglycerol cholinephosphotransferase; PDH: pyruvate dehydrogenase; PLC: phospholipase C; PLD: phospholipase D; PUFA: polyunsaturated fatty acids; SAD: stearoyl-ACP desaturase; SLE: steroleosin ; TAG: triacylglycerol; TF: transcription factor; UFA: unsaturated fatty acid. qRT-PCR validation of the expression patterns of key DEGs involved in oil accumulation To experimentally validate our RNA-seq data, we selected 13 unigenes associated with FA biosynthesis and TAG assembly, in particular those related to ALA, for qRT-PCR validation. The unigenes selected were CL2824.Contig25, encoding SAD; CL15521.Contig3, encoding FAD2; CL1023.Contig4, encoding FAD3; CL5945.Contig8, encoding FAD6; Unigene21286, encoding FAD7/8; CL5087.Contig1, encoding DGAT; Page 18/34 Page 18/34 Unigene24505, encoding PDAT; Unigene5123, encoding OLE; CL18131.Contig3, encoding CLE; and Unigene19272, encoding SLE. The expression levels of these unigenes were quantified using qRT-PCR in the kernel, testa and pericarp at 35, 49, 63, 77, 91, and 119 DAF (18 samples in total). Total RNA was isolated from each tissue at each time point using RNA Exaction Kits (E.Z.N.A. HP Plant RNA Kit, Omega Bio-Tek). First-strand cDNA was prepared from 1 μg of total RNA per sample using a FastKing RT Kit with gDNase (Tiangen). Specific primers were designed for each of the 13 unigenes (Additional file 21: Table S6). PCRs were performed on an ABI StepOnePlus Real-Time PCR System (Applied Biosystems), following the manufacturer’s instructions. Each 20 µL reaction mixture contained 10 μl of TB Green Premix Ex Taq II (Tli RNaseH Plus) (Takara), 0.8 μl of each primer (10 μM), 0.3 μl of cDNA template (1 μg), and 8.1 μl of RNase-free water. PCRs for each gene were performed in triplicate, with the following thermal cycling conditions: 95°C for 30 s; 40 cycles of 95°C for 5 s and 64°C for 30s; and 95°C for 15 s. Primer specificity was confirmed by melting curve analysis. The relative expression levels of the tested genes were calculated using the 2-ΔΔCt method [63], with the actin genes as internal controls. Unigene24505, encoding PDAT; Unigene5123, encoding OLE; CL18131.Contig3, encoding CLE; and Unigene19272, encoding SLE. The expression levels of these unigenes were quantified using qRT-PCR in the kernel, testa and pericarp at 35, 49, 63, 77, 91, and 119 DAF (18 samples in total). Total RNA was isolated from each tissue at each time point using RNA Exaction Kits (E.Z.N.A. HP Plant RNA Kit, Omega Bio-Tek). First-strand cDNA was prepared from 1 μg of total RNA per sample using a FastKing RT Kit with gDNase (Tiangen). Specific primers were designed for each of the 13 unigenes (Additional file 21: Table S6). PCRs were performed on an ABI StepOnePlus Real-Time PCR System (Applied Biosystems), following the manufacturer’s instructions. Ethics approval and consent to participate Not applicable. Consent for publication Declarations Ethics approval and consent to participate Competing interests The authors declare that they have no competing interests. Authors¢ contributions Y-HH and J-HY designed all the experiments. S-YY collected all samples. S-YY and XZ analyzed all phenotypic and molecular data. XZ quantified FA composition, RNA extraction, performed qRT-PCR experiment. YZ cultivated tree peony plants. S-YY and L-BH carried out transcriptome data analysis. Y-PL and Z-JY assisted with the RNA extraction and qRT-PCR experiment. X-XZ assisted with transcriptome data analysis. S-YY and XZ drafted the manuscript. JY and YH managed the experiments, organized the manuscript and revised manuscript. All authors read and approved the final manuscript. S-YY and XZ contributed equally to this work. Consent for publication Page 19/34 Page 19/34 Not applicable. Funding This research was funded by the Chenshan Key Scientific Research Projects of Shanghai Municipal Administration of Landscaping and City Appearance (G172401, G182406, G182407, G192411 and G192419), Science and Technology Service Network Plan of Chinese Academy of Sciences (KFJ-STS- QYZD-035). Funders provided financial support only, they hand no role in the design of the study, analysis and interpretation of data, decision to publish, or the preparation of the manuscript. Acknowledgements The authors thank Plant Metabolic Platform of Shanghai Key Laboratory of Plant Functional Genomics and Resources and Dr. Yu Kong for helps with detecting fatty acids by GC-MS. 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Heatmap showing correlations among samples. Samples are plotted along the x- and y-axes. Higher correlations are indicated by darker blues. Additional file 2 Figure S1. Heatmap showing correlations among samples. Samples are plotted along the x- and y-axes. Higher correlations are indicated by darker blues. Page 24/34 Page 24/34 Page 24/34 Additional file 3 Figure S2 Venn diagrams showing the numbers of expressed genes unique and shared among the seed kernel, seed testa, and fruit pericarp of Paeonia ostii at six different developmental stages. Additional file 3 Figure S2 Venn diagrams showing the numbers of expressed genes unique and shared among the seed kernel, seed testa, and fruit pericarp of Paeonia ostii at six different developmental stages. Additional file 4 Table S2. Total number of the differentially expressed genes (DEGs) among the three Paeonia ostii tissues at various time points. Additional file 4 Table S2. Total number of the differentially expressed genes (DEGs) among the three Paeonia ostii tissues at various time points. Additional file 5 Figure S3. Heatmap showing the hierarchical clustering of the differentially expressed genes. The samples compared are plotted along the x-axis; the DEGs are plotted along the y-axis. The color of each intercept represents the log2-transformed fold change value (high: red, low: blue). Additional file 6 Figure S4. GO classification of differentially expressed genes. The number of DEGs is plotted along the x-axis; the GO terms is plotted along the y-axis. a T1 vs. T2 in the kernel. b T1 vs. T2 in the testa. c T1 vs. T2 in the pericarp. Additional file 7 Figure S5. KEGG pathways enriched in the genes differentially expressed in the developing kernel at various time points. The enrichment factor is plotted on the x-axis; pathway names are shown on the y-axis. The color of each dot reflects the Qvalue, while the size of the dot represents the number of DEGs. a T1 vs. T3. b T1 vs. T4. c T1 vs. T5. d T1 vs. T6. Additional file 8 Figure S6. KEGG pathways enriched in the genes differentially expressed in the developing testa at various time points. Supplementary Materials a Kernel. b Testa. c Pericarp. Additional file 20 Figure S15. Quantitative real-time PCR validation of oil-associated genes. a Kernel. b Testa. c Pericarp. Additional file 21 Table S6. Primer sequences used for qRT-PCR. Additional file 21 Table S6. Primer sequences used for qRT-PCR. Supplementary Materials Heat map showing the expression patterns of the unigenes associated with lipid metabolism during the development of the seed kernel (k), seed testa (t), and fruit pericarp (p) of Paeonia ostii. Labels on the y-axis indicate which plant was used (CS0009 or CS0016), the tissue (k, t, or p), and the developmental period (35–119 days after fertilization, DAF). Additional file 13 Figure S9. Heat map showing the expression patterns of the unigenes associated with lipid metabolism during the development of the seed kernel (k), seed testa (t), and fruit pericarp (p) of Paeonia ostii. Labels on the y-axis indicate which plant was used (CS0009 or CS0016), the tissue (k, t, or p), and the developmental period (35–119 days after fertilization, DAF). Additional file 14 Figure S10. Phylogenetic relationships among ω-3 FADs from Paeonia ostii and Arabidopsis thaliana. Additional file 14 Figure S10. Phylogenetic relationships among ω-3 FADs from Paeonia ostii and Arabidopsis thaliana. Additional file 15 Table S5. Annotations and FPKM values for the 125 transcription factors associated with FA biosynthesis during the development of the three Paeonia ostii tissues. Additional file 15 Table S5. Annotations and FPKM values for the 125 transcription factors associated with FA biosynthesis during the development of the three Paeonia ostii tissues. Additional file 15 Table S5. Annotations and FPKM values for the 125 transcription factors associated with FA biosynthesis during the development of the three Paeonia ostii tissues. Additional file 16 Figure S11. The relative expression levels of genes associated with key TFs. a Kernel. b Testa. c Pericarp. Additional file 16 Figure S11. The relative expression levels of genes associated with key TFs. a Kernel. b Testa. c Pericarp. Additional file 17 Figure S12. Quantitative real-time PCR validation of ACCase genes. a Kernel. b Testa. c Pericarp. Additional file 17 Figure S12. Quantitative real-time PCR validation of ACCase genes. a Kernel. b Testa. c Pericarp. Additional file 18 Figure S13. Quantitative real-time PCR validation of FAD genes. a Kernel. b Testa. c Pericarp. Additional file 18 Figure S13. Quantitative real-time PCR validation of FAD genes. a Kernel. b Testa. c Pericarp. Additional file 19 Figure S14. Quantitative real-time PCR validation of PDAT and DGAT genes. a Kernel. b Testa. c Pericarp. Additional file 19 Figure S14. Quantitative real-time PCR validation of PDAT and DGAT genes. a Kernel. b Testa. c Pericarp. Additional file 20 Figure S15. Quantitative real-time PCR validation of oil-associated genes. Supplementary Materials The enrichment factor is plotted on the x-axis; pathway names are shown on the y-axis. The color of each dot reflects the Qvalue, while the size of the dot represents the number of DEGs. a T1 vs. T4. b T1 vs. T5. c T1 vs. T6. Additional file 9 Figure S7. KEGG pathways enriched in the genes differentially expressed in the developing pericarp at various time points. a The number of genes associated with various KEGG pathways differentially expressed between T1 and T2. b The KEGG pathways overrepresented in the genes differentially expressed between T1 and T2. The color of each dot reflects the Qvalue, while the size of the dot represents the number of DEGs. c The up- and downregulated genes in T2 as compared to T1 that were associated with each KEGG pathway. Additional file 10 Figure S8. KEGG pathways enriched in the genes differentially expressed in the developing pericarp at various time points. The enrichment factor is plotted on the x-axis; pathway names are shown on the y-axis. The color of each dot reflects the Qvalue, while the size of the dot represents the number of DEGs. a T1 vs. T3. b T1 vs. T4. c T1 vs. T5. d T1 vs. T6. Additional file 10 Figure S8. KEGG pathways enriched in the genes differentially expressed in the developing pericarp at various time points. The enrichment factor is plotted on the x-axis; pathway names are shown on the y-axis. The color of each dot reflects the Qvalue, while the size of the dot represents the number of DEGs. a T1 vs. T3. b T1 vs. T4. c T1 vs. T5. d T1 vs. T6. Additional file 11 Table S3. The 1373 unigenes related to lipid metabolism identified in this study, annotated using Nr, Nt, Swiss-Prot, KEGG, and GO. Additional file 11 Table S3. The 1373 unigenes related to lipid metabolism identified in this study, annotated using Nr, Nt, Swiss-Prot, KEGG, and GO. Additional file 12 Table S4. The relative expression (in FPKM) of Paeonia ostii genes associated with 10 lipid metabolism pathways. Additional file 12 Table S4. The relative expression (in FPKM) of Paeonia ostii genes associated with 10 lipid metabolism pathways. Page 25/34 Page 25/34 Page 25/34 Additional file 13 Figure S9. Figures Page 26/34 Page 26/34 Figure 1 Development of fruits and seeds in Paeonia ostii. a Fruit development. b Seed and pod development. c Mature fruit. d Longitudinal sections showing seed development and embryogenesis. DAF, days after fertilization. Figure 1 Development of fruits and seeds in Paeonia ostii. a Fruit development. b Seed and pod development. c Mature fruit. d Longitudinal sections showing seed development and embryogenesis. DAF, days after fertilization. Development of fruits and seeds in Paeonia ostii. a Fruit development. b Seed and pod development. c Mature fruit. d Longitudinal sections showing seed development and embryogenesis. DAF, days after fertilization. Page 27/34 Figure 2 Fatty acid (FA) composition of Paeonia ostii fruits and seeds during d testa. c Fruit pericarp. ALA, α-linolenic acid; LA, linoleic acid; OA, oleic a acid. Figure 2 Fatty acid (FA) composition of Paeonia ostii fruits and seeds during development. a Seed kernel. b Seed testa. c Fruit pericarp. ALA, α-linolenic acid; LA, linoleic acid; OA, oleic acid; PA, palmic acid; SA, stearic acid. Figure 2 Fatty acid (FA) composition of Paeonia ostii fruits and seeds during development. a Seed kernel. b Seed testa. c Fruit pericarp. ALA, α-linolenic acid; LA, linoleic acid; OA, oleic acid; PA, palmic acid; SA, stearic acid. Page 28/34 Page 28/34 Figure 3 Figure 3 Figure 3 Figure 3 KEGG pathways enriched in the genes differentially expressed during the development of the Paeonia ostii seed kernel. a The number of genes differentially expressed between 35 and 49 DAF associated with various KEGG pathways. b The KEGG pathways overrepresented in the genes differentially expressed between 35 and 49 DAF. The color of each dot reflects the Qvalue, while the size of the dot represents the number of DEGs. c The up- and downregulated genes at 49 DAF, as compared to 35 DAF, that were associated with each KEGG pathway. d The up- and downregulated genes at 63 DAF, as compared to 35 DAF, that were associated with each KEGG pathway. Page 29/34 Figure 4 Figure 4 Figure 4 Figure 4 Figure 4 KEGG pathways enriched in the genes differentially expressed during the development of the Paeonia ostii seed testa. a The KEGG pathways overrepresented in the genes differentially expressed between 35 and 49 DAF. The color of each dot reflects the Qvalue, while the size of the dot represents the number of DEGs. b The up- and downregulated genes at 49 DAF, as compared to 35 DAF, associated with each KEGG pathway. c The KEGG pathways overrepresented in the genes differentially expressed between 35 DAF and 63 DAF. d The up- and downregulated genes at 63 DAF, as compared to 35 DAF, associated with each KEGG pathway. KEGG pathways enriched in the genes differentially expressed during the development of the Paeonia ostii seed testa. a The KEGG pathways overrepresented in the genes differentially expressed between 35 and 49 DAF. The color of each dot reflects the Qvalue, while the size of the dot represents the number of DEGs. b The up- and downregulated genes at 49 DAF, as compared to 35 DAF, associated with each KEGG pathway. c The KEGG pathways overrepresented in the genes differentially expressed between 35 DAF and 63 DAF. d The up- and downregulated genes at 63 DAF, as compared to 35 DAF, associated with each KEGG pathway. Page 30/34 Figure 5 Figure 5 Genes putatively associated with the lipid metabolism in Paeonia ostii. a Lipid metabolism in P. ostii, showing putative unigenes associated with FA biosynthesis, TAG biosynthesis, and oil formation. b Expression patterns of key genes associated with ALA biosynthesis in the P. ostii seed kernel. Figure 5 Genes putatively associated with the lipid metabolism in Paeonia ostii. a Lipid metabolism in P. ostii, showing putative unigenes associated with FA biosynthesis, TAG biosynthesis, and oil formation. b Expression patterns of key genes associated with ALA biosynthesis in the P. ostii seed kernel. Figure 5 Figure 5 Genes putatively associated with the lipid metabolism in Paeonia ostii. a Lipid metabolism in P. ostii, showing putative unigenes associated with FA biosynthesis, TAG biosynthesis, and oil formation. b Expression patterns of key genes associated with ALA biosynthesis in the P. ostii seed kernel. Page 31/34 Page 31/34 Figure 6 Expression profiles of Paeonia ostii unigenes that are also transcription factors (TFs). a The of unigenes among TF families. Figure 4 b, c, and d The relative expression levels of genes associate TFs in the developing kernel (b), testa (c), and pericarp (d). Figure 7 Quantitative real-time PCR validation of fatty acid desaturase genes. a Kernel. b Testa. c Pericarp. Actin was used as reference genes. Expression values were normalized such that the expression level at 21 DAF was set to 1. Values shown are means of three biological replicates; error bars indicate standard deviation. One-way ANOVAs were used to compare means among groups, and p value <0.005 was calculated in the bar chart are shown in Figure 7. Quantitative real-time PCR validation of fatty acid desaturase genes. a Kernel. b Testa. c Pericarp. Actin was used as reference genes. Expression values were normalized such that the expression level at 21 DAF was set to 1. Values shown are means of three biological replicates; error bars indicate standard deviation. One-way ANOVAs were used to compare means among groups, and p value <0.005 was calculated in the bar chart are shown in Figure 7. Figure 6 Expression profiles of Paeonia ostii unigenes that are also transcription factors (TFs). a The distribution of unigenes among TF families. b, c, and d The relative expression levels of genes associated with key TFs in the developing kernel (b), testa (c), and pericarp (d). Page 32/34 Page 32/34 Figure 7 Quantitative real-time PCR validation of fatty acid desaturase genes. a Kernel. b Testa. c Pericarp. Actin was used as reference genes. Expression values were normalized such that the expression level at 21 DAF was set to 1. Values shown are means of three biological replicates; error bars indicate standard deviation. One-way ANOVAs were used to compare means among groups, and p value <0.005 was calculated in the bar chart are shown in Figure 7. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Additionalfile2FigureS1.pdf Additionalfile14FigureS10.pdf Additionalfile12TableS4.xlsx Additionalfile15TableS5.xls Page 33/34 Page 33/34 Page 33/34 Additionalfile10FigureS8.pdf Additionalfile16FigureS11.pdf Additionalfile6FigureS4.pdf Additionalfile9FigureS7.pdf Additionalfile5FigureS3.pdf Additionalfile19FigureS14.pdf Additionalfile21TableS6.pdf Additionalfile18FigureS13.pdf Additionalfile20FigureS15.pdf Additionalfile17FigureS12.pdf Additionalfile13FigureS9.pdf Additionalfile8FigureS6.pdf Additionalfile3FigureS2.pdf Page 34/34
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Author Correction: IFNγ-Stat1 axis drives aging-associated loss of intestinal tissue homeostasis and regeneration
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Author Correction: IFNγ-Stat1 axis drives aging-a intestinal tissue homeostasis and regeneration Omid Omrani , Anna Krepelova, Seyed Mohammad Mahdi Rasa, Dovydas Sirvinskas, Jing Lu, Francesco Annunziata, George Garside, Seerat Bajwa, Susanne Reinhardt, Lisa Adam, Sandra Käppel, Nadia Ducano, Daniela Donna, Alessandro Ori, Salvatore Oliviero , Karl Lenhard Rudolph & Francesco Neri Correction to: Nature Communications https://doi.org/10.1038/s41467-023-41683-y, published online 30 September 2023 https://doi.org/10.1038/s41467-023-42168-8 Check for updates In this article the author name Dovydas Sirvinskas was incorrectly written as Dovydas Syr- vinskas. The original article has been corrected. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2023 Corrections&amendments Corrections&amendments Author Correction: IFNγ-Stat1 axis drives aging-associated loss of intestinal tissue homeostasis and regeneration Author Correction: IFNγ-Stat1 axis drives aging-associated loss of intestinal tissue homeostasis and regeneration nature communications (2023) 14:6302 | 1 1
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A new strategy of keratoplasty: laminating and splitting the donor cornea.
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Íîâàÿ ñòðàòåãèÿ êåðàòîïëàñòèêè: ðàññëîåíèå è ðàçäåëåíèå ðîãîâèöû äîíîðà Î.Ã. Îãàíåñÿí — ä-ð ìåä. íàóê, âåäóùèé íàó÷íûé ñîòðóäíèê îòäåëà òðàâìàòîëîãèè è ðåêîíñòðóêòèâíîé õèðóðãèè Ï.Â. Ìàêàðîâ — ä-ð ìåä. íàóê, âåäóùèé íàó÷íûé ñîòðóäíèê îòäåëà òðàâìàòîëîãèè è ðåêîíñòðóêòèâíîé õèðóðãèè À.À. Ãðäèêàíÿí — àñïèðàíò îòäåëà òðàâìàòîëîãèè è ðåêîíñòðóêòèâíîé õèðóðãèè Â.Ð. Ãåòàäàðÿí — àñïèðàíò îòäåëà òðàâìàòîëîãèè è ðåêîíñòðóêòèâíîé õèðóðãèè ФГБУ «Московский НИИ глазных болезней им. Гельмгольца» Минздрава России, 105062, Москва, ул. Садовая-Черногрязская, д. 14/19 Î.Ã. Îãàíåñÿí — ä-ð ìåä. íàóê, âåäóùèé íàó÷íûé ñîòðóäíèê îòäåëà òðàâìàòîëîãèè è ðåêîíñòðóêòèâíîé õèðóðãèè Ï.Â. Ìàêàðîâ — ä-ð ìåä. íàóê, âåäóùèé íàó÷íûé ñîòðóäíèê îòäåëà òðàâìàòîëîãèè è ðåêîíñòðóêòèâíîé õèðóðãèè À.À. Ãðäèêàíÿí — àñïèðàíò îòäåëà òðàâìàòîëîãèè è ðåêîíñòðóêòèâíîé õèðóðãèè Â.Ð. Ãåòàäàðÿí — àñïèðàíò îòäåëà òðàâìàòîëîãèè è ðåêîíñòðóêòèâíîé õèðóðãèè ФГБУ «Московский НИИ глазных болезней им. Гельмгольца» Минздрава России, 105062, Москва, ул. Садовая-Черногрязская, д. 14/19 Цель работы — описать первый случай применения одной донорской роговицы для 5 последовательных транс- плантаций в течение одного дня у пациентов с эндотелиальной дистрофией и кератоконусом. Материал и методы. Полностью отслоенная от стромы донора десцеметова мембрана (ДМ) с эндотелием иссекалась циркулярным и далее прямым высекателем роговицы донора с эндотелиальной стороны, с формированием 4 частичных десцеме- тотрансплантатов треугольной формы (1/4 десцеметотрансплантата). Строма роговицы донора иссекалась циркулярным высекателем заданного диаметра. Одному пациенту с кератоконусом была проведена глубокая перед- няя послойная кератопластика (DALK) по методике Big Bubble. Затем каждый из ¼ десцеметотрансплантатов был последовательно пересажен по методике трансплантации десцеметовой мембраны с эндотелием (DMEK) 4 пациентам с дистрофией Фукса и артифакией. Результаты. Во время и после хирургических операций не наблюда- лось каких-либо осложнений, требующих дополнительного вмешательства. Через 6 мес после DALK максимально корригируемая острота зрения (МКОЗ) составила 0,66, а после 4 операций ¼ DMEK МКОЗ составила 0,95 ± 0,10 (от 0,8 до 1,0). Плотность эндотелиальных клеток (ПЭК) через 6 мес составила 2839 и 1680 ± 254 клеток/мм2 (от 1492 до 2039 клеток/мм2) соответственно. Заключение. Одна донорская роговица может быть использована для трансплантации минимум 5 пациентам. Несмотря на то, что выполнение DALK и нескольких операций по методике ¼ DMEK в течение одного дня технически сложно, такой подход может способствовать кардиналь- ному сокращению дефицита донорской ткани. Подобный подход требует строгого подбора пациентов и большого хирургического опыта. ючевые слова: роговица донора, кератопластика, трансплантация роговицы, транспл цеметовой мембраны, DMEK, Q-DMEK, ¼ DMEK. Для цитирования: Оганесян О.Г., Макаров П.В., Грдиканян А.А., Гетадарян В.Р. Новая стратегия керато- пластики: расслоение и разделение роговицы донора. Российский офтальмологический журнал. 2018; 11 (3):11-8. Клинические исследования Клинические исследования 11 © О.Г. Оганесян, П.В. Макаров, А.А. Грдиканян, В.Р. Гетадарян ФГБУ «Московский НИИ глазных болезней им. Гельмгольца» Минздрава России Для цитирования: Оганесян О.Г., Макаров П.В., Грдиканян А.А., Гетадарян В.Р. Новая стратегия керато- пластики: расслоение и разделение роговицы донора. Российский офтальмологический журнал. 2018; 11 (3):11-8. doi: 10.21516/2072-0076-2018-11-3-11-18 Î.Ã. Îãàíåñÿí — ä-ð ìåä. íàóê, âåäóùèé íàó÷íûé ñîòðóäíèê îòäåëà òðàâìàòîëîãèè è ðåêîíñòðóêòèâíîé õèðóðãèè Ï.Â. Ìàêàðîâ — ä-ð ìåä. íàóê, âåäóùèé íàó÷íûé ñîòðóäíèê îòäåëà òðàâìàòîëîãèè è ðåêîíñòðóêòèâíîé õèðóðãèè À.À. Ãðäèêàíÿí — àñïèðàíò îòäåëà òðàâìàòîëîãèè è ðåêîíñòðóêòèâíîé õèðóðãèè Â.Ð. Ãåòàäàðÿí — àñïèðàíò îòäåëà òðàâìàòîëîãèè è ðåêîíñòðóêòèâíîé õèðóðãèè ФГБУ «Московский НИИ глазных болезней им. Гельмгольца» Минздрава России, 105062, Москва, ул. Садовая-Черногрязская, д. 14/19 Íîâàÿ ñòðàòåãèÿ êåðàòîïëàñòèêè: ðàññëîåíèå è ðàçäåëåíèå ðîãîâèöû äîíîðà doi: 10.21516/2072-0076-2018-11-3-11-18 В последние годы различные методы передней и задней послойной кератопластики стали первой операцией выбора при лечении патологии рого- вицы. Благодаря быстрой реабилитации, высоким функциональным результатам и закрытому харак- теру вмешательства, отмечается ежегодный рост числа выполняемых эндотелиальных кератопластик с десцеметорексисом (DSEK) и трансплантаций десцеметовой мембраны с эндотелием (DMEK). Все чаще эти операции выполняются на более ранних стадиях развития заболеваний. По мере увеличения продолжительности жизни населения растет также и число пациентов, нуждающихся в пересадке рогови- цы. Параллельно увеличивается возраст доноров, а количество интактных донорских роговиц при этом уменьшается по причине увеличивающегося количе- ства хирургических вмешательств (факоэмульсифика- ция, имплантация интраокулярных линз, кросслин- кинг, имплантация интрастромальных сегментов, колец и инлаев, рефракционная хирургия и др.). Все эти факторы, очевидно, будут усугублять имею- щийся дефицит донорской ткани, особенно интакт- ной. По некоторым данным, в настоящее время при- мерно 2 % роговиц, поступающих в банк тканей, ранее перенесли те или иные рефракционные операции [1]. скопию глаза на щелевой лампе (Opton 30 SL-M, Германия), оценку состояния заднего сегмента глаза, кератоанализирование (Galilei 6, Ziemer Ophthalmic Systems AG, Швейцария). Аналогичные обследова- ния, а также измерение ПЭК (Confoscan 4, Nidek Co. Ltd., Япония) проводились во всех 5 случаях через 3 и 6 мес после операции. Минимальный срок на- блюдения всех пациентов составил 6 мес. скопию глаза на щелевой лампе (Opton 30 SL-M, Германия), оценку состояния заднего сегмента глаза, кератоанализирование (Galilei 6, Ziemer Ophthalmic Systems AG, Швейцария). Аналогичные обследова- ния, а также измерение ПЭК (Confoscan 4, Nidek Co. Ltd., Япония) проводились во всех 5 случаях через 3 и 6 мес после операции. Минимальный срок на- блюдения всех пациентов составил 6 мес. Операция DALK проводилась по очередности первой и под общей анестезией. Затем поочеред- но выполнялись операции ¼ DMЕK под местной инстилляционной анестезией. Формирование всех трансплантатов и все хирургические вмешательства выполнены одним хирургом. Все пациенты давали добровольное информиро- ванное согласие. Исследования проводились в соот- ветствии с положениями Хельсинкской декларации. Подготовка трансплантатов. Для выполнения 5 операций использована одна роговица донора 48 лет, мужского пола, с ПЭК = 2774 клеток/мм2. На сегодняшний день во многих странах мира наблюдается нехватка донорского материала. В раз- вивающихся странах ежегодная потребность в до- норской ткани достигает 300 тыс. роговиц, при этом доступно только 15 тыс. роговиц [2, 3]. Отслаивание десцеметовой мембраны (ДМ) с эндотелием выполняли по ранее описанной методи- ке [5]. Вкратце техника операции была следующей. Новая стратегия кератопластики: расслоение и разделение роговицы донора 12 Íîâàÿ ñòðàòåãèÿ êåðàòîïëàñòèêè: ðàññëîåíèå è ðàçäåëåíèå ðîãîâèöû äîíîðà После аспирационной фиксации корнеосклераль- ного диска донора эндотелием вверх ДМ с пери- ферической тканью, включающей трабекулярную сеть, полностью отслаивалась от задней поверх- ности стромы (рис. 1, А). После окрашивания ДМ 0,06 % раствором трипанового синего (Vision Blue, DORC International, Нидерланды) последняя поме- щалась на мягкую контактную линзу, в свою очередь расположенную в высекателе донорской роговицы (Barron vacuum donor Punch; Katena Products Inc, США) (рис. 1, Б). По данным Федеральной службы государствен- ной статистики, в 2015 г. в России было проведено 3453 кератопластики, в том числе 155 операций у детей (сведения с официального сайта Федеральной службы государственной статистики РФ: http://www. gks.ru/). Очевидно, что для увеличения количества и доступности донорской ткани требуется системный подход, поддержка государства и общества, внуши- тельные инвестиции, время и т. д. На наш взгляд, наиболее быстрым и очевидным способом увеличе- ния количества трансплантаций является рациональ- ное и экономное использование донорской ткани, внедрение современных методов кератопластики, а также индивидуальный подход к хирургии роговицы в каждом клиническом случае [4]. Циркулярным стандартным высекателем Barron диаметром 9,5 мм вырезали ДМ с эндотелиальной стороны вместе с контактной линзой. Не перемещая ДМ с подлежащей контактной линзой с помощью разработанного и изготовленного на заказ высека- теля (заявка на полезную модель, регистрационный № 2017137958) (рис. 1, В, 2) разрезали ДМ с эндо- телиальной стороны вместе с подлежащей контакт- ной линзой на 4 равные четверти (рис. 1, Г—Е). Все 4 части ДМ помещали во флакон, где они самопро- извольно сворачивали в рулон эндотелием наружу и свободно плавали в консерванте. ЦЕЛЬ работы — описать первый случай при- менения одной донорской роговицы для 5 по- следовательных трансплантаций в течение одного операционного дня у пациентов с эндотелиальной дистрофией и кератоконусом. Ðîññèéñêèé îôòàëüìîëîãè÷åñêèé æóðíàë, 2018; 3: 11-18 МАТЕРИАЛ И МЕТОДЫ А Г Б Д В Е А Г Б Д А Г Б Д В Е Д Е Рис. 1. Этапы формирования частичных (1/4) десцеметотрансплантатов. А — отслоение десцеметовой мембраны с каймой трабеку- лярной сети от корнеосклерального диска донора. Б — отслоенная десцеметова мембрана с каймой трабекулярной сети расположена на контактной линзе в высекателе Barron. В — десцеметова мембрана с подлежащей контактной линзой разделяется на 2 половины разработанным высекателем (вид сверху) с полукруглым лезвием (черные стрелки). Г — после 90-градусного поворота режущего блока каждая половина десцеметовой мембраны (1/2) затем снова разделена на 2 половины тем же высекателем. После двойного взаимно перпендикулярного разреза получены 4 равные части контактной линзы (Д) и 4 десцеметотрансплантата для ¼ DMEK (Е). Fig. 1. Images of surgical steps of Q-grafts preparation. А — DM stripping with the adjacent trabecular meshwork from the corneoscleral button. Б — isolated DM with adjacent trabecular meshwork was placed onto contact lens which was located on the cutting block of Barron punch. В — the DM with underlying contact lens was first divided into 2 equal halves with a custom made single used punch for linear cutting of DM (view from above) with semicircular blade (black arrows). Г — after a 90-degree turn of the cutting block, each half of DM was then again divided into 2 equal halves with the same custom-made punch. After a double cut 4 pieces of contact lens (Д) and 4 pieces of Q-grafts of DM for Q-DMEK were obtained (Е). g. 1. Images of surgical steps of Q grafts preparation. А DM stripping with the adjacent trabecular meshwork from the corneoscleral utton. Б — isolated DM with adjacent trabecular meshwork was placed onto contact lens which was located on the cutting block of Barron unch. В — the DM with underlying contact lens was first divided into 2 equal halves with a custom made single used punch for linear cutting of M (view from above) with semicircular blade (black arrows). Г — after a 90-degree turn of the cutting block, each half of DM was then again vided into 2 equal halves with the same custom-made punch. After a double cut 4 pieces of contact lens (Д) and 4 pieces of Q-grafts of DM or Q-DMEK were obtained (Е) Рис. 2. Разработанный высекатель для линейного прорезывания десцеметовой мем- браны. A — продольный вид высекателя с полукруглым лезвием (черные стрелки). Ðîññèéñêèé îôòàëüìîëîãè÷åñêèé æóðíàë, 2018; 3: 11-18 МАТЕРИАЛ И МЕТОДЫ Б — поперечный вид высекателя с полукруглым лезвием (черные стрелки). Fig. 2. Custom-made punch for linear cutting of DM. А — longitudinal view of punch with semicircular blade (black arrows). Б — transverse view of punch with semicircular blade (black arrows). А Б 30G формировали канал длиной 2–3 мм, в который вводили каню- лю (Sarnicola, ASICO, Inc, США) для DALK диаметром 27G, при- соединенную к шприцу объемом 5 мл. Канюлю проводили к центру роговицы максимально близко к ДМ и, надавливая на поршень шприца, производили пневмати- зацию роговицы до формирования «большого пузыря» диаметром меньше диаметра кератэктомии. Не извлекая канюли, выполняли парацентез и частично опорож- няли переднюю камеру глаза. После этого продолжали введение воздуха в роговицу до достижения диаметра «большого пузыря», диаметра кератэктомии (рис. 3, А). Лезвием осуществляли вскрытие «большого пузыря» и вводили в его вный вискоэластик (Healon GV, AMO, чная часть стромы удалялась тупоко- охирургическими ножницами после ием крест-накрест.Поверхность ДМ мывалась от вискоэластика (рис. 3, Б). 30G формировали канал длиной 2–3 мм, в который вводили каню- лю (Sarnicola, ASICO, Inc, США) для DALK диаметром 27G, при- соединенную к шприцу объемом 5 мл. Канюлю проводили к центру роговицы максимально близко к ДМ и, надавливая на поршень шприца, производили пневмати- зацию роговицы до формирования «большого пузыря» диаметром меньше диаметра кератэктомии. Не извлекая канюли, выполняли парацентез и частично опорож- няли переднюю камеру глаза. После этого продолжали введение воздуха в роговицу до достижения диаметра «большого пузыря», диаметра кератэктомии (рис. 3, А). Лезвием осуществляли вскрытие «большого пузыря» и вводили в его полость когезивный вискоэластик (Healon GV, AMO, США). Остаточная часть стромы удалялась тупоко- нечными микрохирургическими ножницами после ее разреза лезвием крест-накрест.Поверхность ДМ тщательно промывалась от вискоэластика (рис. 3, Б). десцеметовой мем- ерные стрелки). Б — ки). al view of punch with h semicircular blade Б А Б Рис. 2. Разработанный высекатель для линейного прорезывания десцеметовой мем- браны. A — продольный вид высекателя с полукруглым лезвием (черные стрелки). Б — поперечный вид высекателя с полукруглым лезвием (черные стрелки). Fig. 2. Custom-made punch for linear cutting of DM. А — longitudinal view of punch with semicircular blade (black arrows). Б — transverse view of punch with semicircular blade (black arrows). роговицы реципиента вакуумным трепаном Hes- sburg — Barron (Katena Products Inc, США) диаметром 9 мм делали насечку роговицы глубиной до 80 % от толщины стромы роговицы. Новая стратегия кератопластики: расслоение и разделение роговицы донора МАТЕРИАЛ И МЕТОДЫ В течение одного операционного дня мы по- следовательно выполнили глубокую переднюю послойную кератопластику (DALK) пациенту с ке- ратоконусом (КК) и 4 операции методикой ¼ DMEK 4 пациентам с дистрофией Фукса и артифакией. В послеоперационном периоде фиксировалось коли- чество и характер осложнений, показатели макси- мально корригированной остроты зрения (МКОЗ) и плотности эндотелиальных клеток (ПЭК) спустя 6 мес после операции. Оставшийся корнеосклеральный диск без ДМ после маркировки центра и 4 основных меридианов переносили в пустой контейнер для донорской рого- вицы и помещали на хранение в холодильную камеру. На случай выбраковки донорской ткани в про- цессе формирования трансплантатов либо интраопе- рационных осложнений резервная роговичная ткань имелась в наличии. Глубокая передняя послойная кератопластика. Операцию DALK выполняли методикой «большого пузыря», описанной M. Anwar и K. Teichmann [6]. После маркировки центра и 4 основных меридианов Предоперационное обследование пациентов включало определение МКОЗ на проекторе опто- типов (SZP 350, Zeiss, Германия) и внутриглазного давления (iCare, Finland Oy, Финляндия), биомикро- Ðîññèéñêèé îôòàëüìîëîãè÷åñêèé æóðíàë, 2018; 3: 11-18 Рис. 1. Этапы формирования частичных (1/4) десцеметотрансплантатов. А — отслоение десцеметовой мембраны с каймой трабеку- лярной сети от корнеосклерального диска донора. Б — отслоенная десцеметова мембрана с каймой трабекулярной сети расположена на контактной линзе в высекателе Barron. В — десцеметова мембрана с подлежащей контактной линзой разделяется на 2 половины разработанным высекателем (вид сверху) с полукруглым лезвием (черные стрелки). Г — после 90-градусного поворота режущего блока каждая половина десцеметовой мембраны (1/2) затем снова разделена на 2 половины тем же высекателем. После двойного взаимно перпендикулярного разреза получены 4 равные части контактной линзы (Д) и 4 десцеметотрансплантата для ¼ DMEK (Е). Fig. 1. Images of surgical steps of Q-grafts preparation. А — DM stripping with the adjacent trabecular meshwork from the corneoscleral button. Б — isolated DM with adjacent trabecular meshwork was placed onto contact lens which was located on the cutting block of Barron punch. В — the DM with underlying contact lens was first divided into 2 equal halves with a custom made single used punch for linear cutting of DM (view from above) with semicircular blade (black arrows). Г — after a 90-degree turn of the cutting block, each half of DM was then again divided into 2 equal halves with the same custom-made punch. After a double cut 4 pieces of contact lens (Д) and 4 pieces of Q-grafts of DM for Q-DMEK were obtained (Е). Новая стратегия кератопластики: расслоение и разделение роговицы донора 14 РЕЗУЛЬТАТЫ Возраст пациента, перенесшего операцию DALK, составлял 52 года. МКОЗ до операции со- ставляла 0,16, а через 6 мес после операции она улучшилась до 0,66. На момент последнего осмотра у пациента не наблюдалось никаких осложнений, трансплантат сохранял прозрачность. Конфокальная микроскопия показала ПЭК = 2839 клеток/мм2. Частичная трансплантация десцеметовой мембра- ны с эндотелием (1/4 DMEK). Техника ¼ DMEK во всех 4 случаях была идентичной и в целом аналогичной тех- нике стандартной DMEK, описанной нами ранее [7]. После выполнения единственного парацен- теза в меридиане 10 ч выполняли десцеметорексис (7–8 мм) под воздухом при помощи обратного крюч- ка Sinskey (DORC International, Нидерланды). Лим- бальный тоннельный разрез шириной 1,6 мм выпол- няли в меридиане 12 ч с помощью кератома (MANI inc., Япония). Частичные десцеметотрансплантаты окрашивали 0,06 % раствором трипанового синего и аспирировали в браунюлю диаметром 18G (Apexmed International B.V., Нидерланды), прикрепленную к шприцу объемом 2 мл и заполненную раствором BSS («Способ трансплантации десцеметовой мембра- ны — микроинвазивная десцеметопластика», патент РФ № 2394532 от 20.07.2010). Путем ирригации трансплантат вводили в переднюю камеру глаза реципиента через сформированный разрез в мери- диане 12 ч. Манипулируя ab externo, трансплантат центрировали, раскрывали рулон эндотелием вниз, и переднюю камеру полностью заполняли возду- хом, который оставляли не менее чем на 2 ч. Через 2 ч после операции часть воздуха выпускалась через парацентез под биомикроскопом. Средний возраст пациентов, перенесших опе- рации ¼DMEK, составлял 69,0 ± 1,1 года (от 52 до 70 лет). Средняя МКОЗ перед операцией составляла 0,35 ± 0,10, а через 6 мес после ¼ DMEK повыси- лась до 0,95 ± 0,10. На момент последнего осмотра все роговицы сохраняли прозрачность. Через 6 мес после операции средняя ПЭК с дооперационных 2774 клеток/мм2 уменьшилась до 1680 ± 254 клеток/мм2 (от 1492 до 2039 клеток/мм2). Частичное неприлегание десцеметотран- сплантата имело место в 2 из 4 случаев ¼ DMEK. Тем не менее ни в одном случае не потребовалось повторного введения воздуха в переднюю камеру. Обследование под щелевым биомикроскопом через 6 мес после операции показало полное прилегание всех частичных десцеметотрансплантатов. Других осложнений, таких как реакция отторжения, несо- стоятельность эндотелия трансплантата или синдром Urrets-Zavalia, за весь период наблюдения не зафик- сировано (рис. 4, 5). Рис. 3. Этапы глубокой передней послойной кератопластики. A — формирование big bubble, равного диаметру трепанационного от- верстия. Б — обнаженная десцеметова мембрана реципиента после полной стромэктомии. В — строма донора фиксирована в ложе 8 узловыми швами. Fig. 3. Images of surgical steps of DALK. МАТЕРИАЛ И МЕТОДЫ После послойной ке- ратэктомии с помощью одноразовой иглы диаметром роговицы реципиента вакуумным трепаном Hes- sburg — Barron (Katena Products Inc, США) диаметром 9 мм делали насечку роговицы глубиной до 80 % от толщины стромы роговицы. После послойной ке- ратэктомии с помощью одноразовой иглы диаметром 13 Ðîññèéñêèé îôòàëüìîëîãè÷åñêèé æóðíàë, 2018; 3: 11-18 Корнеосклеральный диск донора без ДМ распо- лагали в высекателе Barron и вырезали трансплантат диаметром 9 мм, который фиксировали в ложе ро- говицы реципиента 8 узловыми (рис. 3, В) и одним непрерывным нейлоновым швом 10/0. Во время и после хирургических операций не отмечалось развития каких-либо осложнений, тре- бовавших вмешательств. После операции во всех слу- чаях назначались глазные капли 0,1 % дексаметазона каждые 4 ч, глазные капли 0,5 % моксифлоксацина гидрохлорида 3 раза в сутки и заменители слезы без консервантов каждые 4 ч. Антибиотикотерапию прекращали через 2 нед после операции, а кратность инстилляций остальных медикаментов постепенно сокращалась в течение 6 мес. В послеоперационном периоде медикаментоз- ная терапия включала глазные капли 0,1 % дексаме- тазона каждые 4 ч, глазные капли 0,5 % моксифлокса- цина гидрохлорида 3 раза в сутки и заменители слезы без консервантов каждые 4 ч. Антибиотикотерапию прекращали через 3 нед после операции, кратность инстилляций остальных препаратов постепенно снижалась в течение 6 мес. Ðîññèéñêèé îôòàëüìîëîãè÷åñêèé æóðíàë, 2018; 3: 11-18 Рис. 3. Этапы глубокой передней послойной кератопластики. A — формирование big bubble, равного диаметру трепанационного от- верстия. Б — обнаженная десцеметова мембрана реципиента после полной стромэктомии. В — строма донора фиксирована в ложе 8 узловыми швами. Fig. 3. Images of surgical steps of DALK. A — formation of a “big bubble” equal to the trephination diameter. Б — the stroma was removed and DM of the host is denuded В — the donor button was sutured in place using 8 interrupted 10-0 monofilament nylon sutures РЕЗУЛЬТАТЫ A — formation of a “big bubble” equal to the trephination diameter. Б — the stroma was removed and DM of the host is denuded. В — the donor button was sutured in place using 8 interrupted 10-0 monofilament nylon sutures. А Б В А Б В А Б Б В А Рис. 3. Этапы глубокой передней послойной кератопластики. A — формирование big bubble, равного диаметру трепанационного от- верстия. Б — обнаженная десцеметова мембрана реципиента после полной стромэктомии. В — строма донора фиксирована в ложе 8 узловыми швами. Fig 3 Images of surgical steps of DALK A formation of a “big bubble” equal to the trephination diameter Б the stroma was removed and 8 узловыми швами. Fig. 3. Images of surgical steps of DALK. A — formation of a “big bubble” equal to the trephination diameter. Б — the stroma was removed and DM of the host is denuded. В — the donor button was sutured in place using 8 interrupted 10-0 monofilament nylon sutures. узловыми швами. ig. 3. Images of surgical steps of DALK. A — formation of a “big bubble” equal to the trephination diameter. Б — M of the host is denuded. В — the donor button was sutured in place using 8 interrupted 10-0 monofilament n Ðîññèéñêèé îôòàëüìîëîãè÷åñêèé æóðíàë, 2018; 3: 11-18 Рис. 4. Клинический случай ¼ DMEK № 1. Биомикроскопическая картина и карта пахи- метрии до операции (A, Г), через 3 дня (Б, Д) и через 6 мес (В, Е) после ¼ DMEK. Карты пахиметрии, а также изображения щелевого биомикроскопа демонстрируют постепенное истончение роговицы. Fig. 4. Q-DMEK case № 1. Slit-lamp images and pachymetry maps preoperatively (A, Г), at 3 days (Б, Д), and at 6 months (В, Е) after Q-DMEK. Pachymetry maps, as well as slit-lamp images show gradual cornea thinning. А Г Б Д В Е Ðîññèéñêèé îôòàëüìîëîãè÷åñêèé æóðíàë, 2018; 3: 11-18 ОБСУЖДЕНИЕ А ОБСУЖДЕНИЕ Первая успешная пересад- ка роговицы была проведена Eduard Zirm в 1905 г. Он так- же первым применил принцип рационального использования донорской ткани, выполнив 2 частичные сквозные кератопла- стики из одной роговицы доно- ра [8]. Есть несколько публика- ций, посвященных использова- нию одной донорской рогови- цы для выполнения нескольких трансплантаций. R. Vajpayee и соавт. [4] описывают примене- ние одного корнеосклерального диска донора для лечения 3 па- циентов, которым выполнили DALK, DSEK и пересадку лим- бальных стволовых клеток. J. Lie и соавт. [9] разработали методику одновременного выполнения 2 операций из одной роговицы донора. Существуют и другие публикации, касающиеся рацио- нального использования донор- ской ткани, указывающие на то, что значительный дефицит до- норских роговиц во многих частях мира является по-настоящему большой проблемой [10–15]. Рас- слоение и разделение донор- ской ткани может являться од- ним из путей сокращения дефицита донорс- кой ткани. Рис. 4. Клинический метрии до операции пахиметрии, а также и истончение роговицы Fig. 4. Q-DMEK case 3 days (Б, Д), and at 6 images show gradual Первая успешная пересад- ка роговицы была проведена Eduard Zirm в 1905 г. Он так- же первым применил принцип рационального использования донорской ткани, выполнив 2 частичные сквозные кератопла- стики из одной роговицы доно- ра [8]. Есть несколько публика- ций, посвященных использова- нию одной донорской рогови- цы для выполнения нескольких трансплантаций. R. Vajpayee и соавт. [4] описывают примене- ние одного корнеосклерального диска донора для лечения 3 па- циентов, которым выполнили DALK, DSEK и пересадку лим- бальных стволовых клеток. J. Lie и соавт. [9] разработали методику одновременного выполнения 2 операций из одной роговицы донора. Существуют и другие публикации, касающиеся рацио- нального использования донор- ской ткани, указывающие на то, что значительный дефицит до- норских роговиц во многих частях мира является по-настоящему большой проблемой [10–15]. Рас- слоение и разделение донор- ской ткани может являться од- ним из путей сокращения деф кой ткани. А Б В Г Д Е Д Е Г Е Д Рис. 4. Клинический случай ¼ DMEK № 1. Биомикроскопическая картина и карта пахи- метрии до операции (A, Г), через 3 дня (Б, Д) и через 6 мес (В, Е) после ¼ DMEK. Карты пахиметрии, а также изображения щелевого биомикроскопа демонстрируют постепенное истончение роговицы. Fig. 4. Q-DMEK case № 1. Slit-lamp images and pachymetry maps preoperatively (A, Г), at 3 days (Б, Д), and at 6 months (В, Е) after Q-DMEK. Pachymetry maps, as well as slit-lamp images show gradual cornea thinning. Рис. 4. Новая стратегия кератопластики: расслоение и разделение роговицы донора Новая стратегия кератопластики: расслоение и разделение роговицы донора 16 ОБСУЖДЕНИЕ Клинический случай ¼ DMEK № 1. Биомикроскопическая картина и карта пахи- метрии до операции (A, Г), через 3 дня (Б, Д) и через 6 мес (В, Е) после ¼ DMEK. Карты пахиметрии, а также изображения щелевого биомикроскопа демонстрируют постепенное истончение роговицы. Fig. 4. Q-DMEK case № 1. Slit-lamp images and pachymetry maps preoperatively (A, Г), at 3 days (Б, Д), and at 6 months (В, Е) after Q-DMEK. Pachymetry maps, as well as slit-lamp images show gradual cornea thinning. МКОЗ и ПЭК, как и стандартная операция DMEK. Стратегия использования одной донорской рогови- цы для проведения нескольких пересадок, впервые реализованная E. Zirm, открывает возможности значительного увеличения количества выполняемых трансплантаций имеющимися в распоряжении объ- емами донорских роговиц. Теоретически можно еще увеличить количество (до 6) проводимых трансплан- таций при условии использования донорской лим- бальной ткани для аллолимбальной трансплантации. Разделение на части донорской ткани активно применяется в других областях клинической транс- плантологии. Например, разделение печени умень- шает потребность в ней примерно на 40 % [16, 17]. Ежегодная потребность развивающихся стран в до- норской роговице составляет примерно 300 тыс., тогда как доступно только 15 тыс. трансплантатов, и почти половина из них не подходит для пересадки [4]. Мы считаем, что следует пересмотреть критерии оценки пригодности донорской ткани для пересад- ки, поскольку на сегодняшний день можно успешно пересаживать как донорскую роговицу целиком, так и отдельные слои, которые ранее выбраковывались [1, 18–25]. Применение ламеллярной кератопла- стики в ургентной хирургии может являться про- явлением рационального использования донорских тканей [26–30]. В рамках настоящего исследования мы опи- сываем первый случай получения 5 трансплантатов из одной донорской роговицы. Метод ¼ DMEK со- вмещает в себе преимущества стандартной DMEK и имплантации ДМ с эндотелием (DMET). Основным преимуществом DMEK по сравнению с другими методами является быстрое восстановление зрения. Основным преимуществом ¼ DMEK, по сравнению с обычным DMEK и ½ DMEK, является максимально эффективное использование донорской роговицы. Кроме того, ¼ DMEK позволяет сохранить больше клеток реципиента и в то же время создает благопри- ятные условия для эндотелизации, поскольку дес- цеметорексис при ¼ DMEK всегда больше размера частичного десцеметотрансплантата. Выполнение Представленные клинические случаи демон- стрируют возможность последовательного проведе- ния 5 операций: DALK и 4 ¼ DMEK в течение одного дня — с использованием одной роговицы донора. Обследование через 6 мес показало, что частичный DMEK позволил достичь таких же показателей 15 Ðîññèéñêèé îôòàëüìîëîãè÷åñêèé æóðíàë, 2018; 3: 11-18 Новая стратегия кератопластики: расслоение и разделение роговицы донора А Г Б Д В Е Рис. 5. ОБСУЖДЕНИЕ Клинический случай ¼ DMEK № 2 (A, Б), № 3 (В, Г) и № 4 (Д, Е). Биомикроскопи- ческая картина до операции (A, В, Д) и спустя 6 мес (Б, Г, Е) после ¼ DMEK. Биомикро- скопическая картина свидетельствует об отсутствии участков неприлегания частичного десцеметотрансплантата и об уменьшении толщины роговицы после операции ¼ DMEK. Fig. 5. Q-DMEK cases № 2 (A, Б), № 3 (В, Г) and № 4 (Д, Е). Slit-lamp images preoperatively (A, В, Д) and at 6 months (Б, Г, Е) after Q-DMEK. Slit-lamp images show absence of any detachment of Q-graft and decreasing the thickness of cornea after surgery. А Б В А Б А Б В В донорского материала, професси- онализма хирургической бригады. Тем не менее, несмотря на слож- ности такой стратегии, ее преиму- щества очевидны. Рациональное использование донорской ткани является одним из важнейших очевидных и доступных способов быстрого увеличения количества пересадок и, следовательно, выле- ченных пациентов. Такой подход с применением расслоения и раз- деления донорской роговицы спо- собен значимо снизить дефицит донорских тканей. Мы считаем, что предварительная подготовка и загрузка частичных десцеме- тотрансплантатов сотрудниками глазных банков может существен- но облегчить методику ¼ DMEK. донорского материала, професси- онализма хирургической бригады. Тем не менее, несмотря на слож- ности такой стратегии, ее преиму- щества очевидны. Рациональное использование донорской ткани является одним из важнейших очевидных и доступных способов быстрого увеличения количества пересадок и, следовательно, выле- ченных пациентов. Такой подход с применением расслоения и раз- деления донорской роговицы спо- собен значимо снизить дефицит донорских тканей. Мы считаем, что предварительная подготовка и загрузка частичных десцеме- тотрансплантатов сотрудниками глазных банков может существен- но облегчить методику ¼ DMEK. В А Б Г Д Е Г Д Е Рис. 5. Клинический случай ¼ DMEK № 2 (A, Б), № 3 (В, Г) и № 4 (Д, Е). Биомикроскопи- ческая картина до операции (A, В, Д) и спустя 6 мес (Б, Г, Е) после ¼ DMEK. Биомикро- скопическая картина свидетельствует об отсутствии участков неприлегания частичного десцеметотрансплантата и об уменьшении толщины роговицы после операции ¼ DMEK. Fig. 5. Q-DMEK cases № 2 (A, Б), № 3 (В, Г) and № 4 (Д, Е). Slit-lamp images preoperatively (A, В, Д) and at 6 months (Б, Г, Е) after Q-DMEK. Slit-lamp images show absence of any detachment of Q-graft and decreasing the thickness of cornea after surgery. Ðîññèéñêèé îôòàëüìîëîãè÷åñêèé æóðíàë, 2018; 3: 11-18 Е Г Д Д Рис. 5. Клинический случай ¼ DMEK № 2 (A, Б), № 3 (В, Г) и № 4 (Д, Е). Биомикроскопи- ческая картина до операции (A, В, Д) и спустя 6 мес (Б, Г, Е) после ¼ DMEK. Биомикро- скопическая картина свидетельствует об отсутствии участков неприлегания частичного десцеметотрансплантата и об уменьшении толщины роговицы после операции ¼ DMEK. Fig. 5. Q-DMEK cases № 2 (A, Б), № 3 (В, Г) and № 4 (Д, Е). Slit-lamp images preoperatively (A, В, Д) and at 6 months (Б, Г, Е) after Q-DMEK. Slit-lamp images show absence of any detachment of Q-graft and decreasing the thickness of cornea after surgery. Конфликт интересов: отсут- ствует. ¼ DMEK является вполне оправданным, так как патологические изменения при дистрофии Фукса в основном затрагивают ограниченную область, расположенную, как правило, в центре роговицы. Для проведения ¼ DMEK необходим строгий подбор пациентов исходя из потенциала остроты зрения, глубины передней камеры, площади и степени эн- дотелиальной дистрофии. Среднее значение МКОЗ всех пациентов с дистрофией Фукса и артифакией перед ¼ DMEK составляло 0,35 ± 0,10 (от 0,2 до 0,4), что указывает на наличие начальных и незначитель- ных изменений в структуре роговицы. Прозрачность финансовой деятельности: никто из авторов не имеет финансовой заинтересованности в представленных материалах или методах. ЗАКЛЮЧЕНИЕ Представленные клиниче- ские случаи показывают, что одна роговица донора может быть успешно пересажена как мини- мум 5 пациентам. Несмотря на то, что последовательное выполне- ние DALK и 4 ¼ DMEK в течение одного операционного дня пред- ставляет технические и логисти- ческие сложности, тем не менее подобная стратегия способствует кардинальному сокращению де- фицита роговичной ткани. При этом необходим строгий выбор пациентов для ¼ DMEK. Прозрачность финансовой деятельности: никто из авторов не имеет финансовой заинтересованности в представленных материалах или методах. Eine erfolgreiche totale keratoplastik. Arch. 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Sci. 2014; 91: 59–62. doi: 10.1097/OPX.0000000000000162 6. Anwar M., Teichmann K.D. Deep lamellar keratoplasty: surgical techniques for anterior lamellar keratoplasty with and without baring of Descemet’s membrane. Cornea. 2002; 21: 374–83 21. Huang T., Wang Y., Hu A., et al. Use of pediatric donor tissue in Descemet’s stripping endothelial keratoplasty. Br. J. Ophthalmol. 2009; 93: 1625–8. doi: 10.1136/bjo.2009.158477 7. Оганесян О.Г., Нероев В.В., Гундорова Р.А., Воробьева М.А. Первый опыт трансплантации десцеметовой мембраны. Офтальмология. 2008; 5 (4): 18–22 Oganesyan O.G., Neroev V.V., Gundorova R.A., Vorobjeva M.A. The first experience of descemet's membrane transplantation. Oftal’mologija. 2008. 5: 18–22 (in Russian) 22. Kim P., Yeung S.N., Lichtinger A., et al. Descemet stripping automated endothelial keratoplasty using infant donor tissue. Cornea. 2012; 31: 52–4. doi: 10.1097/ICO.0b013e31821eeac0 23. Sun Y.X., Hao Y.S., Hong J. Descemet membrane stripping endothelial keratoplasty with neonate donors in two cases. Br. J. Ophthalmol. 2009; 93: 1692–9. http://dx.doi.org/10.1136/ bjo.2009.158188 8. Zirm E. Ðîññèéñêèé îôòàëüìîëîãè÷åñêèé æóðíàë, 2018; 3: 11-18 Новая стратегия кератопластики: расслоение и разделение роговицы донора Прозрачность финансовой деятельности: никто из авторов не имеет финансовой заинтересованности в представленных материалах или методах. Tectonic Descemet stripping automated endothelial keratoplasty for the management of sterile corneal perforations in decompensated corneas. Cornea. 2016; 35: 1516–9. doi: 10.1097/ICO.0000000000001037 16. Otte J.B., de Ville de Goyet J., Alberti D., et al. The concept and technique of the split liver in clinical transplantation. Surgery. 1990; 107: 605–12 17. Cintorino D., Spada M., Gruttadauria S., et al. In situ split liver transplantation for adult and pediatric recipients: an answer to organ shortage. Transplant Proc. 2006; 38: 1096–8. doi:10.1016/j. transproceed.2006.02.146 29. Tu E.Y. Descemet membrane endothelial keratoplasty patch for persistent corneal hydrops. Cornea. 2017; 36: 1559–61. doi: 10.1097/ICO.0000000000001351 30. Kolomeyer A.M., Chu D.S. Descemet stripping endothelial keratoplasty in a patient with keratoglobus and chronic hydrops secondary to a spontaneous Descemet membrane tear. Case Reports in Ophthalmol. Med. 2013; ID 697403. doi: 10.1155/2013/ 697403 18. Armour R., Ousley P., Wall J., et al. Endothelial keratoplasty using donor tissue not suitable for full-thickness penetrating keratoplasty. Cornea. 2007; 26: 515–9. doi:10.1097/ICO.0b013e3180335511 19. Moshirfar M., Khalifa Y., Davis D., et al. Descemet stripping automated endothelial keratoplasty using donor corneas with Поступила: 19.12.2017 Новая стратегия кератопластики: расслоение и разделение роговицы донора A new strategy of keratoplasty: laminating and splitting the donor cornea A new strategy of keratoplasty: laminating and splitting the donor cornea O.G. Oganesyan — Dr. Med. Sci., leading researcher, department of eye traumatology and reconstructive surgery P.V. Makarov — Dr. Med. Sci., leading researcher, department of eye traumatology and reconstructive surgery d k h d d f l d O.G. Oganesyan — Dr. Med. Sci., leading researcher, department of eye traumatology and reconstructive surgery P.V. Makarov — Dr. Med. Sci., leading researcher, department of eye traumatology and reconstructive surgery À.À. Grdikanyan — PhD student, department of eye traumatology and reconstructive surgery V.R. Getadaryan — PhD student, department of eye traumatology and reconstructive surgery À.À. Grdikanyan — PhD student, department of eye traumatology and reconstructive surgery V.R. Getadaryan — PhD student, department of eye traumatology and reconstructive surgery Moscow Helmholtz Research Institute of Eye Diseases, 14/19, Sadovaya-Chernogryazskaya St., 105062, Moscow, Russia oftalmolog@mail.ru Moscow Helmholtz Research Institute of Eye Diseases, 14/19, Sadovaya-Chernogryazskaya St., 105062, Moscow, Russia oftalmolog@mail.ru Purpose: to report the first case of the same day use of a single donor cornea tissue for 5 consecutive transplantations in patients with endothelial dystrophy and keratoconus. Material and methods. Ðîññèéñêèé îôòàëüìîëîãè÷åñêèé æóðíàë, 2018; 3: 11-18 Для контактов: Оганесян Оганес Георгиевич E-mail: oftalmolog@mail.ru Прозрачность финансовой деятельности: никто из авторов не имеет финансовой заинтересованности в представленных материалах или методах. The Descemet membrane (DM) and the endothelium were fully detached from the stroma of the donor on the endothelial side, using first a circular and then a direct corneal cutter, forming four partial triangle-shaped Descemet grafts (each constituted a 1/4 Descemet graft). The stroma of the donor's cornea was separated by a circular cutter of a preset diameter. One patient with keratoconus underwent deep anterior lamellar keratoplasty (DALK) using the big-bubble technique. Then, each of the four Descemet grafts was sequen- tially transplanted by the Descemet membrane endothelial keratoplasty (DMEK) method to 4 patients with Fuchs dystrophy and pseudophakia. Results. During and after the surgery, there were no complications requiring additional intervention. Six months after DALK, the best corrected visual acuity (BCVA) was 0.66. Six months after four operations each using ¼ DMEK the average BCVA was 0.95 ± 0.1 (from 0.8 to 1.0) and the endothelial cell density was, respectively, 2839 after DALK and 1680 ± 254 cells/mm2 (from 1492 to 2039 cells/mm2) after ¼ DMEK. Conclusion. One donor cornea can be successfully transplanted to at least 5 patients. Despite the fact that the implementation of DALK and several operations using the ¼ DMEK technique within one day is technically difficult, this approach can contribute to a drastic reduction in the deficit of donor tissue. This approach requires a thorough selection of patients and an extensive surgical experience. Keywords: donor cornea, keratoplasty, corneal transplantation, Descemet membrane transplantation, DMEK, Q DMEK ¼ DMEK Keywords: donor cornea, keratoplasty, corneal transplantation, Descemet membrane transplantation, DMEK, Q-DMEK, ¼ DMEK The full English version is available online at http://roj.igb.ru/eng/issues The full English version is available online at http://roj.igb.ru/eng/issues The full English version is available online at http://roj.igb.ru/eng/issues For citation: Oganesyan O.G., Makarov P.V., Grdikanyan А.А., Getadaryan V.R. A new strategy of keratoplasty: laminating and splitting the donor cornea. Russian ophthalmological journal. 2018; 11 (3): 11–8 (In Russian). doi: 10.21516/2072-0076-2018-11-3-11-18 Conflict of interests: there is no conflict of interests. Conflict of interests: there is no conflict of interests. Financial disclosure: No author has a financial or property interest in any material or method mentioned. Financial disclosure: No author has a financial or property interest in any material or method mentioned. Для контактов: Оганесян Оганес Георгиевич E-mail: oftalmolog@mail.ru Новая стратегия кератопластики: расслоение и разделение роговицы донора 18 Новая стратегия кератопластики: расслоение и разделение роговицы донора 18
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RNAcentral 2021: secondary structure integration, improved sequence search and new member databases
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RNAcentral 2021: secondary structure integration, improved sequence search and new member nsortium1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,* RNAcentral Consortium1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26 1European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SD, UK, 2Center for non-coding RNA in Technology and Health, Department of Veterinary and Animal Sciences, University of Copenhagen, Copenhagen, DK-1871, Denmark, 3Department of Integrative Biology, University of Texas at Austin, Austin, Texas 78712, USA, 4Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK, 5Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel, 6Department of Biomolecular Engineering, University of California Santa Cruz, Santa Cruz, CA, 95064, USA, 7Department of Haematology, University of Cambridge School of Clinical Medicine, Cambridge, CB2 0AW, UK, 8Universit`a della Calabria, Dipartimento di Biologia, Ecologia e Scienze della Terra, Via Pietro Bucci Cubo 6/C, Rende, CS, 87036, Italy, 9DIANA-LAB, Department of Computer Science and Biomedical Informatics, University of Thessaly, Greece & Hellenic Pasteur Institute, 351 31, Greece, 10China National Center for Bioinformation & National Genomics Data Center, Beijing Institute of Genomics, Chinese Academy of Sciences, 100101, China, 11Cancer Research Institute Ghent (CRIG), 9000 Ghent, Belgium, 12Department of Biomolecular Medicine, Faculty of Medicine and Health Sciences, Ghent University, 9000 Ghent, Belgium, 13Faculty of Biology, Medicine and Health, University of Manchester, Oxford Road, Manchester, M13 9PT, UK, 14Science for Life Laboratory, Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, S-10691, Sweden, 15Department of Biological Sciences, Dartmouth College, Hanover, NH, 03755, USA, 16National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894 USA, 17Center for the Origins of Life, School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30032, USA, 18Department of Genetics, Stanford University, Palo Alto, CA 94303, USA, 19Department of Biochemistry and Functional Genomics, Universit´e de Sherbrooke, Sherbrooke, QCJ1H 5N4, Canada, 20Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA, 21Department of Software Engineering, Faculty of Mathematics and Physics, Charles Univesity, Prague, 11800 Praha 1, Czech Republic, 22Functional Gene Annotation, Preclinical and Fundamental Science, UCL Institute of Cardiovascular Science, University College London, London, WC1E 6BT, UK, 23The Institute of Neuroscience, University of Oregon, Eugene, OR 97403-1254, USA, 24Bioinformatics Group, Department of Computer Science and Interdisciplinary Centre for Bioinformatics, Leipzig University, H¨artelstraße 16–18, 04107, Leipzig, Germany, 25Department of Computational Biology, Adam Mickiewicz University in Poznan, Poznan, 61-614, Poland and 26The Biological Laboratories, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA method that uses consistent, reproducible and rec- ognizable layouts for related RNAs. RNAcentral 2021: secondary structure integration, improved sequence search and new member The sequence similarity search has been updated with a faster in- terface featuring facets for filtering search results by RNA type, organism, source database or any key- word. This sequence search tool is available as a reusable web component, and has been integrated into several RNAcentral member databases, includ- ing Rfam, miRBase and snoDB. To allow for a more fine-grained assignment of RNA types and subtypes, C⃝The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. D212–D220 Nucleic Acids Research, 2021, Vol. 49, Database issue doi: 10.1093/nar/gkaa921 D212–D220 Nucleic Acids Research, 2021, Vol. 49, Database issue doi: 10.1093/nar/gkaa921 Published online 27 October 2020 Received September 15, 2020; Editorial Decision October 01, 2020; Accepted October 05, 2020 *To whom correspondence should be addressed. Tel: +44 1223 492550; Fax: +44 1223 494468; Email: apetrov@ebi.ac.uk RNA 2D STRUCTURE INTEGRATION Since 2017 RNAcentral has included 2D structure informa- tion starting with a tRNA dataset submitted by Genomic tRNA Database (GtRNAdb) (7). However, for the vast ma- jority of RNAcentral sequences no secondary structure is available in the source database (e.g., ENA or RefSeq). In addition, there are accepted layouts and orientations for the display of secondary structures of well-known families (such as rRNA and tRNA) (8,9), but existing automated 2D visualization tools do not account for these layouts, mak- ing it difficult to analyze and compare structured RNAs. As these large families of well-known RNAs constitute the majority of sequences in RNAcentral, we set out to develop a new method for producing 2D structure diagrams in stan- dard orientations called R2DT (RNA 2D Templates) (10). The R2DT software automatically selects the best match- ing template from a library of 3632 2D templates that repre- sent a wide range of RNA types, such as rRNA (both small and large subunit), tRNA, as well as 2675 RNA families from Rfam. A template encapsulates a reference sequence along with cartesian coordinates for each nucleotide and a Since 2017 RNAcentral has included 2D structure informa- tion starting with a tRNA dataset submitted by Genomic tRNA Database (GtRNAdb) (7). However, for the vast ma- jority of RNAcentral sequences no secondary structure is available in the source database (e.g., ENA or RefSeq). In addition, there are accepted layouts and orientations for the display of secondary structures of well-known families (such as rRNA and tRNA) (8,9), but existing automated 2D visualization tools do not account for these layouts, mak- ing it difficult to analyze and compare structured RNAs. As these large families of well-known RNAs constitute the majority of sequences in RNAcentral, we set out to develop a new method for producing 2D structure diagrams in stan- dard orientations called R2DT (RNA 2D Templates) (10). The new search features an updated interface that enables exploring the results using facets, such as species, RNA type and source database (Figure 3). The results can also be filtered by any keyword, similar to the RNAcentral text search, and sorted by E-value, sequence identity, query and target coverage and other parameters. The query sequence is also automatically searched against the Rfam families (3024 as of Rfam 14.2) using Infernal (15). The Rfam results are post-processed to se- lect the top scoring families from the same Rfam clan (16). UPDATED SEQUENCE SIMILARITY SEARCH Since 2015, RNAcentral has been hosting a sequence simi- larity search tool powered by the nhmmer software (14), to enable users to compare any query sequence against a com- prehensive collection of ncRNAs (https://rnacentral.org/ sequence-search). As RNAcentral grew in size, the search time increased and users experienced wait times of up to an hour to get the results. In 2019, an updated version of the search was launched using a scalable cloud infrastructure hosted at the Embassy Cloud platform provided by EMBL- EBI. The searches are executed in parallel and complete more quickly. For example, we repeated all searches submit- ted in 2019 using the new infrastructure and saw a decrease in the average search time from 4.5 min to 13 s, an approxi- mately 20-fold increase in speed. Since the new launch was launched, the number of searches increased from around 600 to 3000 searches per month. 1. Newly integrated 2D structure information 2. Improved sequence similarity search 3. Transition to Sequence Ontology to annotate RNA types INTRODUCTION RNAcentral is the non-coding RNA (ncRNA) sequence database that currently integrates 44 specialist ncRNA databases, known as Expert Databases, to provide uni- fied access to >18 million ncRNA sequences spanning a broad range of functions and species (1). In addition to se- quences, RNAcentral provides a wide range of annotation types, such as genome coordinates, microRNA–target in- teractions (2,3), Gene Ontology (GO) terms (4), orthologs and paralogs (5), RNA family classification from Rfam (6) and more. Data can be accessed via text search, se- quence similarity search, integrated genome browser and bulk data downloads from the FTP archive. The primary goal of RNAcentral is to provide open access to a compre- hensive set of ncRNA sequences for a wide range of species, enabling the users to find what is known about individ- ual sequences or download ncRNA sequences and their ge- nomic locations that can be used for a broad range of stud- ies, such as interpreting the results of RNA-seq experiments or training bioinformatic algorithms. RNAcentral also pro- vides stable accessions for distinct RNA sequences, facilitat- ing the work of other RNA resources. R2DT is now routinely applied to all sequences in RNA- central. In the most recent release (version 16), we generated >13 million 2D structure diagrams, representing the world’s largest collection of RNA 2D structures. The 2D structures are displayed in the sequence report pages and in the text search results (Figure 2). In addition, R2DT is available as a web server (https://rnacentral.org/r2dt) that enables users to submit sequences and generate 2D diagrams. As new templates are added to the R2DT library (e.g., with future Rfam releases), the number and quality of the 2D diagrams will be improved in RNAcentral. We welcome feedback about individual 2D structures to help prioritize improvements in R2DT. RNAcentral continues to grow (Figure 1) with the incor- poration of 16 new Expert Databases since the last publica- tion (1). In this paper, we discuss the new data and focus on the following major new features: ABSTRACT RNAcentral is a comprehensive database of non- coding RNA (ncRNA) sequences that provides a sin- gle access point to 44 RNA resources and >18 mil- lion ncRNA sequences from a wide range of organ- isms and RNA types. RNAcentral now also includes secondary (2D) structure information for >13 million sequences, making RNAcentral the world’s largest RNA 2D structure database. The 2D diagrams are dis- played using R2DT, a new 2D structure visualization Nucleic Acids Research, 2021, Vol. 49, Database issue D213 2D structure. The best-matching templates are selected us- ing the Ribovore (https://github.com/nawrockie/ribovore) and tRNAscan-SE 2.0 (11) software, and are visualized using Traveler (12). The templates ensure that similar se- quences are visualized in consistent, reproducible orienta- tions and can be easily compared across related RNAs. all RNAcentral sequences have been annotated with Sequence Ontology terms. The RNAcentral database continues to grow and provide a central data re- source for the RNA community. RNAcentral is freely available at https://rnacentral.org. A key strength of the method is the ability to visual- ize some of the largest structured RNAs, such as the hu- man large subunit ribosomal rRNAs (LSU) with >5000 nu- cleotides (Figure 2). The LSU templates are displayed using a set of new 3D structure based templates from RiboVision (13). In addition, RiboVision provided a set of 3D structure based small subunit (SSU) rRNA templates that improves the representation of species-specific expansion segments in rRNA. RNA 2D STRUCTURE INTEGRATION For example, a rRNA sequence may match both eukary- otic and bacterial Rfam families, but the clan competi- tion procedure keeps only the top scoring family. In ad- dition, the sequence search is integrated with the R2DT The R2DT software automatically selects the best match- ing template from a library of 3632 2D templates that repre- sent a wide range of RNA types, such as rRNA (both small and large subunit), tRNA, as well as 2675 RNA families from Rfam. A template encapsulates a reference sequence along with cartesian coordinates for each nucleotide and a D214 Nucleic Acids Research, 2021, Vol. 49, Database issue Figure 1. Growth in the number of RNAcentral Expert Databases since its launch in 2014 (for an up-to-date list see https://rnacentral.org/expert-databases). The previous NAR publication is marked with a vertical dashed line. Downloaded from https://academic.oup.com/nar/article/49/D1/D212/5940500 by gues Figure 1. Growth in the number of RNAcentral Expert Databases since its launch in 2014 (for an up-to-date list see https://rnacentral.org/expert-databases). The previous NAR publication is marked with a vertical dashed line. In addition, in response to the COVID-19 pandemic, the cloud-centric approach enabled us to rapidly repur- pose the RNAcentral infrastructure to search Betacoron- avirus genomes instead of ncRNA sequences. The Beta- coronavirus search provides virus-specific facets that en- able filtering the results by virus, such as SARS-CoV or SARS-CoV-2, as well as the country of sample origin. The Betacoronavirus sequence search is available at https: //covid19sequencesearch.github.io. software described above so that a 2D structure (if avail- able) is visualized alongside similar sequences (Figure 3C). The updated search includes some of the most frequently requested features that were not available previously. For example, a batch search mode enables users to submit a FASTA file with up to 50 sequences in order to launch multiple searches simultaneously. Users can also down- load the results in several formats, including plain text and JSON. software described above so that a 2D structure (if avail- able) is visualized alongside similar sequences (Figure 3C). The updated search includes some of the most frequently requested features that were not available previously. For example, a batch search mode enables users to submit a FASTA file with up to 50 sequences in order to launch multiple searches simultaneously. Users can also down- load the results in several formats, including plain text and JSON. RNA 2D STRUCTURE INTEGRATION The new interface is implemented as a reusable web component, enabling other RNAcentral Expert Databases or anyone else to include it in their websites to pro- vide sequence similarity search to their users. The em- beddable component is available at https://github.com/ rnacentral/rnacentral-sequence-search-embed. It can be in- tegrated into any website with a few lines of code. The component is highly customizable, for example, it is pos- sible to select a subset of RNAcentral sequences to be searched or adjust the widget appearance to match the host website. Figure 2. (A) The RNAcentral text search results include simplified thumbnails representing the 2D structures. (B) A 2D structure of the human LSU rRNA displayed on the sequence report page for URS000075EC78 9606. REFINED RNA TYPE ASSIGNMENT USING SE- QUENCE ONTOLOGY The result- classification system that combines the information about the INSDC RNA types submitted by member databases, Rfam annotations and other information to expand the SO term coverage to the entire set of sequences found in RNAcentral. For example, for rRNA sequences, the R2DT rRNA template matches are used to transfer the corre- sponding SO term to the sequence, enabling the classifi- cation of rRNA subclasses. Consequently, an Arabidopsis thaliana sequence URS0000AF5D55 3702 previously an- notated as misc RNA in ENA is now assigned the SO term for 25S LSU rRNA due to matches to the eukary- otic large subunit (LSU) rRNA Rfam model (RF02543) and an eukaryotic LSU R2DT template. For the ‘other’ and ‘misc RNA’ INSDC sequence classes, we use Rfam family annotations to assign the corresponding SO term to the se- quences. For all remaining sequences, we map the INSDC RNA types to the SO terms using the mapping developed by the SO and the RefSeq groups (https://github.com/The- Sequence-Ontology/SO-Ontologies/issues/378). The result- REFINED RNA TYPE ASSIGNMENT USING SE- QUENCE ONTOLOGY Downloaded from https://academic.oup.com/nar/article/49/D1/D212/5940500 by guest on 19 January 2022 Downloaded from https://academic.oup.com/nar/article/49/D1/D212/5940500 by gue Figure 3. New RNAcentral sequence similarity search interface. (A) Query sequence, (B) Identical sequence found in RNAcentral, (C) Secondary structur visualized with R2DT, (D) Rfam classification, including Rfam family annotations and an alignment between the query and the Rfam model, (E) Simila sequences found in RNAcentral that can be filtered using facets, such as RNA types and Organisms, sorted or downloaded. Figure 3. New RNAcentral sequence similarity search interface. (A) Query sequence, (B) Identical sequence found in RNAcentral, (C) Secondary structure visualized with R2DT, (D) Rfam classification, including Rfam family annotations and an alignment between the query and the Rfam model, (E) Similar sequences found in RNAcentral that can be filtered using facets, such as RNA types and Organisms, sorted or downloaded. Figure 3. New RNAcentral sequence similarity search interface. (A) Query sequence, (B) Identical sequence found in RNAcentral, (C) Secondary structure visualized with R2DT, (D) Rfam classification, including Rfam family annotations and an alignment between the query and the Rfam model, (E) Similar sequences found in RNAcentral that can be filtered using facets, such as RNA types and Organisms, sorted or downloaded. ing distribution of RNAcentral sequences by SO terms is shown in Figure 4. ing distribution of RNAcentral sequences by SO terms is shown in Figure 4. classification system that combines the information about the INSDC RNA types submitted by member databases, Rfam annotations and other information to expand the SO term coverage to the entire set of sequences found in RNAcentral. For example, for rRNA sequences, the R2DT rRNA template matches are used to transfer the corre- sponding SO term to the sequence, enabling the classifi- cation of rRNA subclasses. Consequently, an Arabidopsis thaliana sequence URS0000AF5D55 3702 previously an- notated as misc RNA in ENA is now assigned the SO term for 25S LSU rRNA due to matches to the eukary- otic large subunit (LSU) rRNA Rfam model (RF02543) and an eukaryotic LSU R2DT template. For the ‘other’ and ‘misc RNA’ INSDC sequence classes, we use Rfam family annotations to assign the corresponding SO term to the se- quences. For all remaining sequences, we map the INSDC RNA types to the SO terms using the mapping developed by the SO and the RefSeq groups (https://github.com/The- Sequence-Ontology/SO-Ontologies/issues/378). REFINED RNA TYPE ASSIGNMENT USING SE- QUENCE ONTOLOGY Since its inception, RNAcentral has used the INSDC feature table (http://www.insdc.org/files/feature table.html) and ncRNA vocabulary (http://www.insdc.org/rna vocab. html) to annotate sequences with different RNA types. However, the INSDC classification lacks precision and does not distinguish between different rRNA types, such as SSU, LSU or 5S rRNAs, simply grouping them in a single cate- gory. Similarly, there were no specific terms for precursor microRNAs to separate them from other RNA precursors and maintain their connection with mature microRNAs. However, the Sequence Ontology (SO) (19) provides more granular terms for rRNAs, microRNAs and other RNA types. The search has been integrated into Rfam (6), miRBase (17) and snoDB (18). For example, when a user enters a query sequence in Rfam, it is not only annotated with Rfam families but also searched against a comprehensive set of se- quences from RNAcentral. If a query comes from an RNA sequence not represented in Rfam, the results will include hits from RNAcentral, and if a query matches Rfam, the users will get additional information about matching se- quences and can explore them using the facets. yp Several member databases, such as FlyBase or miRBase, already provide SO terms for their sequences. However, <10% of sequences have been annotated with SO terms at the time of submission to RNAcentral. We implemented a Nucleic Acids Research, 2021, Vol. 49, Database issue D215 D215 Downloaded from https://academic.oup.com/nar/article/49/D1/D212/5940500 by gue Downloaded from https://academic.oup.com/nar/article/49/D1/D212/5940500 by guest on 19 January 2022 e RNAcentral text search results include simplified thumbnails representing the 2D structures (B) A 2D structur Figure 2. (A) The RNAcentral text search results include simplified thumbnails representing the 2D structures. (B) A 2D structure of the human LSU rRNA displayed on the sequence report page for URS000075EC78 9606. Figure 2. (A) The RNAcentral text search results include simplified thumbnails representing the 2D structures. (B) A 2D structure of the human LSU rRNA displayed on the sequence report page for URS000075EC78 9606. D216 Nucleic Acids Research, 2021, Vol. 49, Database issue Figure 3. New RNAcentral sequence similarity search interface. (A) Query sequence, (B) Identical sequence found in RNAcentral, (C) Secondary structure visualized with R2DT, (D) Rfam classification, including Rfam family annotations and an alignment between the query and the Rfam model, (E) Similar sequences found in RNAcentral that can be filtered using facets, such as RNA types and Organisms, sorted or downloaded. NEW DATA AND ANNOTATIONS Since the last publication, the number of imported databases increased from 28 to 44 databases, integrating 16 additional resources listed in Table 1. To provide detailed human ncRNA annotations, we im- ported data from LncBook (20) and snoDB (18) that host a variety of annotations for lncRNAs and snoRNAs, respec- tively. GeneCards (21) and MalaCards (22) have also been included into RNAcentral. GeneCards is a human gene knowledgebase, which aims to consolidate information about all human genes, coding and non-coding. MalaCards is an integrated database of human diseases and their an- notations. MalaCards uses text mining and manual cura- tion to associate human ncRNAs with information about diseases and lists the supporting literature. Notably, snoDB Nucleic Acids Research, 2021, Vol. 49, Database issue D217 Figure 4. The distribution of RNAcentral sequences by the SO terms. Downloaded from https://academic.oup.com/nar/article/49/D1/D212/5940500 by gue Figure 4. The distribution of RNAcentral sequences by the SO terms. described. All GO Consortium ncRNA annotations are available in RNAcentral, as well as via the GO browsers QuickGO and AmiGO and in other major resources includ- ing Ensembl, NCBI Gene, miRBase and the web service PSICQUIC. and GeneCards are also using RNAcentral as a data source. GeneCards also used RNAcentral to produce a compre- hensive and non-redundant gene-centric view of ncRNAs, which is available at the ‘GeneCards ncRNAs’ track hub at the UCSC genome browser (23). Several RNA type specific databases have been included, such as 5SRNAdb with 5S rRNAs (30), snoRNA Database with archaeal snoRNAs (31,32), MirGeneDB with mature and precursor microRNAs (33), as well as CRW with 5S, SSU and LSU rRNAs (8). A broad range of prokaryotic ncRNAs has been incorporated from the ZWD database (34), which includes high-quality sequence alignments for structured RNAs discovered in a diverse range of habitats and organisms. We completed the integration of all model organism databases forming the Alliance of Genome Resources (24) by importing ZFIN (25), a model organism database that hosts a wide array of expertly curated, organized and cross- referenced research data for zebrafish (Danio rerio). In or- der to provide genomic annotations for a broad range of organisms, we also imported ncRNAs from Ensembl Fungi, Metazoa and Protists (26). ( ) We have added several new sources of functional annota- tions. NEW DATA AND ANNOTATIONS We have integrated IntAct (27) bringing in 1152 inter- molecular interactions for 382 RNAs, with the majority of data points coming from human and yeast (168 and 114 an- notated RNAs, respectively). As curators continue to anno- tate additional interactions in IntAct, the new data will au- tomatically flow into RNAcentral. We have also integrated microRNA–lncRNA interactions from LncBase v2 (3). We have also imported the Conserved RNA Structure (CRS) resource that computationally screened the hu- man centered 100-way vertebrate sequence alignment from UCSC Genome Browser for conserved RNA secondary structures with CMfinder (35). We have integrated CRSs with a false discovery rate lower or equal to 10% in 29 ver- tebrate species and excluded matches to known structured RNAs from Rfam. In addition to the automatic GO annotations created by RNAcentral, over 3400 ncRNAs currently are asso- ciated with GO terms, following the manual curation of research articles by the GO Consortium (1). Over 80% of these, 17 000 annotations capture the cellular role of human and mouse ncRNAs; microRNAs are the most commonly curated ncRNA. The majority of these anno- tations describe ‘gene silencing by miRNA’ and ‘mRNA binding’ and include the target of the ncRNA in the an- notation extension field. However, downstream processes such as ‘regulation of epithelial-to-mesenchymal transition’ and ‘regulation of inflammatory response’ (28,29) are also Significant data updates A number of previously integrated resources have provided significant updates in the last 2 years. Recent changes in SILVA (36) allowed us to integrate the SILVA-based in- ferred bacterial taxonomy into RNAcentral, which is dis- played on the sequence report pages. FlyBase (37) ncRNA annotations have been continuously updated within RNAcentral. Notably, FlyBase now reflects gene model annotations for Drosophila melanogaster only, D218 Nucleic Acids Research, 2021, Vol. 49, Database issue Table 1. Sixteen new member databases incorporated into RNAcentral in releases 11–16 Database Description Number of annotated sequences 5SrRNAdb 5SrRNAdb is an information resource for 5S ribosomal RNAs. 11 415 CRS Conserved RNA structures (CRS) are structured elements of RNA molecules that are conserved across vertebrate species. 250 867 CRW CRW Site provides comparative sequence and structure information for ribosomal, intron and other RNAs. 948 Ensembl Fungi, Metazoa, Protists The Ensembl Genomes divisions complement the Ensembl database. 16 331; 64 237; 5652 GeneCards GeneCards is a searchable, integrative database that provides comprehensive, user-friendly information on all annotated and predicted human genes. 250 702 IntAct IntAct provides a freely available, open source database system and analysis tools for molecular interaction data. All interactions are derived from literature curation or direct user submissions. 382 LncBase LncBase is a database of experimentally verified and computationally predicted microRNA targets on lncRNAs. 1337 LncBook LncBook is a curated knowledgebase of human lncRNAs. 268 848 MalaCards MalaCards integrates manually curated and text-mining sources to associate genes, including ncRNAs, with diseases, and lists the supporting evidence. 34 087 MirGeneDB MirGeneDB is a curated microRNA gene database covering 45 metazoan organisms. 29 681 snoDB snoDB is an interactive database of human snoRNA sequences, abundance and interactions. 1984 snoRNA Database The snoRNA Database is a curated collection of archaeal snoRNAs maintained by the Lowe Lab at UC Santa Cruz. 727 ZFIN The Zebrafish Information Network (ZFIN) is the database of genetic and genomic data for the zebrafish as a model organism. 1060 ZWD ZWD is a collection of non-coding RNA alignments maintained by Zasha Weinberg. 47 998 Total: 986 256 Table 1. Sixteen new member databases incorporated into RNAcentral in releases 11–16 Number of annotated sequences provements have been made to uniquely link LNCipedia en- tries with those of other databases such as Ensembl (45) and HGNC (38). meaning that ncRNA data for non-melanogaster genomes are no longer submitted by FlyBase to RNAcentral. OTHER IMPROVEMENTS / q The HUGO Gene Nomenclature Committee (HGNC) (38) is the only organization with the authority to approve human gene symbols, including for ncRNA genes. Since January 2019, the HGNC has primarily worked on expand- ing its lncRNA dataset and has approved 528 new gene symbols, representing an increase of 11% for these genes. Note that the HGNC only provides one name per lncRNA gene without naming separate non-coding isoforms. Where possible, lncRNA genes have been named based on func- tional data from publications. Recent examples include CHASERR (39), MYOPARR (40) and CEROX1 (41,42). Where no published data are available, the HGNC prior- itizes naming lncRNA genes that have been manually an- notated by both the RefSeq and Ensembl-Havana projects. These lncRNA genes are named based on genomic context using a systematic schema, as outlined in (43). The HGNC has also increased its small nuclear RNA dataset by 13% and its transfer RNA dataset by 2.5%. The RNAcentral website has been continuously updated with new features, such as the inclusion of the information about paralogs and orthologs from the Ensembl Compara pipeline (5). To increase discoverability with search engines, automatically generated summaries have been added for all sequences. The RNAcentral users can also display the miR- Base word clouds (17) based on literature mining, which al- lows the users to see related terms at a glance. For example, microRNA mir-100 (URS000054969A 9606) is associated with cancer, with this term prominently featured in the word cloud. Following user requests, RNAcentral now hosts a public Postgres database that provides the same data as the RNA- central website. The database is meant to help users who would like to access RNAcentral programmatically or are interested in tasks that are not yet supported by the web- site. The connection details, example queries and a sample Python script can be found in (46) and at https://rnacentral. org/help/public-database. With the most recent release of lncRNA database LNCi- pedia (version 5.2), significant efforts have been made to ex- pand the functional annotation of lncRNAs in the database (44). By combining manual and programmatical curation of thousands of lncRNA papers in PubMed, 2482 PubMed articles were associated with lncRNAs in LNCipedia. As a result, LNCipedia currently contains 1555 unique lncRNA genes with at least one published article. In addition, im- Significant data updates How- ever, ncRNA annotations for other Drosophila species are still available in RNAcentral as they are imported directly from NCBI/RefSeq and the ENA. REFERENCES 1. RNAcentral Consortium (2019) RNAcentral: a hub of information for non-coding RNA sequences. Nucleic Acids Res., 47, D221–D229. 1. RNAcentral Consortium (2019) RNAcentral: a hub of information for non-coding RNA sequences. Nucleic Acids Res., 47, D221–D229. 2. Karagkouni,D., Paraskevopoulou,M.D., Chatzopoulos,S., Vlachos,I.S., Tastsoglou,S., Kanellos,I., Papadimitriou,D., Kavakiotis,I., Maniou,S., Skoufos,G. et al. (2018) DIANA-TarBase v8: a decade-long collection of experimentally supported miRNA–gene interactions. Nucleic Acids Res., 46, D239–D245. 3. Paraskevopoulou,M.D., Vlachos,I.S., Karagkouni,D., Georgakilas,G., Kanellos,I., Vergoulis,T., Zagganas,K., Tsanakas,P., Floros,E., Dalamagas,T. et al. (2016) DIANA-LncBase v2: indexing microRNA targets on non-coding transcripts. 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The improved sequence search is faster and more user-friendly, and the embeddable search component is available for use on any website, enabling an ecosystem of RNAcentral member databases to reuse the resources in a cost-efficient way. The SO integration enables more granular annotation of ncRNAs and powers new ways of discovering the data using text search. The development of the next versions of RNAcentral is underway, focus- ing on the gene-centric organization of ncRNA transcripts and automatic incorporation of the latest scientific litera- ture using text mining. We aim to continue integrating ad- ditional member databases, with 12 databases pending im- port, and we invite the developers of RNA databases wish- ing to join the RNAcentral Consortium to get in touch at https://rnacentral.org/contact. FUNDING Braschi,B., Denny,P., Gray,K., Jones,T., Seal,R., Tweedie,S., Yates,B. and Bruford,E. (2019) Genenames.org: the HGNC and VGNC resources in 2019. 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Uji Coba Pembuatan Kue Kering Sagu Dengan Penambahan Tepung Tulang Ikan Dan Daya Terimanya
Jurnal Kesehatan Masyarakat Perkotaan
2,021
cc-by
5,294
Abstrak Latar belakang : Salah satu jenis kue kering adalah Kue kering sagu yang dibuat dengan bahan dasar tepung sagu. Penelitian ini dilakukan untuk memanfaatkan limbah tulang ikan yang masih memiliki zat gizi khususnya kalsium yang cukup tinggi yang ditambahkan pada pembuatan kue kering sagu Tujuan : Mengetahui pengaruh penambahan tepung tulang ikan tongkol terhadap sifat organoleptik (warna, tekstur, aroma dan rasa), daya terima dan kalsium pada kue kering sagu. Metode : Penelitian ini bersifat eksperimental, dengan melakukan penambahan tepung tulang ikan tongkol pada kue kering sagu 0%, 10%, 15% dan 20%. Penelitian ini menggunakan uji Analysis of variance dilanjutkan menggunakan uji Duncan bila p- Value <0,005. Hasil : Ada pengaruh penambahan tepung tulang ikan tongkol terhadap mutu hedonik aspek warna, aroma, rasa dan tingkat kesukaan kue kering sagu, tidak berpengaruh terhadap mutu hedonik aspek tekstur kue kering sagu. Kue kering sagu terpilih yaitu T2 (10% tepung tulang ikan tongkol) dengan nilai energi total yaitu 368 kkal, protein 10 gram, karbohidrat 68 gram, lemak total 6 gram dan kalsium sebesar 0.1 gram dengan menggunakan metode AAS. Kesimpulan : Penambahan tepung tulang ikan tongkol berpengaruh terhadap aspek warna, aroma, rasa dan tingkat kesukaan secara keseluruhan. Penambahan tepung tulang ikan tongkol tidak berpengaruh terhadap aspek tekstur kue kering sagu. Aktivitas uji kalsium sebesar 0.1 gram dalam 100 gram bahan. Saran: analisis kelayakan usaha pembuatan kue kering sagu dengan penambahan tepung tulang ikan 10% dan dilakukan diuji daya terima untuk snack pasien ginjal. Kata Kunci : kue kering sagu, tepung tulang ikan tongkol, sifat organoleptik Jurnal Kesehatan Masyarakat Perkotaan Volume 2, No.1; Maret 2022 Jurnal Kesehatan Masyarakat Perkotaan Volume 2, No.1; Maret 2022 p-ISSN: 2776-0952 e-ISSN : 2776-0944 Hal 46-60 p-ISSN: 2776-0952 e-ISSN : 2776-0944 Hal 46-60 Uji Coba Pembuatan Kue Kering Sagu Dengan Penambahan Tepung Tulang Ikan Dan Daya Terimanya 1)2)Program Studi Gizi, Fakultas Kesehatan, Universitas Mohammad Husni Thamrin Correspondence Author: alfasiregar07@gmail.com DOI: https://doi.org/10.37012/jkmp.v1i1.1184 Parlin Dwiyana1), Elfa Siregar2)*) Abstrak Open Journal System (OJS): journal.thamrin.ac.id http://journal.thamrin.ac.id/index.php/jkmp/article/view/74 Jurnal Kesehatan Masyarakat Perkotaan p-ISSN: 2776-0952 e-ISSN : 2776-0944 Hal 46-60 p-ISSN: 2776-0952 e-ISSN : 2776-0944 Hal 46-60 2͕ Conclusion: The addition of mackarel tuna bone meal has an effect on aspects of color, aroma, taste, and overall level of preference. The addition of mackarel tuna bone meal did not affect the texture aspect of sago cookies. Calcium test activity is 0.1 grams in 100 grams of material. Suggestion: business feasibility analysis of making sago cookies with the addition of 10% fish bone meal and tested for acceptability of snacks for kidney patients. Keywords: sago pastries, tuna bone meal, organoleptic properties Open Journal System (OJS): journal.thamrin.ac.id Abstrack Background of the research: One type of pastry is sago cookies made with sago flour as the basic ingredient. This research was conducted to utilize fish bone waste which still has nutrients, especially calcium which is quite high and is added to the manufacture of sago pastries. Objective: To determine the effect of adding mackarel tuna bone meal on organoleptic properties (color, texture, aroma, and taste), acceptability, and calcium in sago pastries. Methods: This research is experimental, by adding mackarel tuna bone meal to sago pastries of 0%, 10%, 15%, and 20%. This study uses the Analysis of variance test followed by Duncan's test if the p-Value <0.005. Result: There is an effect of adding mackarel tuna bone meal to the hedonic quality of the color, aroma, taste, and preference level of sago pastries, does not affect the hedonic quality of the texture aspect of sago pastries. The selected sago cookies were T2 (10% mackarel tuna bone meal) with a total energy value of 368 kcal, 10 grams of protein, 68 grams of carbohydrates, 6 grams of total fat, and 0.1 grams of calcium using the AAS method. 46 Open Journal System (OJS): journal.thamrin.ac.id http://journal.thamrin.ac.id/index.php/jkmp/article/view/74 METODE PELAKSANAAN METODE PELAKSANAAN Penelitian ini merupakan penelitian eksperimental yang bertujuan untuk mengetahui komposisi tepung tulang ikan terhadap sifat organoleptik kue kering. Variable bebas dalam penelitian ini yaitu komposisi tepung tulang ikan tongkol. Sedangkan varabel terikat dalam penelitian ini yaitu sifat organoleptik kue kering. Penelitian dilakukan di laboratorium gizi universitas MH.Thamrin Jakarta pada bulan Juli-Agustus 2020. Rancangan percobaan yang digunakan untuk penelitian ini adalah Rancangan Acak Lengkap (RAL) dengan 3 kali perlakuan diulang sebanyak 2 kali. Tujuan dilakukan 3 perlakuan yaitu untuk mendapatkan sifat organoleptik kue kering yang optimal. Perlakuan 1 yaitu dengan penambahan tepung tulang tongkol sebesar 10%. Perlakuan 2 yaitu dengan penambahan tepung tulang tongkol 15%. Perlakuan 3 yaitu dengan penambahan tepung tulang tongkol sebesar 20%. Data yang telah dikumpulkan kemudian dianalisis dengan software analisis data. Penelitian ini menggunakan Rancangan Acak Lengkap (RAL). Dengan 2 kali ulangan. Uji organoleptik dianalisis dengan sidik garam (ANOVA), menunjukkan pengaruh perlakuan nyata, maka dilanjutkan dengan Duncan’s Multiple Range Test untuk mencari keberadaan perbedaan dari perlakuan yang ada. PENDAHULUAN Tulang merupakan salah satu bagian dari tubuh makhluk hidup yang paling banyak mengandung kalsium. Diperkirakan 99% kalsium terdapat di dalam jaringan keras yaitu tulang dan gigi dan 1% kalsium terdapat pada darah dan jaringan lunak. Tanpa 1% kalsium, otot akan mengalami gangguan kontraksi, darah akan sulit membeku, rangsangan saraf akan terganggu dalam pengahantarnya. Untuk memenuhi kebutuhan 1% kalsium,, tubuh mendapatkannya dari makanan yang dimakan atau dari tulang, karena mayoritas mineral dan vitamin tidak dapat di produksi sendiri oleh tubuh. Kandungan kalsium dan fosfor pada tulang cukup tinggi sehingga berpotensi untuk mencukupi asupan kalsium. Tulang ikan dalam bentuk tepung ikan memiliki kandungan kalsium yang cukup tinggi, pada ikan manyung sebesar 12,8 mg/100 gram, pada ikan mata besar 15,2 mg/100 gram (Iwansyah, dkk., 2008). Dalam Almatsier (2004), mengkonsumsi ikan dengan tulangnya merupakan salah satu sumber kalsium yang baik, sehingga tulang ikan mempunyai potensi sebagai alternatif bahan makanan kaya kalsium. Selama ini tulang ikan hanya menjadi limbah perikanan dapat mencemari lingkungan. Tulang ikan dapat dimanfaatkan setelah diolah menjadi tepung tulang ikan misalkan dibuat menjadi kue kering salah satu nya adalah kue kering sagu. Kue kering sagu adalah jenis kue yang memanfaatkan sagu atau tapioka sebagai salah satu komponen penyusun adonannya. Pemanfaatan tulang ikan menjadi tepung kemudian diolah lebih lanjut, dapat mengurangi jumlah limbah perikanan dan secara tidak langsung menyumbang asupan kalsium untuk masyarakat Indonesia. Berdasarkan hal tersebut, peneliti ingin melakukan penelitian tentang Uji Coba Pembuatan Kue Kering Sagu dengan Penambahan Tepung Tulang Ikan dan Daya Terimanya. Penelitian ini bertujuan untuk mengetahui pengaruh penambahan tepung tulang ikan pada pembuatan kue kering sagu dan daya terima nya. 47 Open Journal System (OJS): journal.thamrin.ac.id Open Journal System (OJS): journal.thamrin.ac.id ͬ74 p-ISSN: 2776-0952 e-ISSN : 2776-0944 Hal 46-60 p-ISSN: 2776-0952 e-ISSN : 2776-0944 Hal 46-60 p-ISSN: 2776-0952 e-ISSN : 2776-0944 Jurnal Kesehatan Masyarakat Perkotaan Jurnal Kesehatan Masyarakat Perkotaan sŽůƵŵĞ2͕EŽ͘ϭ͖DĂƌĞƚ2Ϭ22 HASIL DAN PEMBAHASAN Penelitian ini dilakukan 2 tahap yaitu tahap pertama penelitian pendahuluan dan tahap kedua penelitian utama. Penelitian pendahuluan pembuatan kue kering sagu dilakukan pada bulan Juli 2020 Penelitian ini betujuan untuk mengetahui persen penambahan tepung tulang ikan sehingga menghasilkan kue kering sagu yang baik dan dapat diterima. Dalam penelitian pendahuluan telah dibuat kue kering sagu dengan penambahan tepung tulang ikan tongkol sebanyak 10% dan 50%, dilakukan uji daya terima dan organoleptik dengan hasil sebagai berikut: Tabel 1 Karakteristik Kue Kering sagu Kriteria 10% 50% Rasa Manis Kurang manis Aroma Tidak beraroma ikan Aroma ikan sangat kuat Warna Krem Cokelat Tekstur Agak keras Sangat keras Tabel 1 Karakteristik Kue Kering sagu 48 Open Journal System (OJS): journal.thamrin.ac.id ͬ74 ͬ74 Jurnal Kesehatan Masyarakat Perkotaan p-ISSN: 2776-0952 e-ISSN : 2776-0944 Tingkat kesukaan Suka Tidak suka sŽůƵŵĞ2͕EŽ͘ϭ͖DĂƌĞƚ2Ϭ22 Hal 46-60 p-ISSN: 2776-0952 e-ISSN : 2776-0944 Hal 46-60 Berdasarkan hasil penelitian pendahuluan maka dapat disimpulkan bahwa kue kering sagu dengan penambahan tepung tulang ikan 10% hasilnya bisa diterima oleh panelis dari pada kue kering dengan penambahan tepung tulang ikan 50%. pada penambahan 50% penilaian panelis tidak suka sehingga dapat disimpulkan penambahan tepung tulang ikan kurang dari 50%. Penelitian utama merupakan lanjutan dari penelitian pendahuluan. Kue kering dibuat dengan konsentrasi penambahan yang telah di identifikasi pada penelitian pendahuluan untuk selanjutnya akan diuji mutu organoleptik dan daya terimanya. Berdasarkan hasil uji pendahuluan konsentrasi yang dipakai untuk penelitian lanjutan adalah 10%, 15%, 20%. Penelitian ini menggunakan kontrol untuk membandingkan pengaruh percobaan terhadap kue kering sagu yang diteliti. Proses penelitian ini meliputi penambahan tepung tulang ikan tongkol terhadap kue kering sagu pada persentase 10 gram, 15 gram, 20 gram dan 0 gram sebagai kontrol. Selanjutnya dilakukan uji organoleptik yaitu uji mutu hedonik dengan kriteria uji warna, tekstur, aroma dan rasa. Prinsip uji mutu hedonik berdasarkan penilaian panelis terhadap sifat organoleptik dengan penganalisaan tingkat kesan (skala mutu hedonik). Pada uji mutu hedonik produk kue kering sagu ditentukan nilai 1 hingga nilai 5, dimana nilai paling rendah yang berarti kualitas kue kering paling jelek, menaik hingga nilai 5 pada skala hedonik dimana kualitas kue kering sagu semakin baik seiring menaiknya penilaian panelis. Uji mutu hedonik pada penelitian kue kering sagu mencakup kriteria uji warna, tekstur, aroma dan rasa. Uji mutu hedonik kue kering sagu dilakukan di ruang kelas yang dikondisikan sebagai laboratorium uji. Pengujian dilakukan oleh panelis agak terlatih dari mahasiswa S1 Gizi semester 6 sebanyak 32 orang. HASIL DAN PEMBAHASAN Panelis melakukan uji mutu hedonik produk kue kering sagu 4 perlakuan 2 ulangan dengan konsentrasi tepung tulang ikan tongkol 0 gram, 10 gram, 15 gram dan 20 gram. Penelitian ini menggunakan Rancangan Acak Lengkap (RAL) dengan 2 kali ulangan. Uji organoleptik dianalisis dengan sidik ragam (ANOVA), menunjukkan beda nyata pada taraf 5%. Jika ANOVA menunjukkan pengaruh perlakuan nyata, maka dilanjutkan dengan Duncan’s Multiple Range Test untuk mencari keberadaan perbedaan dari perlakuan yang 49 Open Journal System (OJS): journal.thamrin.ac.id Open Journal System (OJS): journal.thamrin.ac.id ͬ74 Jurnal Kesehatan Masyarakat Perkotaan p-ISSN: 2776-0952 e-ISSN : 2776-0944 ada. Data yang didapatkan dari uji organoleptik untuk menentukan produk terpilih yang kemudian akan diuji laboratorium yaitu uji aktivitas Proksimat dan Kalsium. sŽůƵŵĞ2͕EŽ͘ϭ͖DĂƌĞƚ2Ϭ22 Hal 46-60 Jurnal Kesehatan Masyarakat Perkotaan sŽůƵŵĞ2͕EŽ͘ϭ͖DĂƌĞƚ2Ϭ22 y 2Ϭ22 2͕ ada. Data yang didapatkan dari uji organoleptik untuk menentukan produk terpilih yang kemudian akan diuji laboratorium yaitu uji aktivitas Proksimat dan Kalsium. ada. Data yang didapatkan dari uji organoleptik untuk menentukan produk terpilih yang kemudian akan diuji laboratorium yaitu uji aktivitas Proksimat dan Kalsium. Warna Warna termasuk dalam penilaian uji organoleptik. Menurut Nurwantoro et al (2016), warna merupakan faktor yang cukup penting untuk sebuah produk, karena dapat mempengaruhi persepsi terhadap rasa dari produk dan menjadikan produk lebih menarik. Hasil uji organoleptik menunjukkan bahwa nilai rata-rata uji mutu hedonik aspek warna pada T1 = 4.58 (Krem kecoklatan), T2 = 3.64 (Agak coklat), T3 = 4.34 (Krem ), T4= 3.63 (agak coklat) Hasil uji anova didapatkan bahwa terdapat pengaruh penambahan teung tulang ikan tongkol terhadap sifat organoleptk mutu hedonik warna produk kue kering sagu karena nilai p value < 0.05 yaitu sebesar 0.000 yang artinya H0 ditolak. Secara deskriptif terlihat adanya pengaruh penambahan tepung tulang ikan tongkol yaitu produk kue kering sagu cenderung agak coklat. Hal ini didukung dengan hasil analisis statistik menunjukkan ada perbedaan yang nyata pada penambahan tulang ikan tongkol terhadap tingkat kesukaan aspek warna kue kering sagu. Untuk mengetahui pengaruh tersebut maka dilakukan uji lanjut Duncan. Berdasarkan hasil uji Duncan terhadap mutu hedonik aspek warna terlihat bahwa T1-T2 dan T1-T3 menunjukkan perbedaan yang nyata. Urutan nilai terendah sampai tertinggi terhadap aspek warna yaitu perlakuan penambahan 20 gram tepung tulang ikan tongkol (T4) memperoleh nilai terendah dengan nilai 3.63 (agak coklat), kemudian dengan tambahan 10 gram tepung tulang ikan tongkol (T2) dan yang tertinggi adalah dengan tambahan 15 gram tepung tulang ikan tongkol (T3) dengan nilai 4.34 (cenderung krem). Sedangkan T2-T3 tidak menunjukkan ada perbedaan yang nyata. p-ISSN: 2776-0952 e-ISSN : 2776-0944 Hal 46-60 p-ISSN: 2776-0952 e-ISSN : 2776-0944 Hal 46-60 Tabel 2. Data Hasil Uji Organoleptik Kriteria Tekstur Konsentrasi Kriteria Uji Tekstur 0 gram T11 2.84a 0 gram T12 2.91a 10 gram T21 3.16a 10 gram T22 3.03a 15 gram T31 2.97a 15 gram T32 2.84a 20 gram T41 3.06a 20 gram T42 2.94a Tabel 2. Data Hasil Uji Organoleptik Kriteria Tekstur Berdasarkan Tabel 2 hasil uji organoleptik tekstur menunjukkan bahwa nilai rata-rata uji mutu hedonik aspek tekstur pada T1 = 2.87 (cenderung agak keras), T2 = 3.09 (cenderung renyah), T3 = 2.90 (cenderung agak keras), T4=3 (cenderung renyah). Hal ini berhubungan dengan penambahan tepung tulang ikan kedalam kue kering, semakin besar penambahan tulang ikan akan menambah kerenyahan kue kering yang dihasilkan. Kerenyahan kue kering disebabkan tepung tulang ikan mengandung protein, dimana protein memiliki gugus hidrofil (Pratiwi, 2008). Gugus hidrofil pada protein jauh lebih banyak dibandingkan banyak dibandingkan pati, sehingga tekstur semakin bertambah gurih dan renyah (Tababaka, 2004). Hasil uji anova didapatkan bahwa terdapat pengaruh penambahan tepung tulang ikan tongkol terhadap sifat organoleptik tekstur produk kue kering sagu karena nilai p value > 0.05 yaitu sebesar 0.928 yang artinya H0 diterima. Secara deskriptif tidak adanya pengaruh penambahan tepung tulang ikan tongkol terhadap tekstur produk kue kering sagu. Tekstur Tekstur merupakan kategori penilaian uji organoleptik mutu hedonik Tekstur kue kering sagu umumnya keras. Penambahan tepung tulang ikan tongkol dengan konsentrasi berbeda pada penelitian ini dilakukan untuk mengetahui pengaruh konsentrasi tepung tulang ikan tongkol yang diberikan terhadap aspek tekstur kue kering sagu. Kategori penilaian tekstur kue kering sagu meliputi keras sampai amat sangat renyah. Hasil uji organoleptik terhadap aspek tekstur dapat dilihat pada Tabel 2. 50 Open Journal System (OJS): journal.thamrin.ac.id ͬ74 Aroma Aroma termasuk dalam penilaian uji organoleptik. Penilaian aroma dilakukan dengan indera penciuman. Aroma kue kering sagu berasal dari aroma khas kelapa sangrai. Penambahan tepung tulang ikan tongkol dengan konsentrasi berbeda pada penelitian ini dilakukan untuk mengetahui pengaruh konsentrasi tepung tulang ikan yang diberikan terhadap aroma. Kategori penilaian aroma kue kering sagu meliputi tidak beraroma ikan sampai aroma ikan amat sangat kuat. Berdasarkan Tabel 3, hasil uji organoleptik menunjukkan bahwa nilai rata-rata uji mutu hedonik aspek aroma pada T1 = 4.77 (agak beraroma ikan), T2 = 3.92 (cenderung aroma ikan), T3 = 3.24 (cenderung aroma ikan), T4=3.04 (cendrung aroma ikan). 51 Open Journal System (OJS): journal.thamrin.ac.id Open Journal System (OJS): journal.thamrin.ac.id ͬ74 Jurnal Kesehatan Masyarakat Perkotaan p-ISSN: 2776-0952 e-ISSN : 2776-0944 sŽůƵŵĞ2͕EŽ͘ϭ͖DĂƌĞƚ2Ϭ22 Hal 46-60 Jurnal Kesehatan Masyarakat Perkotaan sŽůƵŵĞ2͕EŽ͘ϭ͖DĂƌĞƚ2Ϭ22 Jurnal Kesehatan Masyarakat Perkotaan p-ISSN: 2776-0952 e-ISSN : 2776-0944 Hasil uji anova didapatkan bahwa terdapat pengaruh penambahan tepung tulang ikan tongkol terhadap sifat organoleptik aroma produk kue kering sagu karena nilai p value < 0.05 yaitu sebesar 0.000 yang artinya H0 ditolak. Secara deskriptif terlihat bahwa semakin bertambah konsentrasi tepung tulang ikan, produk kue kering sagu cenderung beraroma ikan agak kuat. sŽůƵŵĞ2͕EŽ͘ϭ͖DĂƌĞƚ2Ϭ22 Hal 46-60 p-ISSN: 2776-0952 e-ISSN : 2776-0944 Hal 46-60 y 2Ϭ22 2͕ Hasil uji anova didapatkan bahwa terdapat pengaruh penambahan tepung tulang ikan tongkol terhadap sifat organoleptik aroma produk kue kering sagu karena nilai p value < 0.05 yaitu sebesar 0.000 yang artinya H0 ditolak. Secara deskriptif terlihat bahwa semakin bertambah konsentrasi tepung tulang ikan, produk kue kering sagu cenderung beraroma ikan agak kuat. Hasil uji organoleptik terhadap aspek aroma dapat dilihat pada Tabel 3 Hasil uji organoleptik terhadap aspek aroma dapat dilihat pada Tabel 3 Tabel 3 Data Hasil Uji Organoleptik Kriteria Aroma Konsentrasi Kriteria Uji Aroma 0 gram T11 4.88c 0 gram T12 4.66c 10 gram T21 3.97b 10 gram T22 3.88b 15 gram T31 3.21a 15 gram T32 3.28a 20 gram T41 3.00a 20 gram T42 3.09a Tabel 3 Data Hasil Uji Organoleptik Kriteria Aroma Hasil analisis statistik memunjukkan ada perbedaan yang nyata pada penambahan tulang ikan tongkol terhadap tingkat kesukaan aspek aroma kue kering sagu. Untuk mengetahui pengaruh tersebut maka dilakukan uji lanjut Duncan. Berdasarkan hasil uji Duncan terhadap mutu hedonik aspek aroma terlihat bahwa T1-T2, T1-T3, dan T2-T4 menunjukkan perbedaan yang nyata. Aroma Urutan nilai terendah sampai tertinggi terhadap aspek warna yaitu perlakuan penambahan 20 gram tepung tulang ikan (T4) memperoleh nilai terendah dengan nilai 3.00 (aroma ikan), kemudian dengan tambahan tepung tulang ikan tongkol 10 gram (T2) dan yang tertinggi adalah dengan tanpa tambahan (T1) dengan nilai 4.88 (cenderung agak beraroma ikan). Sedangkan T3-T4 tidak menunjukkan ada perbedaan yang nyata. Rasa Salah satu faktor yang menentukan kualitas makanan adalah kandungan senyawa citarasa. Menurut West, et al., dalam Margaretha dan Edwin (2012) titik perasa dari lidah adalah kemampuan mendeteksi dasar yaitu manis, asam, asin, pahit, trigeminal (astringent, dingin, panas) dan aroma setelah mengkonsumsi senyawa tersebut. Lewat proses pemberian aroma pada suatu produk pangan, lidah dapat mengecap rasa lain sesuai aroma yang diberikan (Midayanto dan Yuwono, 2014). Dalam pengujian ini dilakukan terhadap kriteria uji rasa 52 Open Journal System (OJS): journal.thamrin.ac.id Open Journal System (OJS): journal.thamrin.ac.id ͬ74 Jurnal Kesehatan Masyarakat Perkotaan p-ISSN: 2776-0952 e-ISSN : 2776-0944 sŽůƵŵĞ2͕EŽ͘ϭ͖DĂƌĞƚ2Ϭ22 Hal 46-60 Jurnal Kesehatan Masyarakat Perkotaan p-ISSN: 2776-0952 e-ISSN : 2776-0944 sŽůƵŵĞ2͕EŽ͘ϭ͖DĂƌĞƚ2Ϭ22 Hal 46-60 p-ISSN: 2776-0952 e-ISSN : 2776-0944 Hal 46-60 p-ISSN: 2776-0952 e-ISSN : 2776-0944 Hal 46-60 y ƚ2Ϭ22 2͕ untuk mutu hedonik yang menyatakan kekhasan rasa kue kering sagu. Hasil uji organoleptik terhadap kriteria uji rasa dapat dilihat pada Tabel 4 untuk mutu hedonik yang menyatakan kekhasan rasa kue kering sagu. Hasil uji organoleptik terhadap kriteria uji rasa dapat dilihat pada Tabel 4 Tabel 4 Data Hasil Uji Organoleptik Kriteria Rasa Tabel 4 Data Hasil Uji Organoleptik Kriteria Rasa Konsentrasi Kriteria Uji Rasa 0 gram T11 3.94bc 0 gram T12 4.19c 10 gram T21 3.53abc 10 gram T22 3.63abc 15 gram T31 3.47abc 15 gram T32 3.09a 20 gram T41 3.22ab 20 gram T42 3.44ab Berdasarkan Tabel 4, hasil uji organoleptik menunjukkan bahwa nilai rata-rata uji mutu hedonik aspek rasa pada T1 = 4.06 (cenderung gurih tidak manis), T2 = 3.58 (cenderung gurih), T3 = 3.28 (cenderung gurih), T4=3.33 (cenderung gurih). Hasil uji anova didapatkan bahwa terdapat pengaruh penambahan tepung tulang ikan tongkol terhadap sifat organoleptik mutu hedonik rasa produk kue kering sagu karena nilai p value > 0.05 yaitu sebesar 0.025 yang artinya H0 ditolak. Secara deskriptif terlihat bahwa penambahan tulang ikan tongkol berpengaruh nyata terhadap aspek rasa kue kering sagu. Hal ini didukung dengan hasil analisis statistik memangmenunjukkan ada perbedaan yang nyata pada penambahan tepung tulang ikan terhadap tingkat kesukaan aspek rasa kue kering sagu. Untuk mengetahui pengaruh tersebut maka dilakukan uji lanjut Duncan, hasilnya terlihat bahwa seluruh perlakuan menunjukkan perbedaan yang tidak nyata, yaitu T1-T2, T1-T3, T1-T4, T2-T3, T2-T4 dan T3-T4. Rasa Urutan nilai terendah sampai tertinggi terhadap aspek tingkat kesukaan rasa yaitu perlakuan dengan tambahan Tepung tulang ikan 15 gram (T3) memperoleh nilai terendah dengan nilai 3.09 (cenderung gurih), kemudian penambahan tepung tulang ikan tongkol 10 gram (P2) dan yang tertinggi adalah dengan tambahan tepung tulang ikan tongkol (P1) dengan nilai 4.19 (cenderung gurih agak manis). Open Journal System (OJS): journal.thamrin.ac.id Jurnal Kesehatan Masyarakat Perkotaan sŽůƵŵĞ2͕EŽ͘ϭ͖DĂƌĞƚ2Ϭ22 2͕ Tingkat kesukaan Tingkat kesukaan meliputi penilaian terhadap aspek penilaian aroma, warna, tekstur dan rasa dari kue kering sagu dengan penambahan tepung tulang ikan tongkol. Oleh karena itu 53 Open Journal System (OJS): journal.thamrin.ac.id ͬ74 Kesehatan Masyarakat Perkotaan p-ISSN: 2776-0952 e-ISSN : 2776-0944 Ğ2͕EŽ͘ϭ͖DĂƌĞƚ2Ϭ22 Hal 46-60 p-ISSN: 2776-0952 e-ISSN : 2776-0944 Hal 46-60 y ƚ2Ϭ22 2͕ dilakukan uji organoleptik untuk mengetahui tingkat kesukaan konsumen terhadap kue kering sagu tersebut dan hasilnya dapat dilihat pada tabel 5 dilakukan uji organoleptik untuk mengetahui tingkat kesukaan konsumen terhadap kue kering sagu tersebut dan hasilnya dapat dilihat pada tabel 5 Tabel 5. Data Hasil Uji Organoleptik Kriteria Tingkat Kesukaan Konsentrasi Kriteria Uji tingkat kesukaan 0 gram T11 2.84de 0 gram T12 3.00e 10 gram T21 2.47bcd 10 gram T22 2.59cde 15 gram T31 2.13abc 15 gram T32 1.88a 20 gram T41 1.69a 20 gram T42 1.97ab Berdasarkan Tabel 5, hasil uji organoleptik mutu hedonik menunjukkan bahwa nilai rata- rata uji mutu hedonik aspek tingkat keseukaan secara keseluruhan pada T1 = 2.92 (cenderung suka), T2 = 2.53 (cenderung suka), T3 = 2.00 (cenderung agak suka), T4=1.83( cenderung tidak suka). Hasil uji anova didapatkan bahwa terdapat pengaruh penambahan tepung tulang ikan tongkol terhadap sifat organoleptik mutu hedonik tingkat kesukaan secara keseluruhan produk kue kering sagu karena nilai p value > 0.05 yaitu sebesar 0.00 yang artinya H0 ditolak. Secara deskriptif terlihat bahwa penambahan tepung tulang ikan tongkol berpengaruh nyata terhadap aspek tingkat kesukaan kue kering sagu. Hal ini didukung dengan analisis statistik memang ada perbedaan yang nyata pada penambahan tepung tulang ikan tongkol terhadap tingkat kesukaan aspek tingkat kesukaan secara kesluruhan. Untuk mengetahui pengaruh tersebut maka dilakukan uji lanjut Duncan, terlihat bahwa seluruh perlakuan menunjukkan perbedaan yang tidak nyata, yaitu T1-T2, T2-T3, T2-T4, dan T3-T4. Urutan nilai terendah sampai tertinggi terhadap aspek tingkat kesukaan rasa yaitu perlakuan dengan tambahan tepung tulang ikan tongkol 20 gram (P4) memperoleh nilai terendah dengan nilai 1.69 (cenderung agak suka), kemudian dengan penambahan tepung tulang ikan tongkol (T2) dan yang tertinggi adalah dengan tanpa tambahan tepung tulang ikan tongkol (T1) dengan nilai 3.00 (cenderung suka). 54 Open Journal System (OJS): journal.thamrin.ac.id Open Journal System (OJS): journal.thamrin.ac.id ͬ74 p-ISSN: 2776-0952 e-ISSN : 2776-0944 p-ISSN: 2776-0952 e-ISSN : 2776-0944 Hal 46-60 p-ISSN: 2776-0952 e-ISSN : 2776-0944 Hal 46-60 Penentuan Produk Terpilih Penentuan produk terpilih ditentukan berdasarkan hasil uji tingkat kesukaan (hedonik) dan seberapa banyak aktivitas Proksimat dan Kalsium yang dapat disumbangkan oleh tepung tulang ikan tongkol ke dalam Kue Kering Sagu. Uji hedonik kue kering sagu meliputi warna, tekstur, aroma dan rasa. Parameter uji hedonik yang digunakan adalah 1 (tidak suka) hingga 5 (amat sangat suka). Nilai rata-rata dari hasil uji tingkat kesukaan (hedonik) tepung tulang ikan tongkol dapat dilihat pada Tabel 6 Tabel 6 Hasil Uji Mutu Hedonik Kue Kering Sagu Tabel 6 Hasil Uji Mutu Hedonik Kue Kering Sagu Perlakuan Kategori Warna Tekstur Aroma Rasa Tingkat kesukaan 0 gram T11 4.47cd 2.84a 4.88c 3.94bc 2.84de 0 gram T12 4.69d 2.91a 4.66c 4.19c 3.00e 10 gram T21 3.84ab 3.16a 3.97c 3.53abc 2.47bcd 10 gram T22 3.44a 3.03a 3.88b 3.63aabc 2.59cde 15 gram T31 4.38bcd 2.97a 3.31a 3.47abc 2.13abc 15 gram T32 4.31bcd 2.84a 3.28a 3.09a 1.88a 20 gram T41 3.88abc 3.06a 3.09a 3.44ab 1.69a 20 gram T41 3.38a 2.94a 3.00a 3.22ab 1.97ab Ket : Huruf yang beda pada baris yang sama menunjukkan perbedaan yang nyata (p<0,05) Ket : Huruf yang beda pada baris yang sama menunjukkan perbedaan yang nyata (p<0,05) Berdasarkan Tabel 6 nilai tertinggi pada kategori penilaian warna adalah T12 (tanpa penambahan tulang ikan tongkol) yaitu 4.69 (cenderung krem kecoklatan ). Nilai tertinggi pada kategori tekstur adalah T21 (10 gram) yaitu 3.16 (cenderung renyah). Nilai tertinggi pada kategori aroma adalah T12 (tanpa penambahan tulang ikan tongkol) yaitu 4.88 (cenderung agak beraroma ikan ). Nilai tertinggi pada kategori rasa adalah T21 (tanpa penambahan tulang ikan tongkol) yaitu 4.19 (cenderung gurih agak manis). Nilai tertinggi dari tingkat kesukaan adalah T12 (tanpa penambahan tepung tulang ikan tongkol) yaitu 3.00 (cenderung suka). Dapat disumpulkan berdasarkan hasil uji mutu hedonik, diketahui produk terpilih adalah T2 yaitu perlakuan dengan penambahan tepung tulang ikan tongkol sebanyak 10% yang memiliki nilai tertinggi dari tingkat kesukaan aspek tekstur dan rasa. Deskripsi sifat fisik produk kue kering sagu terpilih dapat dilihat pada Tabel 7 55 Open Journal System (OJS): journal.thamrin.ac.id Open Journal System (OJS): journal.thamrin.ac.id ͬ74 p-ISSN: 2776-0952 e-ISSN : 2776-0944 Hal 46-60 p-ISSN: 2776-0952 e-ISSN : 2776-0944 Hal 46-60 Tabel 7 Deskripsi Sifat Fisik Kue Kering Sagu Produk Terpilih (T2) Aspek Mutu Hedonik Warna Cenderung krem kecoklatan Tekstur Cenderung renyah Aroma Cenderung agak aroma ikan Rasa Cenderung gurih tidak manis Tingkat kesukaan Cenderung suka Tabel 7 Deskripsi Sifat Fisik Kue Kering Sagu Produk Terpilih (T2) Analisis Zat Gizi Nilai gizi kue kering sagu tertera pada setiap perlakuan berdasarkan bahan dari resep dasar dengan penambahn tepung tulang ikan tongkol. Kebutuhan bahan masing-masing kue kering sagu sesuai perlakuan untuk satu resep dapat dilihat pada Tabel 8 Tabel 8 Kebutuhan Kue Kering Sagu Untuk 1 Resep Tabel 8 Kebutuhan Kue Kering Sagu Untuk 1 Resep Tabel 8 Kebutuhan Kue Kering Sagu Untuk 1 Resep Komposisi Jumlah bahan yang di gunakan untuk satu resep (gram) T1 T2 T3 T4 Tepung sagu 150 150 150 150 Gula 37.5 37.5 37.5 37.5 Rombutter 15 15 15 15 Telur 15 15 15 15 Margarin 45 45 45 45 Kelapa 15 15 15 15 Garam 1.5 1.5 1.5 1.5 Tepung tulang ikan tongkol 0 15 22.5 30 Jumlah Produk 60keping 70 keping 75 keping 80 keping Berat total bahan 279 gr 289 gr 294 gr 299 gr Berdasarkan Tabel 8 satu resep dihasilkan T1 70 keping, T2 81 keping T3 87 keping dan T4 93 keping. Sehingga T1, T2, T3, dan T4 memerlukan 1.5 resep untuk mencapai target panelis 30 panelis. Dengan berat 1 Resep kue kering sagu 240 gram dengan berat 1 keping 6 gram. Analisis zat gizi makro dapat dilihat pada tabel pada tabel 9 dihitung menggunakan data sekunder berdasarkan TKPI tahun 2009. Tabel 9. Zat Gizi Makro dan Kalsium kue kering sagu menurut TKPI Kandungan gizi Per Resep % penambahan tepung tulang ikan 0% 10% 15% 20 % Kalori 321 321 321 321 Karbohidrat 52,91 52,91 52,91 52,91 Protein 1.1 4,25 6,23 7,94 Lemak 13.98 14,54 14.82 15.1 Kalsium - 1.19 mg 1.78 mg 2.38 mg 56 Open Journal System (OJS): journal.thamrin.ac.id ͬ74 Jurnal Kesehatan Masyarakat Perkotaan p-ISSN: 2776-0952 e-ISSN : 2776-0944 sŽůƵŵĞ2͕EŽ͘ϭ͖DĂƌĞƚ2Ϭ22 Hal 46-60 p-ISSN: 2776-0952 e-ISSN : 2776-0944 Hal 46-60 p-ISSN: 2776-0952 e-ISSN : 2776-0944 Hal 46-60 y ƚ2Ϭ22 2͕ Analisis zat Gizi pada penelitian ini untuk produk terpilih di dapatkan dari hasil uji proksimat dan uji kalsium yang dilakukan di laboratorium VICMALAB. Hasil penelitian dapat dilihat pada tabel 10 Tabel 10. Perbandingan nilai gizi makro dan kalsium dengan hasil LAB dan KPI Tabel 10. KESIMPULAN Berdasarkan hasil penelitian dapat disimpulkan bahwa Karakteristik tepung tulang ikan adalah berwarna krem kecoklatan, beraroma ikan agak tajam (amis) dan ada pengaruh penambahan tepung tulang ikan terhadap aspek aroma, warna, rasa dan tingkat kesukaan secara keseluruhan. Berdasarkan hasil uji organoleptic mutu hedonik Warna kue kering sagu (T1 -T4) adalah krem kecoklatan, Tekstur adalah keras cenderung renyah, terhadap Aroma adalah cenderung beraroma ikan, Rasa kue kering sagu adalah gurih cenderung tidak manis, Tingkat kesukaan secara keseluruhan (Aroma, Rasa, Tekstur,dan Warna) kue kering sagu terpilih adalah cenderung suka. Adapun Produk terpilih dari penilaian kesukaan secara keseluruhan (Aroma, Rasa, Tekstur,dan Warna) adalah T2 dengan penambahan tepung tulang ikan 10% dengan nilai gizi produk terpilih adalah sebagai berikut : Energi (368 Kkal), Protein (10 gram), Karbohidrat (68 gram), Lemak (6 gram), dan Kalsium (0,1mg/100gram), dengan Harga jual produk sebesar Rp. 570/ ressp (240gr). Analisis Zat Gizi Perbandingan nilai gizi makro dan kalsium dengan hasil LAB dan KPI Parameter Satuan Unit Result TKPI Kadar air % 13.36 - Kadar Abu % 2.04 - Lemak total % 6 14.54 Protein % 10 4.25 Karbohidrat % 68 52.91 Kalsium mg/100g 0.1 1.19 Energi total Kcal/100g 368 321 Berdasarkan tabel 10 hasil penelitian didapatkan kadar air kue kering yang dihasilkan sebesar 13.36 yang terdapat pada kue kering yang terdapat pada tulang ikan sehibgga kadar air berkurang. Menurut mahani 1999, adanya penambahan tepung tulang ikan berarti terjadi penambahan partikel kalsium yang akan mengikat partikel air yang merupakan bagian dari unsur-unsur air sehingga kadar air berkurang seiring dengan penambahan tepung tulang ikan. Kadar abu terhadap kue kering sebesar 2,04 dalam tulang ikan terkandung sel-sel dalam bentuk garam mineral. Garam mineral merupakan komponen yang terdiri dari kalsium posfat sebanyak 80% dan sisanya terdiri dari kalsium karbonat dan magnesium posfat (Tababaka, 2004), sehingga unsur ini membuat kadar abu padsa kue kering meningkat. Peningkatan kadar abu terhadap produk akibat tepung tulang ikan yang digunakan. Sedangkan, pada uji proksimat dan kalsium yang dilakukan pada produk terpilih pada kue kering sagu dan menggunakan TKPI tidak berbeda jauh. Tabel 11 Hasil Uji Zat Gizi Makro Dalam 30 Gram Kue Kering Sagu Nama bahan Kalori Karbohidrat Protein Lemak Tepung sagu 45.25 10.86 0.06 0.03 Gula 20.6 4.9 0 0 Roombutter 38.95 0.07 0.026 4.27 Margarin 37.8 0.02 0.03 4.25 Kelapa 0.35 0.72 0.05 0.04 Telur 8.07 0.04 0.65 0.56 Tabel 11 Hasil Uji Zat Gizi Makro Dalam 30 Gram Kue Kering Sagu 57 Open Journal System (OJS): journal.thamrin.ac.id ͬ74 p-ISSN: 2776-0952 e-ISSN : 2776-0944 Hal 46-60 p-ISSN: 2776-0952 e-ISSN : 2776-0944 Hal 46-60 Jurnal Kesehatan Masyarakat Perkotaan 2͕ REFERENSI Almatsier, Sunita. 2009. Prinsip dasar ilmu gizi. Gramedia, Jakarta. Almatsier, Sunita. 2009. Prinsip dasar ilmu gizi. Gramedia, Jakarta. Anni Faridah. 2009. Patiseri .Jakarta: Direktorat pembinaan sekolah menengah kejuruan. Anni Faridah. 2009. Patiseri .Jakarta: Direktorat pembinaan sekolah menengah kejuruan. Asni, Yuli. 2004. Studi pembuatan biskuit dengan penambahan tepung tulang ikan patin. Skripsi. Fakultas perikanan dan ilmu kelautan. 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https://www.biorxiv.org/content/biorxiv/early/2017/02/07/106500.full.pdf
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Optimal Response Vigor and Choice Under Non-stationary Outcome Values
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. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint Bernard W. Balleine School of Psychology, UNSW, NSW 2052, Australia Bernard W. Balleine School of Psychology, UNSW, NSW 2052, Australia Richard Nock Data61, CSIRO, NSW 2015, Australia . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint Optimal Response Vigor and Choice Under Non-stationary Outcome Values Amir Dezfouli School of Psychology, UNSW, NSW 2052, Australia Affiliation: School of Psychology, UNSW Email: a.dezfouli@unsw.edu.au Address: School of Psychology, UNSW, NSW 2052, Australia Abstract Within a rational framework a decision-maker selects actions based on the reward-maximisation principle which stipulates they acquire outcomes with the highest values at the lowest cost. Action selection can be divided into two dimen- sions: selecting an action from several alternatives, and choosing its vigor, i.e., how fast the selected action should be executed. Both of these dimensions de- pend on the values of the outcomes, and these values are often affected as more outcomes are consumed, and so are the actions. Despite this, previous works have addressed the computational substrates of optimal actions only in the spe- cific condition that the values of outcomes are constant, and it is still unknown what the optimal actions are when the values of outcomes are non-stationary. Here, based on an optimal control framework, we derive a computational model for optimal actions under non-stationary outcome values. The results imply that even when the values of outcomes are changing, the optimal response rate is constant rather than decreasing. This finding shows that, in contrast to previ- ous theories, the commonly observed changes in the actions cannot be purely attributed to the changes in the outcome values. We then prove that this obser- vation can be explained based on the uncertainty about temporal horizons; e.g., in the case of experimental protocols, the session duration. We further show that when multiple outcomes are available, the model explains probability match- ing as well as maximisation choice strategies. The model provides, therefore, a quantitative analysis of optimal actions and explicit predictions for future test- ing. Keywords: choice, responser vigor, reward learning, optimal actions . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint OPTIMAL RESPONSE VIGOR AND CHOICE 2 Disclosures and Acknowledgments Contents Contents Contents Introduction 4 Model Specification 5 The outcome space . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 The reward . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Cost . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Value . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Results 7 Optimal response vigor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Response vigor when the session duration is known . . . . . . . . . . . . . . . . . . 7 Response vigor when session duration is unknown . . . . . . . . . . . . . . . . . . . 8 Effect of experimental parameters: Relating the model to currently available data . 10 Optimal choice and response vigor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Conservative reward field . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Non-conservative reward field . . . . . . . Contents . . . . . . . . . . . . . . . . . . . . . . . . 15 Discussion 16 References 17 Appendix 19 Value in non-deterministic schedules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Optimal actions in one-dimensional outcome space . . . . . . . . . . . . . . . . . . . . . 21 Theorem 1: Proof . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Theorem 2: Proof and simulation details . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Proof of Theorem 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Simulation details . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Simulation details of Figures 2, 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Optimal actions in multi-dimensional outcome space . . . . . . . . . . . . . . . . . . . . 25 Theorem 3: Proof and simulation details . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Proof of Theorem 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Disclosures and Acknowledgments A.D completed this work when was employed by CSIRO. B.W.B. was supported by a Senior Principal Research Fellowship from the National Health & Medical Research Council of Australia, GNT1079561. All authors contributed in a significant way to the manuscript and that all authors have read and approved the final manuscript. The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. We are grateful to Hadi Lookzadeh and Peter Dayan for helpful discussions. We are grateful to Hadi Lookzadeh and Peter Dayan for helpful discussions. . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint OPTIMAL RESPONSE VIGOR AND CHOICE 3 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint Introduction According to normative theories of decision-making, actions made by humans and ani- mals are chosen with the aim of earning the maximum amount of future reward whilst incurring the lowest cost (Marshall, 1890; von Neumann & Morgenstern, 1947). Within such theories indi- viduals optimize their actions by learning about their surrounding environment so as to satisfy their long-term objectives. The problem of finding the optimal action is, however, argued to have two aspects: (1) choice, i.e., deciding which action to select from several alternatives; and (2) vigor, i.e., deciding how fast the selected action should be executed. For a rat in a Skinner box, for example, the problem of finding the optimal action involves selecting a lever (choice) and de- ciding at what rate to respond on that lever (vigor). High response rates can have high costs (e.g., in terms of energy consumption), whereas a low response rate could have an opportunity cost if the experimental session ends before the animal has earned sufficient reward. Optimal actions provide the right balance between these two factors and, based on the reinforcement-learning framework and methods from optimal control theory, the characteristics of optimal actions and their consistency with various experimental studies have been previously elaborated (Dayan, 2012; Niv, Daw, Joel, & Dayan, 2007; Salimpour & Shadmehr, 2014). These previous models have assumed, however, that outcome values are stationary and do not change on-line over the course of a decision-making session. To see the limitations of such an assumption, imagine the rat is in a Skinner box and has started to earn outcomes (e.g., food pellets) by taking actions. One can assume that, as a result of consuming rewards, the motivation of the animal to earn more food outcomes will decrease (e.g., because of satiety) and, therefore, over time, the outcomes earned will have a lower value. Such changes in value should affect both optimal choice and vigor (Killeen, 1995) but have been largely ignored in the previous models. This is while in most of the experimental and real-world scenarios, outcome values are affected by the history of outcome consumption, a phenomenon known as “law of diminishing marginal utility”1 in the economics literature, and as “drive reduction theory” in psychological accounts of motivation, which indicates that the drive for earning an outcome decreases as the consequence of prior consumption of the outcomes (Hull, 1943; Keramati & Gutkin, 2014). OPTIMAL RESPONSE VIGOR AND CHOICE 4 1Also known as “First Law of Gossen” named for Hermann Heinrich Gossen (1810 – 1858). Contents It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint Contents . 27 Simulation details . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Theorem A2: Definition, proof and simulation details . . . . . . . . . . . . . . . . . . . . 28 Proof of Theorem A2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Simulation details . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Theorem A3: Definition, proof and simulation details . . . . . . . . . . . . . . . . . . . . 29 Proof of Theorem A3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Simulation details . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Introduction Here, building on previous work, we introduce a new concept called reward field, which captures non-stationary outcome values. Using this concept and methods from optimal control theory, we derive the optimal response vigor and choice strategy without assuming that outcome values are stationary. In particular, the results indicate that even when the values of outcomes are changing, the optimal response rate in an instrumental conditioning experiment is a con- stant response rate. This finding rules out previous suggestions that the commonly observed decrease in within-session response rates is due to decreases in outcome value (Killeen, 1995). Instead, we show that decreases in within-session response rates can be explained by uncer- tainty regarding session duration. This later analysis is possible because the session duration is explicitly represented in the current model, which is another dimension in which the current model extends previous work. The framework is then extended to choice situations and specific predictions are made concerning conditions under which the optimal strategy is maximization or probability matching. . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint The outcome space We define the outcome space as a coordinate space with n dimensions, where n is the number of outcomes in the environment. For example, in a concurrent instrumental condition- ing experiment in which the outcomes are water and food pellets, the outcome space will have two dimensions corresponding to water and food pellets. Within the outcome space, the state of the decision-maker at time t is defined by two factors: (i) the amount of earned outcome up to time t, which is denoted by xt and can be thought of as the position of the decision-maker in outcome space; e.g., in the above example, xt = [1,2] would indicate that one unit of water and two units of food pellet have been gained up to time t; and (ii) the outcome rate at time t, denoted by vt, which can be considered the velocity of the decision-maker in the outcome space (vt = dxt/dt); e.g., if a rat is earning two units of water and one unit of food pellet per unit of time, then vt = [2,1]. In general, we assume that the outcome rate cannot be negative (v ≥0), which means that the cumulative number of earned outcomes cannot decrease with time. OPTIMAL RESPONSE VIGOR AND CHOICE 5 The reward We assume that there exists an n-dimensional reward field, denoted by Ax,t, where each element of Ax,t represents the per unit value of each of the outcomes. For example, the element of Ax,t corresponding to food pellets represents the value of one unit of food pellet at time t, given that x units of outcome have been previously consumed. As such, Ax,t is a function of both time and the amount of outcome earned. This represents the fact that (i) the reward value of an outcome can change value as a result of consuming previous outcomes, e.g., due to satiety (depending on x) and (ii) the reward value of an outcome can change purely with the passage of time; e.g., an animal can get hungrier over time causing the reward value of food pellets to increase (depending on t). In general, we assume that Ax,t has two properties: ∂Ax,t ∂x ≤0, ∂Ax,t ∂t ≥0, (1) (1) which entail that (i) the outcome values decrease (or remain constant) as more outcomes are earned, and (2) that outcome values do not decrease with the passage of time. Cost Within the context of instrumental conditioning experiments, previous studies have ex- pressed the cost of earning outcomes as a function of the delay between consecutive responses made to earn outcomes. For example, if a rat is required to make several lever presses to earn outcomes, then the cost will be higher if the delay between lever presses is short. More precisely, if the previous response has occurred τ time steps ago, then the cost of the current lever press will be (Dayan, 2012; Niv et al., 2007): Cτ = a τ +b, (2) (2) where a and b are constants. b is the constant cost of each lever press, which is independent of the delay between lever presses whereas the factor a controls the rate-dependent component . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint OPTIMAL RESPONSE VIGOR AND CHOICE 6 6 of the cost. Previous research has established that predictions derived from this definition of cost are consistent with experimental data (Dayan, 2012; Niv et al., 2007). Note that costs such as basal metabolic rate and the cost of operating the brain, although consuming a high portion of energy produced by the body, are not included in the above definition because they are con- stant and independent of response rate and, therefore, are not directly related to the analysis of response vigor and choice. Here, we express cost as a function of outcome rate rather than the rate of action execu- tion. We define the cost function Kv as the cost paid at each time step for earning outcomes at rate v. In the specific case that the outcome space has one dimension (there is only one out- come), and under ratio schedules of reinforcement (fixed-ratio, variable-ratio, random-ratio) in which the decision-maker is required to perform either precisely or on average k responses to earn one unit of outcome, the cost defined in equation 2 will be equivalent to: Kv = ak2v2 +kbv. (3) (3) Kv = ak2v2 +kbv. See Theorem A1 in Appendix for the proof. This definition of cost implies that it is only a func- tion of outcome rate and is time-independent (∂Kv/∂t = 0). Although, in general, it may seem reasonable to assume that, as time passes within a session, the cost of taking actions will in- crease because of factors such as effector fatigue, here we made a time-independence assump- tion based on previous studies showing that factors such as effector fatigue have a negligible effect on response rate (McSweeney, Hinson, & Cannon, 1996). In general, we assume that at least one response is required to earn an outcome (k > 0), and the cost of earning outcomes is non-zero (a > 0). OPTIMAL RESPONSE VIGOR AND CHOICE 7 7 Value The reward earned in each time step can be calculated as the reward of one unit of each of the outcomes (Ax,t) multiplied by the amount earned from each of the outcomes, which will be v.Ax,t. The cost of earning this amount of reward is Kv, and therefore the net amount of reward earned will be: (4) Lx,v,t = v.Ax,t −Kv. (4) A decision-making session starts at t = 0 and the total duration of that session is T . We denote the total reward gained in this period as S0,T , which is the sum of the net rewards earned at each point in time: S0,T = ZT 0 Lx,v,tdt. (5) (5) The quantity S0,T is called the value function, and the goal of the decision-maker is to find the optimal rate of earning outcomes that yields the highest value. The optimal rates that maximize S0,T can be found using different variational calculus methods, such as the Euler-Lagrange equa- tion or the Hamilton-Jacobi-Bellman equation (Liberzon, 2011). The results presented in the next sections are derived using the Euler-Lagrange equation (see Appendix for details of value function in non-deterministic schedules). . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint Response vigor when the session duration is known. We maintain the following theo- rem: Response vigor when the session duration is known. We maintain the following theo- rem: Theorem 1 If the duration of the session is fixed and the time-dependent change in the reward field is zero (∂Ax,t/∂t = 0), then the optimal outcome rate is constant (dv/dt = 0). If the time- dependent change in the reward field is positive (∂Ax,t/∂t > 0), then the optimal outcome rate will be accelerating (dv/dt > 0). See Appendix for a proof of this theorem. Note that the assumption ∂Ax,t/∂t = 0 implies that the passage of time has no significant effect on the reward value of the outcome; e.g., a rat is not get- ting hungrier during an instrumental conditioning session, which is a reasonable assumption given the short duration of such experiments (typically less than an hour). Within this condi- tion, the above theorem states that the optimal response rate is constant throughout the session, even under conditions in which the reward value of the outcome decreases within the session as a result of earning outcomes, e.g., because of satiety. As an intuitive explanation for why a constant rate is optimal, imagine a decision-maker who chooses a non-constant outcome rate that results in a total of xT outcomes during the session. If, instead of the non-constant rate, the decision-maker selects a constant rate v = xT /T , then the total outcomes earned will be the same as before; however, the cost will be lower because cost is a quadratic function of the out- come rate and, therefore, it is better to earn outcomes at a constant rate. Nevertheless, although this prediction is clear enough, it is not consistent with the experimental results, described next. Within-session pattern of responses. It has been established that in various schedules of reinforcement, including variable-ratio (VR) schedules (McSweeney, Roll, & Weatherly, 1994), the rate of responding within a session has a particular pattern: the response rate reaches its maximum a short time after the session starts (warm-up period), and then gradually decreases toward the end of the session (Figure 1:left panel). Killeen (Killeen, 1994) proposed a mathe- matical description of this phenomenon, which can be expressed as follows (Killeen & Sitomer, 2003): β = r δr +1/α, (6) (6) where β is the response rate, δ is the minimum delay between responses, r is the resulting out- come rate, and α is called specific activation2. 2Note that in the original notation in (Killeen & Sitomer, 2003), α is denoted by a and β is denoted by b. Optimal response vigor In this section we use the model presented above to analyse optimal response vigor when there is one outcome and one response available in the environment. The analysis is divided into two sections. In the first section, we assume that the decision-maker is certain about session duration, i.e., that the session will continue for T time units, and will extend this analysis in the next section to a condition in which the decision-maker assumes a probabilistic distribution of session lengths. OPTIMAL RESPONSE VIGOR AND CHOICE Right panel: The theoretical pattern of within-session responses predicted by the model in different conditions. Please see the text for details of each condition. Figure 1. The pattern of within-session response rates (responses per minute). Left panel: Ex- perimental data. The rate of responding per minute during successive intervals (each interval is 5 minutes) in a variable-ratio (VR15) schedule (k = 15). The figure is adopted from (McSweeney et al., 1994). Right panel: The theoretical pattern of within-session responses predicted by the model in different conditions. Please see the text for details of each condition. response rate time 10 20 30 40 50 60 0 20 40 60 time (min) response rate decreasing reward and unknown session duration fixed reward (known or unknown session duration) known session duration (fixed or decreasing reward) onstant (dv/dt = 0). ward field is positive me rate will be acceler- Note that the assump- ssage of time has no Fig. 1. The pattern of within-session response rates (responses per minute). Left panel: Experimental data. The rate of responding per minute during successive intervals (each interval is 5 minutes) in a variable-ratio (VR15) schedule (k = 15). The figure is adopted from [11]. Right panel: The theoretical pattern of within-session responses predicted by the model in different conditions. Please see the text for details of each condition. Figure 1. The pattern of within-session response rates (responses per minute). Left panel: Ex- perimental data. The rate of responding per minute during successive intervals (each interval is 5 minutes) in a variable-ratio (VR15) schedule (k = 15). The figure is adopted from (McSweeney et al., 1994). Right panel: The theoretical pattern of within-session responses predicted by the model in different conditions. Please see the text for details of each condition. RAFT ; g , ental conditioning ses- ven the short duration an hour). Within this the optimal response even under conditions e decreases within the .g., because of satiety. nstant rate is optimal, non-constant outcome mes during the session. decision-maker selects outcomes earned will will be lower because me rate and therefore that the cause of any decrease in the within-session response rate cannot be due purely to a change in outcome value. OPTIMAL RESPONSE VIGOR AND CHOICE 8 cause a decrease in response rate3. Although this model has been shown to provide a quantita- tive match to the experimental data, it is not consistent with Theorem 1 which posits that, even under conditions in which outcome values are changing within a session, the optimal response rate should not decrease during the session. As a consequence, the present model suggests that the cause of any decrease in the within-session response rate cannot be due purely to a change in outcome value. cause a decrease in response rate3. Although this model has been shown to provide a quantita- tive match to the experimental data, it is not consistent with Theorem 1 which posits that, even under conditions in which outcome values are changing within a session, the optimal response rate should not decrease during the session. As a consequence, the present model suggests that the cause of any decrease in the within-session response rate cannot be due purely to a change in outcome value. Note, however, the optimal response rate advocated by Theorem 1 is not consistent with reports of decreasing response rates across a session, which implies that some of the assump- tions made to develop the model may not be accurate. Although the form of the cost and reward functions is reasonably general, we assumed that the duration of the session, T , is fixed and known by the decision-maker. In the next section we show that relaxing this assumption such that the duration of the session is unknown results much closer concordance between predic- tions from the model and the experimental data. n duration, i.e., that nits, and will extend ondition in which the distribution of session uration is known. We ssion is fixed and the ld is zero (ˆAx,t/ˆt = constant (dv/dt = 0). eward field is positive me rate will be acceler- Note that the assump- assage of time has no response rate time 10 20 30 40 50 60 0 20 40 60 time (min) response rate decreasing reward and unknown session duration fixed reward (known or unknown session duration) known session duration (fixed or decreasing reward) Fig. 1. The pattern of within-session response rates (responses per minute). Left panel: Experimental data. The rate of responding per minute during successive intervals (each interval is 5 minutes) in a variable-ratio (VR15) schedule (k = 15). The figure is adopted from [11]. Response vigor when the session duration is known. We maintain the following theo- rem: The model suggests that as the decision-maker earns outcomes during the session, the value of α gradually declines due to satiety, which will where β is the response rate, δ is the minimum delay between responses, r is the resulting out- come rate, and α is called specific activation2. The model suggests that as the decision-maker earns outcomes during the session, the value of α gradually declines due to satiety, which will . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint DR en established that uding variable-ratio inaccuracies in interval timing in animals [15, 16]. In this condition, a plausible way to calculate optimal response rates is to set an expectation as to how long the session will last 3Here satiety refers to both post-ingestive factors (such as blood glucose level; (Killeen, 1995)) and/or pre-ingestiv actors (for example sensory specific satiety; (McSweeney, 2004)). OPTIMAL RESPONSE VIGOR AND CHOICE Note, however, the optimal response rate advocated by Theorem 1 is not consistent with reports of decreasing re- sponse rates across a session, which implies that some of the assumptions made to develop the model may not be accurate. Although the form of the cost and reward functions is reason- ably general, we assumed that the duration of the session, T, is fixed and known by the decision-maker. In the next section we show that relaxing this assumption such that the duration of the session is unknown results much closer concordance between predictions from the model and the experimental data. Response vigor when session duration is unknown. In this section we assume that the decision-maker is uncertain about the session duration, which can be either because the ses- sion duration is in fact non-deterministic, or because of inherent inaccuracies in interval timing in animals (Gallistel & Gibbon, 2000; Gibbon, 1977). In this condition, a plausible way to cal- culate optimal response rates is to set an expectation as to how long the session will last and to calculate the optimal response rate based on that expectation. Based on this, if t′ time step has passed since the beginning of the session, then the expected session duration is E[T |T > t′] and therefore the value of the rest of the session will be St′,E[T |T >t′]. The following theorem maintains that the optimal rate of outcome delivery that maximizes the value function is a decreasing func- tion of the current time in the session t′, and therefore the optimal response rates will decrease throughout the session. OPTIMAL RESPONSE VIGOR AND CHOICE OPTIMAL RESPONSE VIGOR AND CHOICE 9 is non-zero (p(T ) > 0), (iii) values of outcomes decrease as more are consumed (∂Ax,t/∂x < 0), then the optimal rate of outcome delivery is a decreasing function of t′: is non-zero (p(T ) > 0), (iii) values of outcomes decrease as more are consumed (∂Ax,t/∂x < 0), then the optimal rate of outcome delivery is a decreasing function of t′: dv∗ t′ dt′ < 0. (7) dv∗ t′ dt′ < 0. dv∗ t′ dt′ < 0. (7) See Appendix for the proof of this theorem. Theorem 2 stems from two bases: (i) the opti- mal rate of outcome delivery is a decreasing function of session length, i.e., when the session duration is long the decision-maker can afford to earn outcomes more slowly, and (ii) when the session duration is unknown, expected session duration should increase with the passage of time. This phenomenon, which has been elaborated within the context of delayed gratifica- tion (McGuire & Kable, 2013; Rachlin, 2000), is more significant if the decision-maker assumes a heavy-tail distribution over T . Putting (i) and (ii) together implies that the optimal response rate will decrease with the passage of time. Importantly, this suggests, from a normative perspective, uncertainty about the session duration is necessary in order to explain within-session decreases in response rates. For simulation of the model we characterized the session duration using a Generalized Pareto distribution following (McGuire & Kable, 2013). Simulations of the model are depicted in Figure 1:right panel. Simulations show three different conditions. In condition (i) the ses- sion duration is known, and as the figure shows irrespective of whether the reward of outcomes is decreasing or fixed, the optimal response rate is constant. In condition (ii) session duration is unknown, but the reward of outcomes is constant. Again in this condition the optimal re- sponse rate is constant. In condition (iii) session duration is unknown and the reward decreases as more outcomes are consumed. Only in this condition, consistent with experimental data, re- sponse rates decrease as time passes (see Appendix for details of the simulations). Therefore, the simulations confirm that decreases in the reward of outcomes alone are not sufficient to explain within-session response rates, but by assuming uncertainty about session duration, the pattern of responses will be consistent with the experimental data. RA nt rate. Nevertheless, h, it is not consistent next. p g section we assume that the decision-maker is uncertain about the session duration, which can be either because the session duration is in fact non-deterministic or because of inherent Theorem 2 Assuming St′,E[T |T >t′] is the value function and that (i) the time dependent change in the reward field is zero (∂Ax,t/∂t = 0), (ii) the probability that the session ends at each point in time RA nt rate. Nevertheless, h, it is not consistent next. p g section we assume that the decision-maker is uncertain about the session duration, which can be either because the session duration is in fact non-deterministic, or because of inherent Theorem 2 Assuming St′,E[T |T >t′] is the value function and that (i) the time dependent change in the reward field is zero (∂Ax,t/∂t = 0), (ii) the probability that the session ends at each point in time DRA been established that cluding variable-ratio h h inaccuracies in interval timing in animals [15, 16]. In this condition, a plausible way to calculate optimal response rates is to set an expectation as to how long the session will last 3Here satiety refers to both post-ingestive factors (such as blood glucose level; (Killeen, 1995)) and/or pre-ingestive factors (for example sensory specific satiety; (McSweeney, 2004)). . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint 9 OPTIMAL RESPONSE VIGOR AND CHOICE 10 lasts exactly 30 minutes whereas for another group the session length is uncertain and can end at any time (but ends on average after 30 minutes), then the model predicts that the response rate in the second group will be higher at the start and decrease more sharply than in the first group. This effect is not anticipated by the temporal discounting account of the effect. Effect of experimental parameters: Relating the model to currently available data. Op- timal response rates predicted by the model are affected by experimental parameters (e.g., re- ward magnitude), which can be compared against experimental data. In general, in an instru- mental conditioning experiment, the duration of the session can be divided into three sections: (i) outcome handling/consumption time, which refers to the time that an animal spends con- suming the outcome, (ii) post-reinforcer pause, which refers to the pause that occurs after con- suming the outcome and before starting to make the next response (e.g., lever press). Such a pause is consistently reported in previous studies using an FR schedule, (iii) run time, which refers to the time spent making responses (e..g, lever pressing). Experimental manipulations have been shown to have different effects on the duration of these three sections of the session, and whether each of these sections is included when calculating response rates can affect the re- sults. The predictions of the current model are with regard to response rate; whether manipulat- ing experimental parameters are expected to change the two other measures (outcome handling and post-reinforcer pause) cannot be predicted by the current model. In the following sections, we briefly review the currently available data from instrumental conditioning experiments and their relationship to predictions of the model4. The effect of response cost (a and b). Experimental studies in rats working on a FR sched- ule (Alling & Poling, 1995) indicate that increasing the force required to make responses causes increases in both inter-response time and the post-reinforcement pause. The same trend has been reported in Squirrel monkeys (Adair & Wright, 1976). Consistent with this observation, the present model predicts that increases in the cost of responding, for example by increasing the effort required to press the lever (increases in a and b), lead to a lower rate of earned outcomes and a lower rate of responding (Figure 2). OPTIMAL RESPONSE VIGOR AND CHOICE CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint 10 4Note that, for simplicity, the simulations in this section are made under the assumption that the session duration is fixed. OPTIMAL RESPONSE VIGOR AND CHOICE Note that a similar pattern will be obtained using any other distribution that assigns a non-zero probability to positive values of T . Relationship to temporal discounting. There are, however, alternative explanations available based on changes in outcome value. One candidate explanation is based on the tem- poral discounting of outcome value according to which the value of future rewards is discounted compared to more immediate rewards. Typically, the discount value due to delay is assumed to be a function of the interaction of delay and outcome value. If, at the beginning of the session, outcome values are large (e.g., because a rat is more hungry), then any discount produced by selecting a slow response rate will be larger at that point than later in the session when the value of the outcome is reduced (e.g., due to satiety) and so a delay will have less impact. It could be argued, therefore, that it is less punitive to maintain a high response rate at the beginning of the session to avoid delaying outcomes and then to decrease response rate as time passes within the session. As such, temporal discounting predicts decreases in within-session response rates consistent both with experimental observations and with the argument that outcome value de- creases within the session (e.g., the satiety effect). Prediction. Although plausible, such explanations make very different predictions com- pared to the model. The most direct prediction from the model is that introducing uncertainty into the session duration without altering the average duration should nevertheless lead to a sharper decline in response rate within the session; e.g., if for one group of subjects the session . CC-BY 4.0 International license available under a as not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: ioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . OPTIMAL RESPONSE VIGOR AND CHOICE The reason for this is that, by increasing the cost, the response rate for each outcome should slow in order to compensate for the increase in the cost and so maintain a reasonable gap between the reward and the cost of each outcome. The effect of ratio-requirement (k). Experimental studies mainly imply that the rate of earned outcomes decreases with increases in the ratio-requirement (Aberman & Salamone, 1999; Barofsky & Hurwitz, 1968), which is consistent with the general trend of the optimal rate of out- come earning implied by the present model (see below). Experimental studies on the rate of responding on FR schedules indicate that the post- reinforcer pause increases with increases in the ratio-requirement (Ferster & Skinner, 1957, Fig- ure 23)(Felton & Lyon, 1966; Powell, 1968; Premack, Schaeffer, & Hundt, 1964). In terms of overall response rates, some experiments report that response rates increase with increases in the ratio- requirement up to a point beyond which response rates will start to decline, in rats (Barofsky & Hurwitz, 1968; Kelsey & Allison, 1976; Mazur, 1982), pigeons (Baum, 1993) and mice (Greenwood, Quartermain, Johnson, Cruce, & Hirsch, 1974), although other studies have reported inconsis- tent results in pigeons (Powell, 1968), or a decreasing trend in response rate with increases in the ratio-requirement (Felton & Lyon, 1966; Foster, Blackman, & Temple, 1997). The inconsistency is partly due to the way that response rates are calculated in the different studies; for example . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. OPTIMAL RESPONSE VIGOR AND CHOICE ; https://doi.org/10.1101/106500 doi: bioRxiv preprint OPTIMAL RESPONSE VIGOR AND CHOICE 11 OPTIMAL RESPONSE VIGOR AND CHOICE 10 20 30 40 50 3 4 5 6 7 cost (a.u) inter-response interval Figure 2. Effect of response cost on response rates. Left panel: Empirical data. Inter-response intervals when the force required to make a response is manipulated. Figure is adopted from Adair and Wright (1976). Right panel: Model prediction. Inter-response interval (equal to the inverse of response rates) as a function of cost of responses (b). 10 20 30 40 50 3 4 5 6 7 cost (a.u) inter-response interval Figure 2. Effect of response cost on response rates. Left panel: Empirical data. Inter-response intervals when the force required to make a response is manipulated. Figure is adopted from Adair and Wright (1976). Right panel: Model prediction. Inter-response interval (equal to the inverse of response rates) as a function of cost of responses (b). Figure 2. Effect of response cost on response rates. Left panel: Empirical data. Inter-response intervals when the force required to make a response is manipulated. Figure is adopted from Adair and Wright (1976). Right panel: Model prediction. Inter-response interval (equal to the inverse of response rates) as a function of cost of responses (b). 2.5 5.0 7.5 0 25 50 75 100 ratio requirement (k) response rate 0.5 1.0 1.5 2.0 25 50 75 100 deprivation level (a.u) response rate 0 5 10 15 0.00 0.25 0.50 0.75 1.00 reward magnitude (a.u) response rate Figure 3. Left panel: The effect of ratio-requirement on the response rate. Middle panel: The effect of the initial motivational drive on response rates. Right panel: The effect of the reward magnitude on response rates. 2.5 5.0 7.5 0 25 50 75 100 ratio requirement (k) response rate 0.5 1.0 1.5 2.0 25 50 75 100 deprivation level (a.u) response rate 25 50 75 100 deprivation level (a.u) 0 5 10 15 0.00 0.25 0.50 0.75 1.00 reward magnitude (a.u) response rate Figure 3. Left panel: The effect of ratio-requirement on the response rate. Middle panel: The effect of the initial motivational drive on response rates. Right panel: The effect of the reward magnitude on response rates. in some studies outcome handling and consumption time are not excluded when calculating re- sponse rates (Barofsky & Hurwitz, 1968), in contrast to the other studies (Foster et al., 1997). OPTIMAL RESPONSE VIGOR AND CHOICE 12 pause (see (Pear, 2001, Page 289) for a review of other reinforcer schedules). The model predicts that, with increases in deprivation, the rate of responding and of earned outcomes will increase linearly (Figure 3: middle panel). The rate of increase is, however, negligible when the session duration is long, in which case, even under high deprivation, sufficient time is available to earn sufficient reward and become satiated. This provides a potential reason why the effect of depri- vation on response rate has not previously been observed in experimental data. The effect of reward magnitude. Some studies show that post-reinforcement pauses in- crease as the magnitude of the reward increases (Powell, 1969), whereas other studies suggest that the post-reinforcement pause decreases (Lowe, Davey, & Harzem, 1974), although, in this later study, the magnitude of the reward was manipulated within-session and a follow-up study showed that, at a steady state, the post-reinforcement pause increases with increases in the mag- nitude of the reward (Meunier & Starratt, 1979). Reward magnitude does not, however, have a reliable effect on the overall response rate (Keesey & Kling, 1961; Lowe et al., 1974; Powell, 1969). Regarding predictions from the model, the effect of the reward magnitude on earned outcome and response rates is, again, predicted go take an inverted U-shaped relationship (Figure 3: right panel), and, therefore, depending on the value of the parameters, the predictions of the model are consistent with the experimental data. The model makes a U-shaped prediction because, when the reward magnitude is large then, given high response rates, the animals will become satiated quickly and, therefore, the reward value of future outcomes will decrease substantially if animal maintains this high response rate. As a consequence, under a high reward magnitude condition, increase in reward will cause response rates to decrease. Under a low reward mag- nitude condition, however, satiety has a negligible effect and a high response rate ensures that sufficient reward can be earned before the session ends. OPTIMAL RESPONSE VIGOR AND CHOICE ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint OPTIMAL RESPONSE VIGOR AND CHOICE As a consequence, the experimental data regarding the relationship between response rate and the ratio-requirement is inconclusive. in some studies outcome handling and consumption time are not excluded when calculating re- sponse rates (Barofsky & Hurwitz, 1968), in contrast to the other studies (Foster et al., 1997). As a consequence, the experimental data regarding the relationship between response rate and the ratio-requirement is inconclusive. With regard to this issue, the present model predicts that the relationship between re- sponse rate and the ratio-requirement is an inverted U-shaped pattern (Figure 3: left panel), which is consistent with the studies mentioned previously depending on the value of other ex- perimental parameters. The model makes an inverted U-shaped prediction because, under a low ratio-requirement, the cost is generally low and, therefore, as the ratio-requirement increases, the decision-maker will make more responses to compensate for the drop in the amount of re- ward. In contrast, when the ratio-requirement is high, the cost of earning outcomes is high and the margin between the cost and the reward of each outcome becomes significantly tighter as the ratio-requirement increases. The margin can, however, be loosened by decreasing the response rate. The Effect of deprivation level. Experimental studies that have used FR schedules sug- gest that response rates increase with increases in deprivation (Ferster & Skinner, 1957, Chap- ter 4)(Sidman & Stebbins, 1954). However, such increases are mainly due to decreases in the post-reinforcement pause, and not due to the increases in the actual rate of responding after the . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. OPTIMAL RESPONSE VIGOR AND CHOICE consuming one of the outcomes reduces the amount of future reward produced by the other out- come. Here, if the effect of the outcomes on each other is symmetrical, i.e., consuming outcome O1, reduces the reward elicited by outcome O2 by the same amount that consuming outcome O2 reduces the reward elicited by outcome O1, then it will not matter which outcome is consumed first and the total reward during the session will be independent of the chosen order. As such, the reward field will be conservative. consuming one of the outcomes reduces the amount of future reward produced by the other out- come. Here, if the effect of the outcomes on each other is symmetrical, i.e., consuming outcome O1, reduces the reward elicited by outcome O2 by the same amount that consuming outcome O2 reduces the reward elicited by outcome O1, then it will not matter which outcome is consumed first and the total reward during the session will be independent of the chosen order. As such, the reward field will be conservative. More precisely, a reward field is conservative if there exists a scalar field Dx such that: Ax,t = −∂Dx ∂x . (8) (8) It can be shown that if a reward field satisfies equation 8 then the amount of reward experienced in a session depends on the total number of earned outcomes. Under this condition the optimal response rate will be constant for each outcome relative to the other. Intuitively, this is because, in terms of the total rewards per session, the only thing that matters is the final number of earned outcomes and, therefore, there is no reason why the relative allocation of responses to outcomes should vary within the session. The actual response rate for each outcome will, however, depend on whether the costs of the outcomes are independent, a point elaborated in the next section. Conservative reward field and independent response cost. The costs of various out- comes are independent if the decision-maker can increase their work for one outcome without affecting the cost of other outcomes. As an example, imagine a decision-maker that is using their left hand to make responses that earn one outcome and their right-hand to make responses that earn the second outcome. Optimal choice and response vigor ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint 13 13 Optimal choice and response vigor In this section we address the choice problem, i.e., the case where there are multiple out- comes available in the environment and the decision-maker needs to make a decision about the response rate along each outcome dimension. An example of this situation is a concurrent in- strumental conditioning experiment in which two levers are available and pressing each lever produces an outcome on a ratio schedule. Unlike the case with single outcome environments, the optimal rate of earning outcomes is not necessarily constant and can take different forms depending on whether the reward field is a conservative field or a non-conservative field, and whether the costs of responses along the outcome dimensions are independent of each other. Below, we derive the optimal choice strategy in each condition. Conservative reward field. A reward field is conservative if the amount of reward expe- rienced by consuming different outcomes does not depend on the order of consumption and depends only on the number of each outcome earned by the end of the session. This property holds in two conditions (i) when the value of each outcome is unrelated to the prior consumption of other outcomes; and (ii) the consumption of an outcome affects the value of other outcomes and this effect is symmetrical. As an example of condition (i), imagine an environment with two outcomes in which one of the outcomes only satisfies thirst and the other only satisfies hunger. Here, consumption of one of the outcomes will not affect the amount of reward that will be ex- perienced by consuming the other outcome and, therefore, the total reward during the session does not depend on the order of choosing the outcomes. As an example of condition (ii), imag- ine an environment with two outcomes in which both outcomes satisfy hunger and, therefore, . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. OPTIMAL RESPONSE VIGOR AND CHOICE 14 See Appendix for the proof and for the specification of optimal responses. As an example, imag- ine a concurrent fixed-ratio (FR) schedule in which an animal needs to make k responses on the left lever in order to earn O1, and lk responses on the right lever in order to earn O2, and both outcomes have the same reward properties. According to Theorem 3, the optimal response rate for O1 (the outcome with the lower ratio-requirement) is l times greater than the response rate for the second outcome O2. Figure 4:left panel independent cost condition shows the simulation of the model and the optimal trajectory in the outcome space. As the figure shows, the rate of earning O1 is higher than O2, however, the relative portion of earned outcomes remains the same throughout the session. The above results are generally in line with the probability matching notion, which states that a decision-maker allocates responses to outcomes based on the ratio of responses required for each outcome (Estes, 1950). Probability matching is often argued to violate rational choice theory because, within this theory, it is expected that a decision-maker works exclusively for the outcome with the higher probability (i.e., the lower ratio-requirement). However, based on the model proposed here probability matching is the optimal strategy and therefore consistent with rational decision-making. Conservative reward field and dependent response cost. In this section we assume that the cost of responses for an outcome is affected by the rate of responses for earning other out- comes. In other words, what determines the cost is the delay between subsequent responses either for the same or for a different outcome; i.e., the cost is proportional to the rate of earning all of the outcomes. In instrumental conditioning this assumption entails, for example, that the cost of pressing, say, the right lever is determined by the time that has passed since the last press on either the right or a left lever. In this condition the optimal strategy is maximisation; i.e., to take the action with the higher reward (or lower ratio-requirement) and to stop taking the other action (see Theorem A2 in Appendix). OPTIMAL RESPONSE VIGOR AND CHOICE It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint OPTIMAL RESPONSE VIGOR AND CHOICE In this case, the independence assumption entails that the cost of responding with one or other hand is determined by the response rate on that hand; e.g., the decision-maker can increase or decrease rate of responding on the left hand without affecting the cost of responses on the right hand. More precisely, the independence assumption entails that the Hessian matrix of Kv is diagonal: ∂2Kv ∂vi∂v j = 0,i ̸= j. (9) (9) In this situation even if some of the outcomes have a lower reward or a higher cost (inferior out- comes) compared to other outcomes (superior outcomes), it is still optimal to allocate a portion of responses to the inferior outcomes. This is because responding for inferior outcomes has no effect on the net reward earned from superior outcomes and, therefore, as long as the response rate for inferior outcomes is sufficiently low that the reward earned from them is more than the cost paid, responding for them is justified. The portion of responses allocated to each outcome depends, however, on the cost and reward of each outcome. We maintain the following theorem: In this situation even if some of the outcomes have a lower reward or a higher cost (inferior out- comes) compared to other outcomes (superior outcomes), it is still optimal to allocate a portion of responses to the inferior outcomes. This is because responding for inferior outcomes has no effect on the net reward earned from superior outcomes and, therefore, as long as the response rate for inferior outcomes is sufficiently low that the reward earned from them is more than the cost paid, responding for them is justified. The portion of responses allocated to each outcome depends, however, on the cost and reward of each outcome. We maintain the following theorem: Theorem 3 If (i) the reward field is conservative, i.e., there exists a scalar field Dx such that equa- tion 8 is satisfied, (ii) the time-dependent term of the reward field is zero (∂Ax,t/∂t = 0), and (iii) the cost function satisfies equation 9, then the optimal rate of earning outcome v∗will be constant (dv/dt = 0) and satisfies the following equation: ∂Kv∗ ∂v∗= AT v∗,T . (10) ∂Kv∗ ∂v∗= AT v∗,T . (10) . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. OPTIMAL RESPONSE VIGOR AND CHOICE 15 he structure of such studies cannot be readily related to the model proposed here. the structure of such studies cannot be readily related to the model proposed here. Prediction. One way of testing the above explanation for maximization and matching strategies is to compare the pattern of responses when two different effectors are used to make responses for the outcomes vs. when a single effector is being used to earn both outcomes. In the first condition the costs of the two outcomes are independent of each other whereas in the second condition they are dependent on each other. As a consequence, the theory predicts that, in the first condition, response rates will reflect probability matching whereas in the second con- dition they will reflect the maximization strategy. 0 25 50 75 0 25 50 75 O2(a.u) O1(a.u) independent cost dependent cost 0 30 60 90 0 30 60 90 O2(a.u) O1(a.u) short medium long Fig. 2. Left panel: Optimal trajectory in a conservative reward field. Earning O1 requires k responses and earning O2 requires lk responses. Initially the amount of earned outcome is zero (starting point is at point [0, 0]), and the graph shows the trajectories that the decision-maker takes in two different conditions corresponding to when the costs of outcomes are independent, and when the costs are dependent on each other. Right panel: The optimal trajectories in the outcome space when the reward field is non-conservative. The graph shows the optimal trajectory in the conditions that the session duration is short (T = 7), medium (T = 15.75) and long (T = 23). properties, e.g., consumption of one unit of either O1 or O2 d h g b it h th g t diff and here we extended solution to the proble are two significant di here and previous m although the effect o values on response v [22], the effects of on a session were not ad of changes in outcom has been addressed in role in determining re We address such limit Secondly, previous the task as a semi-M optimal actions that m time (average reward to calculate the optim earned within the ses in the previous works range of instrumental Figure 4. Left panel: Optimal trajectory in a conservative reward field. Earning O1 requires k responses and earning O2 requires lk responses. OPTIMAL RESPONSE VIGOR AND CHOICE Earning O1 requires k responses and earning O2 requires lk responses. Initially the amount of earned outcome is zero (starting point is at point [0, 0]), and the graph shows the trajectories that the decision-maker takes in two different conditions corresponding to when the costs of outcomes are independent, and when the costs are dependent on each other. Right panel: The optimal trajectories in the outcome space when the reward field is non-conservative. The graph shows the optimal trajectory in the conditions that the session duration is short (T = 7), medium (T = 15.75) and long (T = 23). properties, e.g., consumption of one unit of either O1 or O2 has been addressed in role in determining r We address such limi Secondly, previou the task as a semi-M optimal actions that time (average reward to calculate the opti earned within the ses in the previous work f i Figure 4. Left panel: Optimal trajectory in a conservative reward field. Earning O1 requires k responses and earning O2 requires lk responses. Initially the amount of earned outcome is zero (starting point is at point [0,0]), and the graph shows the trajectories that the decision-maker takes in two different conditions corresponding to when the costs of outcomes are independent, and when the costs are dependent on each other. Right panel: The optimal trajectories in the outcome space when the reward field is non-conservative. The graph shows the optimal trajec- tory in the conditions that the session duration is short (T = 7), medium (T = 15.75) and long (T = 23). g y , , y g ent amount of rewards, e.g., one unit of O1 generates more reward than one unit of O2. Within such an environment, if hunger is high then consuming O1 generates significantly more reward than O2 and, therefore, early in the session it is better to allocate more responses to O1, whereas later in the session (when hunger is presumably lower and the difference in the value of the outcomes is small) responses for O2 can gradually increase. If we reverse this order, i.e., first O2 is consumed and then O1, then early consumption of O2 will cause satiety and the decision-maker will lose the chance to experience high reward from O1 when hungry. OPTIMAL RESPONSE VIGOR AND CHOICE As such, the amount of experienced reward depends on the order of con- suming the outcomes and, based on the above explanation, a larger amount of reward can be earned during the session if more responses are allocated to the outcome with the higher reward at the beginning of the session (see Theorem S3 in SI text). Figure 2:right panel shows the simulations of the model under different session durations. In each simulation, at the beginning of the session the initially earned outcomes are zero and each line in the figure shows the trajectory of the amount earned from each outcome during the session. As the figure shows, in all conditions the rate of earning O1 is higher than O2 and this difference is more apparent under long session durations, in which a large amount of reward can be gained during the session and it makes a significant difference to earn schedules, whereas th to the case of interval average reward (as ad the maximization of i.e., the session durat used here explicitly which, as we showed, of responses. ACKNOWLEDGMENT Peter Dayan for helpful Principal Research Fell Research Council of Au 1. von Neumann J, Morgenster university press Princeton) V 2. Marshall A (1890) Principles 3. Niv Y, Daw ND, Joel D, Daya response vigor. Psychophar 4. Dayan P (2012) Instrument neuroscience 35(7):1152–68 5. Salimpour Y, Shadmehr R ( Journal of neuroscience 34( 6. Killeen PR (1995) Economic conditions of varying motivat 431. 7. Keramati M, Gutkin BS (201 lection and physiological sta 8. Hull CL (1943) Principles of 9. McSweeney FK, Hinson JM operant conditioning. Psych 10 Lib D (2011) C l l Non-conservative reward field. A reward filed is non-conservative if the total amount of reward experienced during the session depends on the order of the consumption of the out- comes. Imagine an environment with two outcomes say O1 and O2, where both outcomes have the same motivational properties, e.g., consumption of one unit of either O1 or O2 decreases hunger by one unit, however, they generate different amount of rewards, e.g., one unit of O1 generates more reward than one unit of O2. OPTIMAL RESPONSE VIGOR AND CHOICE The reason is, unlike the case with independent costs, allocating more responses to earn an inferior outcome will increase the cost of earning superior outcomes and, therefore, it is better to pay the cost for the superior outcome only, which requires fewer responses per unit of outcome. For example, under a concurrent FR schedule in which an animal needs to make k re- sponses on the left lever to earn O1, and lk responses on the right lever to earn O2 (O1 and O2 have the same reward properties), the optimal response rate will be a constant response rate on the left lever and a zero response rate on the right lever. Figure 4:left panel dependent cost condition shows a simulation of the model and the optimal trajectory in outcome space, which shows that the subject only earns O1. As such, whether the outcome rate reflects a probability matching or a maximization strat- egy depends on the cost function and, in instrumental conditioning experiments, the cost that reflects the maximization strategy is more readily applicable. Regarding the experimental data, evidence from concurrent instrumental conditioning experiments in pigeons tested using VR schedules (Herrnstein & Loveland, 1975) is in-line with the maximization strategy and consis- tent with predictions from the model. Within the wider scope of decision-making tasks, some studies are consistent with probability matching, whereas other studies provide evidence in-line with the maximization strategy (see (Vulkan, 2000) for a review). In many of these latter studies, however, the decision-making task involved making a single choice (e.g., single button press) with immediate feedback (about whether the choice was rewarded) after which the next trial was initiated and, because such disjointed actions are unlikely to convey a rate-dependent cost, . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint OPTIMAL RESPONSE VIGOR AND CHOICE Initially the amount of earned outcome is zero (starting point is at point [0,0]), and the graph shows the trajectories that the decision-maker takes in two different conditions corresponding to when the costs of outcomes are independent, and when the costs are dependent on each other. Right panel: The optimal trajectories in the outcome space when the reward field is non-conservative. The graph shows the optimal trajec- tory in the conditions that the session duration is short (T = 7), medium (T = 15.75) and long (T = 23). 0 25 50 75 0 25 50 75 O2(a.u) O1(a.u) independent cost dependent cost 0 30 60 90 0 30 60 90 O2(a.u) O1(a.u) short medium long Fig. 2. Left panel: Optimal trajectory in a conservative reward field. Earning O1 requires k responses and earning O2 requires lk responses. Initially the amount of earned outcome is zero (starting point is at point [0, 0]), and the graph shows the trajectories that the decision-maker takes in two different conditions corresponding to when the costs of outcomes are independent, and when the costs are dependent on each other. Right panel: The optimal trajectories in the outcome space when the reward field is non-conservative. The graph shows the optimal trajectory in the conditions that the session duration is short (T = 7), medium (T = 15.75) and long (T = 23). properties, e.g., consumption of one unit of either O1 or O2 has been addressed in role in determining r We address such limi Secondly, previou the task as a semi-M optimal actions that m time (average reward to calculate the opti earned within the ses in the previous work Figure 4. Left panel: Optimal trajectory in a conservative reward field. Earning O1 requires k responses and earning O2 requires lk responses. Initially the amount of earned outcome is zero (starting point is at point [0,0]), and the graph shows the trajectories that the decision-maker takes in two different conditions corresponding to when the costs of outcomes are independent, and when the costs are dependent on each other. Right panel: The optimal trajectories in the outcome space when the reward field is non-conservative. The graph shows the optimal trajec- tory in the conditions that the session duration is short (T = 7), medium (T = 15.75) and long (T = 23). Fig. 2. Left panel: Optimal trajectory in a conservative reward field. Discussion Computational models of action selection are essential for understanding decision- making processes in humans and animals, and here we extended them by providing a general analytical solution to the problem of response vigor and choice. There are two significant differ- ences between the model proposed here and previous models of response vigor (Dayan, 2012; Niv et al., 2007). Firstly, although the effect of between-session changes in outcome values on re- sponse vigor was addressed in previous models (Niv, Joel, & Dayan, 2006), the effects of on-line changes in outcome values within a session were not addressed. On the other hand, the effect of changes in outcome value on the choice between actions has been addressed in some previ- ous models (Keramati & Gutkin, 2014), however their role in determining response vigor has not been investigated. We address such limitations directly in this model. Secondly, previous work conceptualized the structure of the task as a semi-Markov deci- sion process and derived the optimal actions that maximize the average reward per unit of time (average reward). Here, we used a variational analysis to calculate the optimal actions that max- imize the reward earned within the session. One benefit of the approach taken in the previous works is that it extends naturally to a wide range of instrumental conditioning schedules such as interval schedules, whereas the extension of the model proposed here to the case of interval schedules is not trivial. Optimizing the average reward (as adopted in previous work) is equiv- alent to the maximization of reward in an infinite-horizon time scale; i.e., the session duration is unlimited. In contrast, the model used here explicitly represents the duration of the session which, as we showed, plays an important role in the pattern of responses. Relationship to principle of least action. A basic assumption that we made here is that the decision-maker takes actions that yield the highest amount of reward. This reward max- imization assumption has a parallel in physics literature known as the principle of least action, which implies that among all trajectories that a system can take, the true trajectories are the ones that minimize the action. Here action has a different meaning from that used in psychology lit- erature, and it refers to the integral of the Lagrangian (L) along the trajectory. OPTIMAL RESPONSE VIGOR AND CHOICE it makes a significant difference to earn O1 first. Prediction. A test of the above prediction would involve an experiment in which the sub- ject is responding for two food outcomes containing an equal number of calories (and therefore having the same impact on motivation) but with different levels of the desirability (e.g., having different flavors) and, therefore, having a different reward effect. Theorem A3 predicts that, if the session duration is long enough, early in the session the response rate for the outcome with the greater desirability will be higher whereas, later in the session, responses for the other outcome will increase. OPTIMAL RESPONSE VIGOR AND CHOICE Within such an environment, if hunger is high then consuming O1 generates significantly more reward than O2 and, therefore, early in the session it is better to allocate more responses to O1, whereas later in the session (when hunger is pre- sumably lower and the difference in the value of the outcomes is small) responses for O2 can gradually increase. If we reverse this order, i.e., first O2 is consumed and then O1, then early con- sumption of O2 will cause satiety and the decision-maker will lose the chance to experience high reward from O1 when hungry. As such, the amount of experienced reward depends on the order of consuming the outcomes and, based on the above explanation, a larger amount of reward can be earned during the session if more responses are allocated to the outcome with the higher re- ward at the beginning of the session (see Theorem A3 in Appendix). Figure 4:right panel shows the simulations of the model under different session durations. In each simulation, at the begin- ning of the session the initially earned outcomes are zero and each line in the figure shows the trajectory of the amount earned from each outcome during the session. As the figure shows, in all conditions the rate of earning O1 is higher than O2 and this difference is more apparent under long session durations, in which a large amount of reward can be gained during the session and . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint 16 Discussion In the case of a charged particle with charge q and mass m in a magnetic field B, the Lagrangian will be: L = 1 2mv2 + qv.A, (11) (11) where A is the vector potential (B = ∇× A). By comparing equation 11 with equations 4 and 5, we can see that the reward field Ax,t corresponds to the vector potential, and the term Kv corresponds to 1 2mv2 by assuming m = 2ak2, and b = 0. This parallel can provide some in- sights into the properties of the response rates. For example, it can be shown that when the where A is the vector potential (B = ∇× A). By comparing equation 11 with equations 4 and 5, we can see that the reward field Ax,t corresponds to the vector potential, and the term Kv corresponds to 1 2mv2 by assuming m = 2ak2, and b = 0. This parallel can provide some in- sights into the properties of the response rates. For example, it can be shown that when the . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint 17 OPTIMAL RESPONSE VIGOR AND CHOICE 17 Lagrangian is not explicitly dependent on time (time-translational invariance), which here im- plies that ∂Ax,t/∂t = 0, then the Hamiltonian (H , or energy) of the system is conserved. The Hamiltonian in the case of the system defined in equation 4 (with single outcome) is: H = Kv −∂Kv ∂v v = −ak2v2 (using equation 3). = −ak2v2 (using equation 3). = −ak2v2 (using equation 3). Conservation of the Hamiltonian implies that ak2v2 (and therefore v) is constant (response-rate is constant), as stated by Theorem 1. Further exploration of this parallel can be an interesting future direction. References CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint OPTIMAL RESPONSE VIGOR AND CHOICE 18 Killeen, P. R. (1995, nov). Economics, ecologics, and mechanics: The dynamics of responding under conditions of varying motivation. Journal of the Experimental Analysis of Behavior, 64(3), 405–431. Killeen P R & Sitomer M T (2003 apr) MPR Behavioural Processes 62(1 3) 49 64 Killeen, P. R. (1995, nov). Economics, ecologics, and mechanics: The dynamics of responding under conditions of varying motivation. Journal of the Experimental Analysis of Behavior, 64(3), 405–431. Killeen, P. R., & Sitomer, M. T. (2003, apr). MPR. Behavioural Processes, 62(1-3), 49–64. y g f p y f Killeen, P. R., & Sitomer, M. T. (2003, apr). MPR. Behavioural Processes, 62(1-3), 49–64. Killeen, P. R., & Sitomer, M. T. (2003, apr). MPR. Beh Liberzon, D. (2011). Calculus of Variations and Optimal Control Theory: A Concise Introduction. Princeton University Press. Lowe, C. F., Davey, G. C. L., & Harzem, P. (1974). Effects of reinforcement magnitude on interval and rati schedules. Journal of the Experimental analysis of behavior, 22(3), 553–560. Marshall, A. (1890). Principles of Economics. London: Macmillan and Co., Ltd. Mazur, J. E. (1982). A molecular approach to ratio schedule performance. In M. L. Commons, R. J. Her- rnstein, & H. Rachlin (Eds.), Quantitative analyses of behavior. vol. 2: Matching and maximizing accounts. Ballinger. McGuire, J. T., & Kable, J. W. (2013, apr). Rational temporal predictions can underlie apparent failures to delay gratification. 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Behavioral and Brain Sciences, 17, 105–172. . . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint Value in non-deterministic schedules The value of a trajectory in the outcome space is the sum of the net amount of rewards that can be earned during a session. However, the amount of reward earned during a session can be non-deterministic, as for example in the case of VR and RR schedules of reinforcement, ac- tions lead to outcomes probabilistically. Similarly, the cost of earning outcomes will also be non- deterministic, since the number of responses required to earn outcomes is non-deterministic. Let’s denote the cost of earning outcomes under such non-deterministic schedules by K ′ v. Using this, we define the value function as the sum of the expected net amount of rewards that will be earned during a session: S0,T = ZT 0 E[v.Ax,t −K ′ v]dt = ZT 0 Lx,v,tdt, (A12) (A12) where the expectation is w.r.t the number of earned outcomes along each outcome dimension during dt time step. Following the above definition, we have: Lx,v,t = E[v.Ax,t −K ′ v], (A13) (A13) where Lx,v,t is the expected net reward earned in dt time step. In the main text and in the follow- ing sections, E[v] is denoted by v for simplicity of notation. By replacing v by E[v] in equation 4 we get: (A14) Lx,v,t = E[v].Ax,t −KE[v]. (A14) In the main text, equation A14 (equation 4 in the main text) was used instead of equation A13, and the aim of this section is to show that equation A14 and equation A13 are equivalent. We first consider environments with one-dimensional outcome spaces, and then we extend it to the case of environments with multi-dimensional outcome spaces. We maintain the following theorem: Theorem A1 Assume that the cost of one response, given that the delay since the last response is τ, is as follows: Cτ = a/τ+b. (A15) (A15) Furthermore, assume that on average, or exactly, k responses are required to earn one unit of the outcome, and r is the number of outcomes earned. Then we have: Lx,v,t = Er [v]Ax,t −KEr [v], (A16) Kv = vk(kav +b). (A17) Lx,v,t = Er [v]Ax,t −KEr [v], (A16) (A16) where Kv = vk(kav +b). (A17) (A17) Proof. We first provide an intuitive explanation for why the cost defined in equation A15 is the same as the cost defined in equation A17 in the case of FR schedules of reinforcement (i.e., exactly k responses are required to earn an outcome). OPTIMAL RESPONSE VIGOR AND CHOICE 19 OPTIMAL RESPONSE VIGOR AND CHOICE The effect of small sequential changes in fixed-ratio size upon the post-reinforcemen pause. Journal of the Experimental Analysis of Behavior, 11(5), 589–593. Powell, R. W. (1969). The effect of reinforcement magnitude upon responding under fixed-ratio schedules. Journal of the Experimental Analysis of Behavior, 12(4), 605–608. Premack, D., Schaeffer, R. W., & Hundt, A. (1964). Reinforcement of drinking by running: effect of fixed ratio and reinforcement time. Journal of the experimental analysis of behavior, 7(1), 91–96. Rachlin, H. (2000). The Science of Self-Control. Harvard University Press. alimpour, Y., & Shadmehr, R. (2014, jan). Motor costs and the coordination of the two arms. The Journa of neuroscience, 34(5), 1806–18. f Sidman, M., & Stebbins, W. C. (1954). Satiation effects under fixed-ratio schedules of reinforcement. Journal of Comparative and Physiological Psychology, 47(2), 114. f p y g y gy von Neumann, J., & Morgenstern, O. (1947). Theory of games and economic behavior. Princeton university press Princeton. Vulkan, N. (2000). An economist’s perspective on probability matching. Journal of economic surveys, 14(1), 101–118. . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint Value in non-deterministic schedules Earning the outcome at rate v implies that the time it takes to earn the outcome is 1/v, and since k responses have been executed in this period, the delay between responses is: 1 τ = 1 kv , (A18) τ = 1 kv , (A18) . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint OPTIMAL RESPONSE VIGOR AND CHOICE 20 and therefore using equation A15 (equation 2 in the main text), the cost of making one response will be akv+b. Since k responses are required for earning each outcome, the total cost of earning one unit of the outcome will be k times the cost of one response, which will be k(akv +b). Since the total amount of outcome earned is vdt, the total cost per unit of time will be: Kv =k(akv +b)vdt dt =vk(akv +b), (A19) (A19) which is equivalent to equation 3 and A17. which is equivalent to equation 3 and A17. We now show that equation A16 and equation A13 are equivalent in order to prove Theo- rem A1. Equation A13 has two terms. As for the first term, Ax,t can be assumed to be constant in dt time step, and therefore we have: Er [v Ax,t] = Er [v]Ax,t. (A20) (A20) As for the second term we maintain that: As for the second term we maintain that: As for the second term we maintain that: Er [K ′ v] = KEr [v]. (A21) (A21) To show the above relation, assume that r is the number of outcomes earned after making one response. Since according to the definition of RR and VR schedules, out of N responses on av- erage N/k will be rewarded, we have Er [r] = 1/k and the expected rate of outcome earning will be: Er [v] = Er hr τ i = 1 kτ. (A22) (A22) Furthermore, according to equation A15 the cost of one response is a/τ + b, and therefore, the cost per unit of time will be: b Furthermore, according to equation A15 the cost of one response is a/τ + b, and therefore, the cost per unit of time will be: K ′ v = a/τ+b τ . (A23) (A23) Therefore: OPTIMAL RESPONSE VIGOR AND CHOICE 21 which states that E[K ′ v] can be represented as a function of E[v]. To show this, assume ri is the number of outcomes earned after making one response for outcome i, and τi is the delay be- tween responses for outcome i. We have: which states that E[K ′ v] can be represented as a function of E[v]. To show this, assume ri is the number of outcomes earned after making one response for outcome i, and τi is the delay be- tween responses for outcome i. We have: E[vi] = E ·ri τi ¸ = E[ri] τi , (A26) (A26) and therefore τi can be expressed as a function of E[vi]. Next, assume that [Cτ]i is the cost of making one response for outcome i with delay τi between the responses, and τ is a vector con- taining the delay between responses for each outcome (τ = [τ1 ...τn]). In dt time step, dt/τi re- sponses for outcome i are made, and therefore the total cost in dt time period will be [Cτ]idt/τi, which implies that the cost for outcome i per unit of time is [Cτ]i/τi. Given this, the total cost paid for all the outcomes per unit of time will be: E[K ′ v] = X i [Cτ]i τi = X i [Cτ]i E[vi] E[ri] (using equation A26) = KE[v], E[K ′ v] = X i [Cτ]i τi = X i [Cτ]i E[vi] E[ri] (using equation A26) = KE[v], = KE[v], where we used the fact that τ in Cτ can be expressed using E[v] (using equation A26), and there- fore E[K ′ v] can be expressed as a function of E[v], which is denoted by KE[v] (as noted in equa- tion A25). Substituting equation A25,A24 in equation A13 yields equation A14. where we used the fact that τ in Cτ can be expressed using E[v] (using equation A26), and there- fore E[K ′ v] can be expressed as a function of E[v], which is denoted by KE[v] (as noted in equa- tion A25). Substituting equation A25,A24 in equation A13 yields equation A14. Therefore: Er [K ′ v] =a/τ+b τ =Er [v]k(akEr [v]+b) (using equation A22) =KEr [v] (using equation A17), which proves equation A21. Substituting equations A21, A20 in equation A13 yields equa- tion A16, which proves the theorem. which proves equation A21. Substituting equations A21, A20 in equation A13 yields equa- tion A16, which proves the theorem. We now turn to the case of multi-dimensional outcome spaces. The aim is to show equa- tion A13 is equivalent to equation A14. To show this, we first maintain that: E[v.Ax,t] = E[v].Ax,t, (A24) (A24) which holds since Ax,t can be assumed to be constant during dt time step. Next, we show that: E[K ′ v] = KE[v], (A25) (A25) E[K ′ v] = KE[v], . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint OPTIMAL RESPONSE VIGOR AND CHOICE 22 which implies: ∂Kv ∂v ¯¯¯¯ t=T = Ax,t ¯¯ t=T , (A31) which implies: which implies: which implies: ∂Kv ∂v ¯¯¯¯ t=T = Ax,t ¯¯ t=T , (A31) ∂Kv ∂v ¯¯¯¯ t=T = Ax,t ¯¯ t=T , (A31) (A31) where as mentioned T is the total session duration. re as mentioned T is the total session duration. where as mentioned T is the total session duration. ubstituting equation A27 in equation A29 we will have: By substituting equation A27 in equation A29 we will have: By substituting equation A27 in equation A29 we will have: d dt µ −∂Kv ∂v + Ax,t ¶ = v d Ax,t dx . (A32) (A32) The term d Ax,t/dt has two components: the first component is the change in Ax,t due to the change in x and the second component is due to the time-dependent changes in Ax,t: The term d Ax,t/dt has two components: the first component is the change in Ax,t due to the change in x and the second component is due to the time-dependent changes in Ax,t: d Ax,t dt =dx dt ∂Ax,t ∂x + ∂Ax,t ∂t =v ∂Ax,t ∂x + ∂Ax,t ∂t . (A33) (A33) Furthermore we have: Furthermore we have: Furthermore we have: d dt µ∂Kv ∂v ¶ = dv dt ∂2Kv ∂v2 . (A34) d dt µ∂Kv ∂v ¶ = dv dt ∂2Kv ∂v2 . (A34) (A34) Substituting equations A33, A34 in equation A32 yields: Substituting equations A33, A34 in equation A32 yields: Substituting equations A33, A34 in equation A32 yields: dv dt µ∂2Kv ∂v2 ¶ = ∂Ax,t ∂t . (A35) (A35) In the condition that the rate of outcome earning is constant (dv/dt = 0), we have xT = vT , which by substituting in equation A31 yields: In the condition that the rate of outcome earning is constant (dv/dt = 0), we have xT = vT , which by substituting in equation A31 yields: ∂Kv∗ ∂v∗= AT v∗,T . (A36) (A36) The above equation will be used in order to calculate the optimal rate of outcome earning. The above equation will be used in order to calculate the optimal rate of outcome earning. Th 1 P f Optimal actions in one-dimensional outcome space The aim is to derive optimal actions when the outcome space has one dimension. Given the reward field Ax,t, the reward of gaining dx units of outcome will be Ax,tdx, and since dx = vdt, the reward earned in each time step is v Ax,t. Given that Kv is the cost function (the cost paid in each time step), the net reward in each time step can be written as: Lx,v,t = v Ax,t −Kv, (A27) (A27) and based on this, the value, which is the sum of net rewards in each time step, will be: and based on this, the value, which is the sum of net rewards in each time step, will be: S0,T = ZT 0 Lx,v,tdt. (A28) (A28) The optimal rates that maximize S0,T can be found using different variational calculus methods such as the Euler-Lagrange equation, or the Hamilton-Jacobi-Bellman equation (Liber- zon, 2011). Here we use the Euler-Lagrange form, which sets a necessary condition for δS = 0: d dt ∂L ∂v = ∂L ∂x . (A29) (A29) Furthermore, since the end-point of the trajectory is free (the amount of outcomes that can be gained during a session is not limited, but the duration of the session is limited to T ), the optimal trajectory will also satisfy the transversality conditions: Furthermore, since the end-point of the trajectory is free (the amount of outcomes that can be gained during a session is not limited, but the duration of the session is limited to T ), the optimal trajectory will also satisfy the transversality conditions: ∂L ∂v ¯¯¯¯ t=T = 0, (A30) (A30) . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint OPTIMAL RESPONSE VIGOR AND CHOICE 22 OPTIMAL RESPONSE VIGOR AND CHOICE OPTIMAL RESPONSE VIGOR AND CHOICE 23 Theorem 1: Proof The cost function Kv defined in equation 3 satisfies the following relation: ∂2Kv ∂v2 > 0, (A37) (A37) which holds as long as at least one response is required to earn an outcome (k > 0), and the cost of earning outcomes is non-zero (a > 0). which holds as long as at least one response is required to earn an outcome (k > 0), and the cost of earning outcomes is non-zero (a > 0). Assuming that ∂Ax,t/∂t = 0, and given equation A37, the only admissible solution to equa- tion A35 is: dv Assuming that ∂Ax,t/∂t = 0, and given equation A37, the only admissible solution to equa- tion A35 is: dv dv dt = 0. (A38) (A38) thermore, assuming ∂Ax,t/∂t > 0, and given equation A37, the only admissible solution to ation A35 is: dv dt > 0, (A39) dv dt > 0, (A39) which proves Theorem 1. which proves Theorem 1. . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint OPTIMAL RESPONSE VIGOR AND CHOICE 24 24 Simulation details. The simulation of the model depicted in Figure 1:right panel re- quires defining (i) the reward field, (ii) the cost function, and (iii) a probability distribution over the session duration. As for the probability distribution of the session duration, following McGuire et al (McGuire & Kable, 2013), we assumed that T follows a Generalized Pareto distri- bution: /k F(T ) = 1− µ 1+ kt σ ¶−1/k , (A47) (A47) where k is a shape parameter (note that k is not the ratio-requirement here) and σ is a scale parameter, and the third parameter (location µ) was assumed to be zero. Furthermore we have: where k is a shape parameter (note that k is not the ratio-requirement here) and σ is a scale parameter, and the third parameter (location µ) was assumed to be zero. Furthermore we have: F(T |T > t′) = 1− µ 1+ kt σ+kt′ ¶−1/k , (A48) (A48) which has the following expected value: which has the following expected value: E[T |T > t′] = σ+kt′ 1−k , (A49) (A49) which as we expect is an increasing function of t′. For the simulation of the model we assumed that k = 0.9 and σ = 6, which represents that the initial expectation for the session duration is 60 minutes. For the cost function, in all the simulations the cost defined in equation 3 was used, which is equivalent to the cost function used in the previous works (Dayan, 2012; Niv et al., 2007). For the definition of the reward field, we used the framework provided by Keramati et al (Keramati & Gutkin, 2014), which provides a computational model for how the values of out- comes change with the consumptions of the outcomes. They suggested that the dependency of the reward field on the amount of outcome earned is indirect and it is through the motivational drive. They conceptualized the motivational drive as the deviations of the internal states of a decision-maker from their homeostatic set-points. For example, let’s assume that there is only one internal state, say hunger, where H denotes its homeostatic set-point (which corresponds to the deprivation level, assuming that initial value of x is zero), and there is an outcome which consuming each unit of it satisfies l units of the internal state. Theorem 2: Proof and simulation details ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint Theorem 2: Proof and simulation details Proof of Theorem 2. In order to prove the theorem, we first provide a lemma. Assuming that the total session duration (T ) has the probability density function f (T ) and that f (T ) > 0, here we show that the expectation of the total session duration never decreases as time passes throughout the session. Lemma 1 Let’s denote the expectation of the session duration at time t′ with T ′ T ′ = E[T |T > t′], (A40) (A40) and assume T has the following probability density function: and assume T has the following probability density function: T ∼f (T ), f (T ) > 0. (A41) (A41) f ( ), f ( ) ( ) Then: ∂T ′ ∂t′ > 0. (A42) Then: ∂T ′ > 0 (A42) Then: Then: ∂T ′ ∂t′ > 0. (A42) ∂T ′ ∂t′ > 0. (A42) (A42) Proof. We have: ∂T ′ ∂t′ = ∂E[T |T > t′] ∂t′ = f (T ) 1−F(T ) ¡ E[T |T > t′]−t′¢ > 0, (A4 Proof. We have: Proof. We have: ∂T ′ ∂t′ = ∂E[T |T > t′] ∂t′ = f (T ) 1−F(T ) ¡ E[T |T > t′]−t′¢ > 0, (A43) (A43) where F(T ) is the cumulative distribution function of T . Based on the above lemma, we show that the optimal response rate is a decreasing func- tion of t′. Based on equation A31, the optimal response rate satisfies the following equation: ∂Kv ∂v ¯¯¯¯ t=T ′ = Ax,t ¯¯ t=T ′ . (A44) (A44) Taking the derivative w.r.t to t′ we get: Taking the derivative w.r.t to t′ we get: Taking the derivative w.r.t to t′ we get: Taking the derivative w.r.t to t′ we get: dv dt′ µ∂2Kv ∂v2 ¶ = ∂T ′ ∂t′ µ v ∂Ax,t ∂x + ∂Ax,t ∂T ′ ¶ . (A45) (A45) Theorem 2 assumes that ∂Ax,t/∂x < 0 and ∂Ax,t/∂T ′ = 0, which given equations A42,A37, and that v > 0 yields: dv∗ dt′ < 0, (A46) (A46) which implies that the rate of earning outcomes decreases as time passes within a session. . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. OPTIMAL RESPONSE VIGOR AND CHOICE 25 Equation A52 was used in the simulations of the “decreasing reward and unknown session dura- tion” condition in Figure 1:right panel. The simulation of this condition was done using param- eters k = 15, l = 0.1, a = 0.002, b = 0.1, H = 80. As for T , in each time t′ within the session, the expected session duration (E[T |T > t′]) was calculated using equation A49, and was used as T in equation A52. Equation A52 was used in the simulations of the “decreasing reward and unknown session dura- tion” condition in Figure 1:right panel. The simulation of this condition was done using param- eters k = 15, l = 0.1, a = 0.002, b = 0.1, H = 80. As for T , in each time t′ within the session, the expected session duration (E[T |T > t′]) was calculated using equation A49, and was used as T in equation A52. For the “known session duration (fixed or decreasing reward)” condition in Figure 1:right panel, the same parameters as the previous condition were used, but the session duration was fixed to T = 60. For the “fixed reward (known or unknown session duration)” condition, we assumed that the reward field is independent of the amount of reward earned: Ax,t = lH. (A53) (A53) Given the above reward field, the optimal rate of outcome earning is: Given the above reward field, the optimal rate of outcome earning is: v∗= Hl −bk 2ak2 . (A54) (A54) The simulation of this condition was done using parameters k = 15, l = 0.1, a = 0.002, b = 0.1, H = 40. Note that in this condition the response rate was independent of the session duration. The response rates in all the conditions were obtained by multiplying the outcome rates by k (since k responses are required to earn one unit of outcome). The simulation of this condition was done using parameters k = 15, l = 0.1, a = 0.002, b = 0.1, H = 40. Note that in this condition the response rate was independent of the session duration. The response rates in all the conditions were obtained by multiplying the outcome rates by k (since k responses are required to earn one unit of outcome). Simulation details of Figures 2, 3. OPTIMAL RESPONSE VIGOR AND CHOICE In this condition, the motivational drive at point x, denoted by Dx, will be: Dx = 1 2(H −lx)2. (A50) (A50) Keramati et al (Keramati & Gutkin, 2014) showed that such a definition of the motivational drive has implications that are consistent with the behavioral evidence. According to the framework, the reward generated by earning δx units of the outcome is proportional to the change in the motivational drive, which can be expressed as: Ax,t = −∂Dx ∂x = l(H −lx). (A51) (A51) As equation A51 suggests, with earning more outcomes (increase in x) Ax,t decreases. Given the above reward field, the optimal response rate of outcome earning, obtained by equation A36, will b As equation A51 suggests, with earning more outcomes (increase in x) Ax,t decreases. Given the above reward field, the optimal response rate of outcome earning, obtained by equation A36, will b be: v∗= Hl −bk T l2 +2ak2 . (A52) (A52) . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint OPTIMAL RESPONSE VIGOR AND CHOICE The simulation depicted in Figure 2 and Figure 3 are using equation A52 with the following parameters (note that the optimal response rates were obtained by multiplying v∗by k). For Figure 2:right panel simulation parameters are T = 50, k = 1, l = 1, a = 1, H = 8. Parameter b is varied between 3 to 7 in order to generate the plot. In Figure 3:left panel simulation parameters are T = 50, l = 1, a = 0.3, b = 0.05, H = 100. Parameter k was varied between 1 to 100 in order to generate the plot. In Figure 3:middle panel simulation parameters are T = 50, k = 1, l = 1, a = 0.3, b = 0.05. Parameter H was varied between 10 to 100 in order to generate the plot. In Figure 3:right panel simulation parameters are T = 50, k = 1, a = 0.1, b = 0.1, H = 100. Parameter l was varied between 0 to 1 in order to generate the plot. Optimal actions in multi-dimensional outcome space The aim of this section is to derive the optimal actions in the condition that the outcome space is multi-dimensional. Optimal trajectory will satisfy the Euler-Lagrange equation along each outcome dimension: d ∂ ∂ d dt ∂L ∂v = ∂L ∂x , (A55) (A55) where: Lx,v,t = Ax,t.v−Kv. (A56) (A56) Furthermore since the end point of the trajectory is free (the total amount of outcomes is not fixed) we have: ∂L ¯ Furthermore since the end point of the trajectory is free (the total amount of outcomes is not fixed) we have: ∂L ¯ ∂L ∂v ¯¯¯¯ t=T = 0. (A57) (A57) Using equation A55, A56 we have: d dt µ d dv(−Kv +v.Ax,t) ¶ = d(v.Ax,t) dx . (A58) (A58) . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint OPTIMAL RESPONSE VIGOR AND CHOICE 26 For the right hand side of the above equation we have: For the right hand side of the above equation we have: d(v.Ax,t) dx = v⊺∂Ax,t ∂x . (A59) We also have: d Ax,t dt = ∂Ax,t ∂t + ∂Ax,t ∂x v, (A60) d(v.Ax,t) dx = v⊺∂Ax,t ∂x . (A59) d(v.Ax,t) dx = v⊺∂Ax,t ∂x . ( (A59) dx ∂x We also have: d Ax,t dt = ∂Ax,t ∂t + ∂Ax,t ∂x v, (A60) We also have: d Ax,t dt = ∂Ax,t ∂t + ∂Ax,t ∂x v, (A60) (A60) which by substitution into equation A58 yields: which by substitution into equation A58 yields: which by substitution into equation A58 yields: d dt ∂Kv ∂v = ∂Ax,t ∂t + à ∂Ax,t ∂x − ∂A⊺ x,t ∂x ! v. (A61) (A61) We now provide two lemmas, which will be used in the proof of the following theorems. We now provide two lemmas, which will be used in the proof of the following theorems. where: Lemma 2 Assume that H is the Hessian matrix of Kv, i.e., [H]i,j = K 2 v ∂vi∂v j , (A62) [H]i,j = K 2 v ∂vi∂v j , [H]i,j = K 2 v ∂vi∂v j , (A62 (A62) and furthermore assume that the cost of earning outcomes along each dimension is independent of the outcome rate on the other dimensions, i.e., and furthermore assume that the cost of earning outcomes along each dimension is independent of the outcome rate on the other dimensions, i.e., and furthermore assume that the cost of earning outcomes along each dimension is independent of the outcome rate on the other dimensions, i.e., Hi,j = 0,i ̸= j. (A63) Hi,j = 0,i ̸= j. (A63) (A63) Then: Proof. Using equation A66 we get: [M]i,j = ∂[Ax,t]i ∂x j − ∂[Ax,t]j ∂xi = −∂2Dx ∂x j∂xi + ∂2Dx ∂xi∂x j = −∂2Dx ∂xi∂x j + ∂2Dx ∂xi∂x j (using Schwarz’s theorem) = 0 = 0. Note that the use of Schwarz’s theorem is based on the assumption that Dx is twice differentiable, which holds in the circumstances that we consider here. Note that the use of Schwarz’s theorem is based on the assumption that Dx is twice differentiable, which holds in the circumstances that we consider here. Theorem 3: Proof and simulation details Proof of Theorem 3. Theorem 3 assumes that (i) the costs of earning outcomes are inde- pendent (equation A63), (ii) the reward field is conservative (equation A66), and (iii) the reward field is independent of time (∂Ax,t/∂t = 0). Based on Lemma 2, Lemma 3 and equation A61 we have: 2 dv dt ⊙ µ∂2Kv ∂v2 ¶ = 0. (A68) (A68) Given that equation A37 holds along each outcome dimension (∂2Kv/∂v2 > 0), the only admis- sible solution to equation A68 is dv/dt = 0, which shows that the optimal rate of earning out- comes is constant. Since the optimal rate is constant, we have xT = T v∗, which by substituting in boundary conditions implied by equation A57 yields equation 10: ∂Kv∗ ∂v∗= AT v∗,T , (A69) (A69) Then: Then: d dt ∂Kv ∂v = dv dt ⊙ µ∂2Kv ∂v2 ¶ , (A64) (A64) where ∂2Kv/∂v2 represents the diagonal terms of H, and ⊙is entrywise Hadamard product. where ∂Kv/∂v represents the diagonal terms of H, and ⊙is entrywise Hadamard product. Proof. Using equation A63 we have: Proof. Using equation A63 we have: Proof. Using equation A63 we have: d dt ∂Kv ∂v = dv dt H = dv dt ⊙ µ∂2Kv ∂v2 ¶ , d dt ∂Kv ∂v = dv dt H = dv dt ⊙ µ∂2Kv ∂v2 ¶ , (A65) (A65) where the last equation comes from the fact that H is a diagonal matrix. Lemma 3 Assuming that the reward field is conservative, i.e., Ax,t = −∂Dx ∂x , (A66) then: M = ∂Ax,t ∂x − ∂A⊺ x,t ∂x = 0. (A67) then: M = ∂Ax,t ∂x − ∂A⊺ x,t ∂x = 0. (A67) (A67) . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint OPTIMAL RESPONSE VIGOR AND CHOICE OPTIMAL RESPONSE VIGOR AND CHOICE 27 Proof. Using equation A66 we get: OPTIMAL RESPONSE VIGOR AND CHOICE 28 The above equation was used as the reward field in the simulations. As for the cost function, earning one unit of O1 requires k responses on the left lever, and earning one unit of O2 requires lk responses on the right lever. Based on this and using equation 3, the cost function will be: Kv = v1[ak2v1 +kb]+ v2[ak2l2v2 +klb], (A72) (A72) where v1 is the rate of earning O1 and v2 is the rate of earning O2. Using Theorem 3, the optimal response rate will be: Using Theorem 3, the optimal response rate will be: Using Theorem 3, the optimal response rate will be: Using Theorem 3, the optimal response rate will be: response rate =   for left lever z }| { kl2H T l2 +2ak2l2 +T , for right lever z }| { klH T l2 +2ak2l2 +T  , (A73) (A73) where as mentioned in the main text “left lever” is the response that should be taken for earning O1, and “right lever” is the response that should be taken for earning O2. Parameters used for simulations are k = 1, l = 2, a = 1, b = 0, H = 100. where as mentioned in the main text “left lever” is the response that should be taken for earning O1, and “right lever” is the response that should be taken for earning O2. Parameters used for simulations are k = 1, l = 2, a = 1, b = 0, H = 100. which completes the proof the theorem. which completes the proof the theorem. Simulation details. For the simulation of the model in Figure 4:left panel “independent cost” condition, it is assumed that the two outcomes have the same reward effect, but earning the second outcome requires l times more responses. Following Keramati et al (Keramati & Gutkin, 2014), since the two outcomes have the same reward properties we defined the motivational drive as follows: Dx = 1 2(H −x1 −x2)2, (A70) (A70) where as mentioned Dx is the motivational drive and it represents the deviations of the internal state of the decision-maker from its homeostatic set-point (H). x1 is the amount of O1 earned and x2 is the amount of O2 earned, and the current motivational drive for earning outcomes de- pends on the difference between the total amount of earned outcomes (x1 +x2) and the homeo- static set-point (H). Given the motivational drive, the amount of reward generated by consuming each out- come will be equal to the amount of change in the motivational drive due to the consumption of the outcomes (equation 8), and therefore, we have: Ax,t = −∂Dx ∂x = [H −x1 −x2,H −x1 −x2]. (A71) (A71) . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint Using the boundary condition mentioned in equation A57 we have: Parameters used for simulations are k = 1, l = 2, a = 1, b = 0, H = 100. Parameters used for simulations are k = 1, l = 2, a = 1, b = 0, H = 100. OPTIMAL RESPONSE VIGOR AND CHOICE OPTIMAL RESPONSE VIGOR AND CHOICE 29 Theorem A2: Definition, proof and simulation details Proof of Theorem A2. The aim of this section is to derive optimal actions in the condi- tions that the costs of earning outcomes are dependent on each other. In this condition, one can assume what determines the cost is the delay between subsequent responses, either for the same or for a different outcome, i.e., the cost is proportional to the rate of earning all of the outcomes. In particular, if for earning O1, k responses are required and for earning O2, lk responses are required (l ̸= 1), then the delay between subsequent responses (τ) will be 1/(kv1 +lkv2). Given equation 2, the cost of earning one unit of O1 will be k[a(kv1+lkv2)+b], and the cost of earning one unit of O2 will be kl[a(kv1 +lkv2)+b]. Such a cost function can be achieved by defining the cost as follows: Kv = v1[ak(kv1 +lkv2)+kb]+ v2[akl(kv1 +lkv2)+klb]. (A74) (A74) In the following theorem, we maintain that given the above cost function, the optimal actions are to make no response for O2, and to make responses for O1 at a constant rate. In the following theorem, we maintain that given the above cost function, the optimal actions are to make no response for O2, and to make responses for O1 at a constant rate. Theorem A2 Given the cost function defined in equation A74 and assuming that the two out- comes have the same reward properties, i.e.,: [Ax,t]1 = [Ax,t]2. (A75) (A75) Then the optimal actions satisfy the following equations: Then the optimal actions satisfy the following equations: Then the optimal actions satisfy the following equations: p fy f g q dv1 dt = 0, v2 = 0. (A76) dv1 dt = 0, v2 = 0. (A76) (A76) Proof. By substituting equation A74 in equation A56 we have: Proof. By substituting equation A74 in equation A56 we have: uting equation A74 in equation A56 we have: L = −v1 [ak(kv1 +lkv2)+kb)]−v2 [akl(kv1 +lkv2)+klb]+ v1[Ax,t]1 + v2[Ax,t]2. (A77) (A77) . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint Using the boundary condition mentioned in equation A57 we have: Using the boundary condition mentioned in equation A57 we have: Using the boundary condition mentioned in equation A57 we have: [AxT ,T ]1 −2ak2lv2 −2ak2v1 −bk = 0, [AxT ,T ]2 −2ak2l2v2 −2ak2lv1 −bkl = 0. (A78) (A78) v1 = −lv2 −b/(2ak), (A79) (A79) which is not admissible given constraints v1 ≥0 and v2 ≥0, and therefore we assume either v1 or v2 will be equal to zero. The trajectory will have a higher value by setting v2 to zero since O2 has a higher cost, and therefore the optimal solution will be v2 = 0. Since v2 = 0 the problem de- generates to a one-dimensional problem, in which according to Theorem 1 the optimal response rate is constant, and therefore the rate of responding for O1 will be constant, which proves the theorem. Simulation details. For the simulation of the model in Figure 4:left panel “dependent cost” condition, it is assumed that k responses on the left lever are required to earn O1 and lk response are required on the right lever to earn O2. Similar to the “independent cost” condition mentioned in the previous section, the reward field was assumed as follows: Ax,t = −∂Dx ∂x = [H −x1 −x2,(H −x1 −x2)]. (A80) (A80) Since the response rate for one of the outcomes will be zero (according to Theorem A2), the problem degenerates to an environment with one action and one outcome. Using Theorem 1, and equation A36 the optimal response rate will be: Since the response rate for one of the outcomes will be zero (according to Theorem A2), the problem degenerates to an environment with one action and one outcome. Using Theorem 1, and equation A36 the optimal response rate will be: response rate =  k H −bk T +2ak2 | {z } for left lever , 0 |{z} for right lever  . (A81) Parameters used for simulations are k = 1, l = 2, a = 1, b = 0, H = 100. response rate =  k H −bk T +2ak2 | {z } for left lever , 0 |{z} for right lever  . (A81) (A81) Parameters used for simulations are k = 1, l = 2, a = 1, b = 0, H = 100. Parameters used for simulations are k = 1, l = 2, a = 1, b = 0, H = 100. OPTIMAL RESPONSE VIGOR AND CHOICE 30 In this condition, ∂[Ax,t]1/∂x2 = −1 and ∂[Ax,t]2/∂x1 = −l, and therefore the reward of the second outcome due to the consumption of the first outcome decreases more sharply than the reward of the first outcome would, due to the consumption of the second outcome. We have: M = ∂Ax,t ∂x − ∂A⊺ x,t ∂x = · 0 l −1 1−l 0 ¸ , (A84) (A84) and as long as l ̸= 1 then M ̸= 0, and therefore the reward field is non-conservative, because if it was conservative then according to Lemma 3 we should have M = 0. If the reward field is non-conservative, i.e., there does not exist a scalar field Dx such that Ax,t satisfies equation 8, then the optimal response rates are as follows: early in the session the decision-maker exclusively works for the outcome with the higher reward value (O1) and, when the time remaining in the session is less than the threshold (Tc), the decision-maker then gradu- ally starts working for the outcome with the lower reward value (O2). More precisely we maintain the following theorem: Theorem A3 If the reward field follows equation A83, ∂Ax,t/∂t = 0, and the cost is as follows: Kv = 1 2mv2 1 + 1 2mv2 2, (A85) (A85) then the optimal trajectory in the outcome space will be: then the optimal trajectory in the outcome space will be: n the optimal trajectory in the outcome space will be: [v1,v2] = (h 0, H(l−1) T l−Tc i , T −t > Tc arc of a circle T −t ≤Tc , (A86) (A86) where Tc = m arctan(1/l) l −1 , m = 2ak2. (A87) Tc = m arctan(1/l) l −1 , m = 2ak2. (A87) (A87) Proof. We have: ∂Ax,t ∂x − ∂A⊺ x,t ∂x = · 0 l −1 1−l 0 ¸ , (A88) (A88) and based on equations A63, A85, A61 we get: dv1 dt = l −1 m v2, dv2 dt = −l −1 m v1. (A89) (A89) Defining w = (l −1)/m, the solution to the above set of differential equations has the form: x = £ q1 +r/w sin(wt +α),q2 +r/w cos(wt +α) ¤ , (A90) (A90) which is an arc of a circle centered at [q1,q2], and r and α are free parameters. Theorem A3: Definition, proof and simulation details Proof of Theorem A3. The aim of Theorem A3 is to derive optimal actions when the re- ward field is non-conservative and the costs of actions are independent. An example of a non- conservative reward field is when the amount of reward that consuming an outcome produces is greater or smaller than the change in the motivational drive. For example, assume that there are two outcomes available, and the consumption of both outcomes has a similar effect on the motivational drive: 1 Dx = 1 2(H −x1 −x2)2, (A82) (A82) but the reward that the second outcome generates is l times larger (l ̸= 1) than the change it creates in the motivational drive: but the reward that the second outcome generates is l times larger (l ̸= 1) than the change it creates in the motivational drive: Ax,t = · −∂Dx ∂x1 ,−l ∂Dx ∂x2 ¸ = [H −x1 −x2,l(H −x1 −x2)]. (A83) (A83) . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint where Tc = m arctan(1/l) l −1 , (A93) (A93) and therefore Tc is independent of H (the initial motivational drive). As such if T ≤Tc (equa- tion A92) then the optimal trajectory will be an arc of a circle starting from the origin. Otherwise, if T > Tc, the optimal trajectory will be composed of two segments. In the first segment, v1 will take the boundary condition v1 = 0 and the decision-maker earns only the second outcome (the outcome with the higher reward effect). The first segment continues until the remaining time in the session satisfies equation A92 (the remaining time is less than Tc), after which the second segment starts, which is an arc of a circle defined by equation A90. The rate of earning the second outcome, v2, in the first segment of the trajectory (when v1 = 0) can be obtained by calculating the rates at the beginning of the circular segment. The initial rate at the start of the circular segment is as follows: r = H(l −1) T l −Tc , r = H(l −1) T l −Tc , (A94) (A94) which implies that at the first segment of the trajectory we have: [v1,v2] = · 0, H(l −1) T l −Tc ¸ , (A95) (A95) which completes the proof of Theorem A3. which completes the proof of Theorem A3. It is interesting to mention that there is a parallel between the trajectory that a decision- maker takes in the outcome space, and the motion of a charged particle in a magnetic field. In the case that the outcome space is three dimensional, using equation A61 the optimal path in the outcome space satisfies the following properties: m dv dt = à ∂Ax,t ∂x − ∂A⊺ x,t ∂x ! v = −v×B, (A96) (A96) = −v×B, where × is the cross product, B is the curl of the reward field (B = curlAx,t), and m = 2ak2. The equation A96 in fact lays out the motion of a unit charged particle (negatively charged) with mass m in a magnetic field with magnitude B. where × is the cross product, B is the curl of the reward field (B = curlAx,t), and m = 2ak2. The equation A96 in fact lays out the motion of a unit charged particle (negatively charged) with mass m in a magnetic field with magnitude B. Simulation details. OPTIMAL RESPONSE VIGOR AND CHOICE The parameters can be determined using the boundary condition imposed by equation A57, and also assuming that the initial position is x = 0. The boundary condition in equation A57 implies: mv = Ax,t ¯¯ t=T = hp 2Dx,l p 2Dx i , (A91) (A91) . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 7, 2017. ; https://doi.org/10.1101/106500 doi: bioRxiv preprint OPTIMAL RESPONSE VIGOR AND CHOICE 31 which implies that at the end of the trajectory the rate of earning the second outcome is l times larger than the first outcome. Therefore, the general form of the trajectory will be an arc starting from the origin and ending along the above direction. Given the constraint that v ≥0 only the solutions in which q2 ≤0 are acceptable ones (i.e., the center of the circle is below the x-axis). Solving equation A90 for q2 ≤0 we get: (A92) T ≤Tc, (A92) T ≤Tc, where Simulations shown in Figure 4:right panel are based on Theorem A3, and the parameters used are k = 1, l = 1.1, a = 1, b = 0, H = 100, m = 2ak2.
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Quantifying dynamic mechanisms of auto-regulation in Escherichia coli with synthetic promoter in response to varying external phosphate levels
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Quantifying dynamic mechanisms of auto-regulation in Escherichia coli with synthetic promoter in response to varying external phosphate levels Cansu Uluşeker1,2, Jesús Torres-Bacete3, José L. García4,5, Martin M. Hanczyc   1,6, Juan Nogales3 & Ozan Kahramanoğulları7 Received: 2 May 2018 Accepted: 13 December 2018 Published: xx xx xxxx Escherichia coli have developed one of the most efficient regulatory response mechanisms to phosphate starvation. The machinery involves a cascade with a two-component system (TCS) that relays the external signal to the genetic circuit, resulting in a feedback response. Achieving a quantitative understanding of this system has implications in synthetic biology and biotechnology, for example, in applications for wastewater treatment. To this aim, we present a computational model and experimental results with a detailed description of the TCS, consisting of PhoR and PhoB, together with the mechanisms of gene expression. The model is parameterised within the feasible range, and fitted to the dynamic response of our experimental data on PhoB as well as PhoA, the product of this network that is used in alkaline phosphatase production. Deterministic and stochastic simulations with our model predict the regulation dynamics in higher external phosphate concentrations while reproducing the experimental observations. In a cycle of simulations and experimental verification, our model predicts and explores phenotypes with various synthetic promoter designs that can optimise the inorganic phosphate intake in E. coli. Sensitivity analysis demonstrates that the Pho-controlled genes have a significant influence over the phosphate response. Together with experimental findings, our model should thus provide insights for the investigations on engineering new sensors and regulators for living technologies. Mechanisms of inorganic phosphate intake within the context of cellular processes is a topic of extensive research effort, also due to potential applications in enhanced biological phosphorus removal (EBPR) from wastewater. Phosphorous, which is one of the major causes of water quality problems, occurs in wastewater almost solely in the form of phosphates such as inorganic phosphate (Pi). Microorganisms, which are key players in bioremedia- tion, have potential to treat large amounts of the pollutants and hold promise for renewable sources1. An in-depth understanding of the mechanisms controlling such processes should pave the way for metabolic engineering to lead to improvements in wastewater treatment.h p The physiological characteristics of Pi transport in E. coli have been extensively studied2–6. Nonetheless, the regulatory interactions that control the Pi transport are complex, and they involve two major phosphate transport systems, depending on external Pi levels. The low affinity phosphate inorganic transport (Pit) system depends on the proton motive force; it is a coupled transporter of two different ions through the membrane2,7. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 2 May 2018 Accepted: 13 December 2018 Published: xx xx xxxx Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w www.nature.com/scientificreports/ Consequently, the tanscription factor PhoB does not become phosphorylated by PhoR. Right: Due to the ABC transporter activity, the external Pi binds to the PstS component of the ABC transporter. It is then translocated to the inner membrane domain of the transporter through PstC and PstA. Following this, PstB changes its conformation by consuming ATP. Pi is internalised and released to the cytosol. When the Pi influx through the ABC transporter decreases, PhoU does not stabilise PhoR, which is depicted with the red bar next to PhoU. As a result of this, PhoR becomes free to perform its auto-kinase-phosphotransferase activity, whereby it phosphorylates PhoB. Phosphorylated PhoB then forms a dimer to act as a transcription factor for the operons, resulting in PhoA, PhoB and PhoR expression. specific transport (Pst) system, on the other hand, is Pi-repressible, and it is induced when the external Pi concen- tration is depleted8–10. p Mechanistically, Pi signalling associated with the Pst system in E. coli, is a negative process, that is, excessive Pi is required for turning the system off. Activation is the default state and occurs in conditions of Pi limitation2,8,11. Signal transduction by environmental Pi requires seven proteins, which are thought to interact in a membrane associated signalling complex. These Pi signalling proteins include2,8. • Four components of the ABC transporter Pst (PstSCAB), which consist of an extracellular binding protein (PstS), two transmembrane proteins (PstC, PstA) that form the transmembrane domain (TMD), and a dimer of cytosolic peripheral proteins (PstB), i.e., the nucleotide-binding domain; • Four components of the ABC transporter Pst (PstSCAB), which consist of an extracellular binding protein (PstS), two transmembrane proteins (PstC, PstA) that form the transmembrane domain (TMD), and a dimer of cytosolic peripheral proteins (PstB), i.e., the nucleotide-binding domain; y g • Two that are members of the large family of two component systems (TCS) that perform both positive and negative regulation, a sensor histidine kinase PhoR and a transcriptional response regulator PhoB; The chaperone like PhoR/PhoB inhibitor protein PhoU • Two that are members of the large family of two component systems (TCS) that perform both positive and negative regulation, a sensor histidine kinase PhoR and a transcriptional response regulator PhoB; The chaperone like PhoR/PhoB inhibitory protein PhoU g g p p g • The chaperone-like PhoR/PhoB inhibitory protein PhoU. g g p p g The chaperone-like PhoR/PhoB inhibitory protein PhoU. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. The transmembrane signal transduction due to external Pi levels and the Pho regulon activity when extracellular inorganic phosphate (Pi) is in excess (left) and when it is depleted (right). The ABC transporter consists of the extracellular domain PstS, transmembrane domains PstA and PstC, and intracellular domains PstB (see the main text for the description). Left: when external Pi is in excess, the ABC transporter activity is limited as the cell does not consume ATP for the Pi transport. According to the current model in the literature, PhoR assesses Pi availability by monitoring the activity of Pst transporter, and relays the signal from PstB via PhoU to PhoR. When the Pi influx is increased, PhoU stabilises PhoR, which is depicted with the green bars around PhoU. This prevents PhoR dimers from autphosphorylating (red). Consequently, the tanscription factor PhoB does not become phosphorylated by PhoR. Right: Due to the ABC transporter activity, the external Pi binds to the PstS component of the ABC transporter. It is then translocated to the inner membrane domain of the transporter through PstC and PstA. Following this, PstB changes its conformation by consuming ATP. Pi is internalised and released to the cytosol. When the Pi influx through the ABC transporter decreases, PhoU does not stabilise PhoR, which is depicted with the red bar next to PhoU. As a result of this, PhoR becomes free to perform its auto-kinase-phosphotransferase activity, whereby it phosphorylates PhoB. Phosphorylated PhoB then forms a dimer to act as a transcription factor for the operons, resulting in PhoA, PhoB and PhoR expression. Figure 1. The transmembrane signal transduction due to external Pi levels and the Pho regulon activity when extracellular inorganic phosphate (Pi) is in excess (left) and when it is depleted (right). The ABC transporter consists of the extracellular domain PstS, transmembrane domains PstA and PstC, and intracellular domains PstB (see the main text for the description). Left: when external Pi is in excess, the ABC transporter activity is limited as the cell does not consume ATP for the Pi transport. According to the current model in the literature, PhoR assesses Pi availability by monitoring the activity of Pst transporter, and relays the signal from PstB via PhoU to PhoR. When the Pi influx is increased, PhoU stabilises PhoR, which is depicted with the green bars around PhoU. This prevents PhoR dimers from autphosphorylating (red). Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w Quantifying dynamic mechanisms of auto-regulation in Escherichia coli with synthetic promoter in response to varying external phosphate levels Cansu Uluşeker1,2, Jesús Torres-Bacete3, José L. García4,5, Martin M. Hanczyc   1,6, Juan Nogales3 & Ozan Kahramanoğulları7 The phosphate 1University of Trento, Centre for Integrative Biology, Trento, 38123, Italy. 2The Microsoft Research – University of Trento Centre for Computational and Systems Biology, Rovereto, 38068, Italy. 3Centro Nacional de Biotecnología (CNB-CSIC), Systems Biology Department, Madrid, 28049, Spain. 4Centro de Investigaciones Biológicas (CIB-CSIC), Microbial and Plant Biotechnology Department, Madrid, 28040, Spain. 5Institute for Integrative Systems Biology (I2Sysbio-CSIC-UV), Applied Systems Biology and Synthetic Biology Department, Paterna, 46980, Spain. 6Chemical and Biological Engineering, University of New Mexico, Albuquerque, NM, 87131, USA. 7University of Trento, Department of Mathematics, Trento, 38123, Italy. Correspondence and requests for materials should be addressed to O.K. (email: ozan.kahramanogullari@unitn.it) 1 Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w www.nature.com/scientificreports/ Previous studies have shown that the TCS plays a central role in sensing the Pi level in the environ- ment and regulating the expression of genes that are directly involved in the intake2,12. g g p g y Although much is known about the molecular aspects of this signal transduction pathway, a comprehensive and structured mechanistic model of the Pho regulon is currently unavailable3. Here, we investigate the relation- ship between Pi-starvation, the TCS signalling, and promoter activity by using a combination of wet lab exper- iments and dynamic modelling. This work introduces the proteins involved in phosphate starvation response mechanism. Our model describes the phosphate starvation response at the genetic level by considering all system variables that affect the phosphate starvation response. We consider components where phosphate starvation response of TCS model is described in terms of their interactions. The model is composed of a set of ordinary differential equations and the corresponding stochastic model that are derived from a chemical reaction network. The model provides a quantitative description of how different processes interact to form a positively-regulated biological control system. The mechanism is based on an E. coli Pi-starvation signalling system. Our model dis- plays how specific TCS proteins work together to provide gene expression and increase in Pi intake, and provides a set up for identifying a wide family of promoter mechanisms that potentially have synthetic applications.i p y g y p p y y pp In recent years, significant advances have been made in understanding the role and the structure of TCS and signalling mechanisms. It is known that most bacteria species have more than 10 different TCS13. Moreover, in E. coli, 30 sensor kinases and 32 response regulators have been found14. Although there is large qualitative knowledge, especially for E. coli, quantitative research is still scarce. A model for PhoR/PhoB signal transduction was set up by Van Dien and Keasling earlier3, whereby the authors have determined a model for the induction of PhoA. Models for other signal transduction systems in E. coli are described by Wong et al., Fisher et al., and Koh et al.15–17. Recently, research groups have only just begun to use modelling in combination with experimental data to analyse the TCS especially PhoR and PhoB4,18. Gao et al. built quantitative analyses of PhoR and PhoB protein concentrations and activities. They showed that the experimental data and protein expression levels of TCSs matched. www.nature.com/scientificreports/ Figure 1 displays a schematic representation of the system in the starvation condition. When Pi is limited in quantity outside the cell, PstS binds to the Pi following its diffusion to the cell surface2,8,12. The transmembrane domain of the ABC transporter, that is, PstC and PstA are integral membrane proteins that span the entirety of the membrane. They regulate the translocation of Pi from PstS to the inner membrane2,3,8,12. Pi intake happens with the conformational changes in the PstB as a result of ATP binding, also known as ATP-switch model. This way, the ABC transporter provides the required increase in the amount of phosphate in the cell2,8.h p p q p p The low activity of the ABC transporter PstSCAB causes, in a number of steps, the autophosphorylation of the sensor kinase PhoR, which relays the signal to the transcription factor PhoB. The current evidence suggests that PhoR and eventually PhoB assess Pi availability by monitoring the activity of Pst transporter via PhoU11,12. In mechanistic terms, when there is sufficient Pi flux, PhoU stabilizes PhoR. The resulting stable conformation prevents PhoR from auto-phosphorylation. This, in return, prevents the down-stream signalling that would oth- erwise result in the expression of the genes that feedback to the Pst system for further Pi intake. In fact, when PhoU is deleted, PstB does not only continue to spend ATP and transport Pi, but PhoR acts as a constitutive PhoB kinase, leading to high expression of the Pho regulon genes2,8, and thereby, activating the expression of the Pst sys- tem. On the other hand, when external Pi is limited, PhoU does not stabilise PhoR. Consequently, PhoR is freed to bind phosphoryl groups and passes to the active state. Active PhoR phosphorylates PhoB. Phosphorylated PhoB then acts as a transcription factor for the operon. Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w 2 www.nature.com/scientificreports/ All experimental evidence indicates that Pho regulon is controlled by external phosphate limitation rather than internal3. When the surrounding environment has abundant phosphate, E. coli uses as few resources as possible to facilitate the phosphate intake. However, when Pi becomes low outside the cell, it turns into a growth limiting factor and the cell spends energy to up-regulate the expression of target genes that are used to acquire phosphate. www.nature.com/scientificreports/ Recently, Gao and Stock et al.19 built a quantitative analyses of TCS switch off mechanism. However, to the best of our knowledge, there has been no focus on developing genetically encoded signal transduction path- ways leading to transcription factors. y g p In this work, we have developed an approach combining wet-lab experiments and modelling to show that rates of histidine kinase and promoter activities can be used to tune TCS detection thresholds. We have described the steps in the design of a synthetic biological system based on the use of TCS. This should constitute a contri- bution to the research on genetically modified bacteria that detect environmental changes and respond to higher inorganic phosphate levels. In order to better understand the quantitative analyses of protein concentrations in response to modifications and activities in the environment, we have focused on the activation response of the TCS components PhoB and PhoR. Our model should thus help to better understand the dynamic behaviour of system activation, and to quantitatively evaluate the role of phosphatase activity under varying external Pi conditions. The model includes TCS members and activation of the Pho regulon promoters pPhoB and pPhoA. Experimental data is used to fit the parameters to the feasible physiological range given in the literature, and to determine the relative sensitivity to the parameters. The simulations with our model provide a dynamic descrip- tion of the mechanisms. With a combination of wet-lab experiments and computer simulations, we use our model to quantify dynamic mechanisms of auto-regulation in E. coli in response to varying external phosphate levels, and explore and verify emerging phenotypes with synthetic promoters. Simulations with the model do not only reproduce our experimental measurements, but also predict phenotypes with various synthetic promoter designs that can optimise the Pi intake in E. coli. Sensitivity analysis on the parameters demonstrate the influence on the expression of Pho-controlled genes and the gain of the system under variations in transcription efficiency in response to external phosphate concentration. The model can thus serve as a virtual lab, and can be used to test various promoter designs for enhanced Pi intake in biotechnology applications for phosphate sequestration. Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w Results Ph h The deterministic ODE and stochastic simulations are performed by applying the standard translation from CRNs based on stoichiometry. The blue color denotes the proteins, orange denotes the promoters in their active and inactive forms, and purple denotes the mRNA molecules. Filled arrowheads denote the reversible reactions. The red arrows denote degradation reactions, the green arrows denote complexations, the black arrows denote phosphorylation and dephosphorylation, and the blue arrows denote the transcription and translation reactions. The model species that are plotted in C and D are distinguished with frames. (C) The dynamics of the highlighted species of the control model in panel B as a result of the fitting procedure together with the experimental data, as described in Methods, are plotted. In the first of the two steps, the blue curves are obtained by using only the experimental values of PhoA levels. In the second step, that delivers the control model, the red curves are obtained by using the experimental values of both PhoA and PhoB levels. Inclusion of the PhoB data highlights the contribution of the feedback mechanism to the response dynamics, which is otherwise not represented. The stochastic dynamics, plotted in grey, display the fluctuations in the control model that are due to small molecule numbers and are not observable in the deterministic simulations. (D) The response of the control model to variations in the external Pi levels, which are represented as fold change factors, applied to the autophosphorylation propensities of PhoR. A higher external Pi concentration corresponds to a smaller factor and vice versa. Figure 2. Schematic representation of the experimental data and the control model, and its dynamics in response to varying external Pi concentrations. (A) The data for the PhoA and PhoB expression are obtained using PCR amplified DNA from E. coli MG1655 genome and transcriptionally fused to the translational coupler BCD2 and the fluorescent ms-fgfp gene. (B) The control model has been obtained from the experimental data and the chemical reaction network (CRN) described in Methods by applying a fitting procedure with the physiological ranges obtained from the literature in Table 1, and verified by sensitivity analysis. The deterministic ODE and stochastic simulations are performed by applying the standard translation from CRNs based on stoichiometry. The blue color denotes the proteins, orange denotes the promoters in their active and inactive forms, and purple denotes the mRNA molecules. Filled arrowheads denote the reversible reactions. Results Ph h The red arrows denote degradation reactions, the green arrows denote complexations, the black arrows denote phosphorylation and dephosphorylation, and the blue arrows denote the transcription and translation reactions. The model species that are plotted in C and D are distinguished with frames. (C) The dynamics of the highlighted species of the control model in panel B as a result of the fitting procedure together with the experimental data, as described in Methods, are plotted. In the first of the two steps, the blue curves are obtained by using only the experimental values of PhoA levels. In the second step, that delivers the control model, the red curves are obtained by using the experimental values of both PhoA and PhoB levels. Inclusion of the PhoB data highlights the contribution of the feedback mechanism to the response dynamics, which is otherwise not represented. The stochastic dynamics, plotted in grey, display the fluctuations in the control model that are due to small molecule numbers and are not observable in the deterministic simulations. (D) The response of the control model to variations in the external Pi levels, which are represented as fold change factors, applied to the autophosphorylation propensities of PhoR. A higher external Pi concentration corresponds to a smaller factor and vice versa. procedure. The rates in Table 1 are obtained as a result of this fitting procedure that delivered the control model parameters, whereby we have enforced the displayed physiologically boundaries. The difference in the phenotype between red and blue curves in Fig. 2C should thus highlight the role of the feedback of the PhoB and PhoR expression to the system.i procedure. The rates in Table 1 are obtained as a result of this fitting procedure that delivered the control model parameters, whereby we have enforced the displayed physiologically boundaries. The difference in the phenotype between red and blue curves in Fig. 2C should thus highlight the role of the feedback of the PhoB and PhoR expression to the system.i We have performed a large scale analysis of the system dynamics of the control model in terms of the fitted parameter values within a broader range. We have first analysed the broader effect of the TCS disassociation rates (r3r, r5r, r7r, r8r) on the dynamics and the PhoB transcription and translation parameters that deliver the expres- sion of PhoB (r14, r15), which is a transcription factor. Results Ph h Phosphate intake at starvation requires rapid activation of PhoB dimers. Figure 2 provides a schematic representation of the control model set up with the experimental data and the formal model. The control model is set up with respect to the initial phosphate starvation in accordance with the experimental data and the parametrisation and fitting procedure described in Methods. In first of the two steps, the blue curves in Figs 2C and S2 are obtained by using only the experimental values of PhoA levels. When external Pi is abundant, the Pst system inhibits the activation of TCS, and consequently the Pi intake; PhoA is then expressed at a basal level. However, when Pi is limiting, inhibition of TCS is relieved, resulting in the activation of PhoA transcription. The alterations of PhoA expression can thus be interpreted to an extent as an indicator for the changes in external phosphate level and Pi intake. In these simulations, in response to external Pi level at 0 μM, the system initiates the activation of PhoR, given by the autophosphorylation of both of the monomers in the stable dimer. This results in the subsequent transfer of phosphoryl groups. As a consequence, the response regulator PhoB rapidly becomes active and dimerises to form active transcription factors. The resulting rapid increase in the promoter activity delivers the mRNA tran- scription, and the consequent experimentally observed levels of PhoA. At a second step in our analysis, to highlight and contrast the role of PhoB dynamics in the feedback mecha- nism, we have refined the model to include the experimental data on PhoB expression. The resulting red curves in Figs 2C and S2 are obtained by using the relative experimental levels of both PhoA and PhoB in the fitting Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223 3 www.nature.com/scientificreports/ Figure 2. Schematic representation of the experimental data and the control model, and its dynamics in response to varying external Pi concentrations. (A) The data for the PhoA and PhoB expression are obtained using PCR amplified DNA from E. coli MG1655 genome and transcriptionally fused to the translational coupler BCD2 and the fluorescent ms-fgfp gene. (B) The control model has been obtained from the experimental data and the chemical reaction network (CRN) described in Methods by applying a fitting procedure with the physiological ranges obtained from the literature in Table 1, and verified by sensitivity analysis. Results Ph h On the other hand, the dissociation rate of the inactive PhoR from the active PhoB, given by r8r, affects the activation of PhoB as a transcription factor in a proportional way, yet its effect on promoter activity is negligible (Fig. S8). As it can be seen from Table 1, there are also other parameters that have an effect on both system dynamics and PhoB activity. Nonetheless, these are not suitable candidates for modification at this first analysis due to their physiologic ranges, and they are discussed below. transcription and translation of PhoB and none on PhoA (Fig. S7). On the other hand, the dissociation rate of the inactive PhoR from the active PhoB, given by r8r, affects the activation of PhoB as a transcription factor in a proportional way, yet its effect on promoter activity is negligible (Fig. S8). As it can be seen from Table 1, there are also other parameters that have an effect on both system dynamics and PhoB activity. Nonetheless, these are not suitable candidates for modification at this first analysis due to their physiologic ranges, and they are discussed below. As it can be observed in Fig. 2, and supported by the analysis above, the simulations that include the PhoB data for parametrisation result in faster response dynamics, measured in terms of the time required to reach a peak state. This can be explained by the self-feeding role of the TCS, and the resulting increased requirement for the active transcription factors to sustain the experimentally observed protein levels due to their feedback to the network: because PhoR and PhoB are encoded by the same operon, not only PhoB levels, but also PhoR levels increase as a result of the changes in the reaction rates. This causes the cell to increase the sensor histidine kinase levels, resulting in a more immediate response. The response time thus decreases with an increase in the amount of sensor histidine kinase as well as an increase in its activity.h y The deterministic ODE simulations lead to observations that describe the average dynamic behaviour of the variable concentrations for the simulated 4.5 hours. To observe the possible fluctuations in the system, we have performed stochastic simulations. This way, we have been able to compare the mean behaviour with the regula- tory dynamics that incorporates the noise due to smaller molecular numbers. Results Ph h The cumulative output of different simulations with fold changes that cover the physiological intervals for r14 and r15 as well as a broad range for r3r, r5r, r7r, and r8r are depicted in Figs S3, S4, S5, S6, S7 and S8, respectively.f In these simulations, the dissociation rates of PhoR and PhoB, that is, r3r and r5r, have transient effects on the steady state concentrations of the complexes formed by these molecules. These rates do not modify the PhoB activity or the levels of PhoA and PhoB as shown in Figs S5 and S6. Variations in the dissociation rate of PhoB, given by r7r, affect the activity of PhoB as a transcription factor, however this has a minor effect on the Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w 4 www.nature.com/scientificreports/ Reaction Number Rate Symbol Fit Value Literature Value References 1 r1 25.3658 10–100 s−1 5 1 reverse r1r 8.1165  − s 10 1 5 2 r2 25.3658 10–100 s−1 5 2 reverse r2r 8.1165 −  s 10 1 5 3 r3 100 100 μM−1 s−1 48 3 reverse r3r 44.9411 N.A. N.A. 4 r4 21.3718 17–23 s−1 5 5 r5 100 100 μM−1 s−1 48 5 reverse r5r 94.9411 N.A. N.A. 6 r6 21.3718 17–23 s−1 5 7 r7 100 100 μM−1 s−1 48 7 reverse r7r 24.9411 N.A. N.A. 8 r8 100 100 μM−1 s−1 48 8 reverse r8r 34.9411 N.A. N.A. 9 r9 12.95  − s 17 1 5 10 r10 10000 10000 μM−1 s−1 6 10 reverse r10r 1000 N.A. 6 11 r11 10000 10000 μM−1 s−1 6 11 reverse 11r 1000 N.A. 6 12 r12 0.0540 0.0025–0.2 s−1 47,49 13 r13 0.0302 0.0006–0.05 s−1 47,50 14 r14 0.130 0.0025–0.2 s−1 47,49 15 r15 0.035 0.0006–0.05 s−1 47,50 16 r16 0.0302 0.0006–0.05 s−1 47,50 17 r17 0.0001 0.000096–0.00079 s−1 47,49,50 18 r18 0.0001 0.000096–0.00079 s−1 47,49,50 19 r19 0.0001 0.000096–0.00079 s−1 47,49,50 20 r20 0.0055 0.0055 s−1 47 21 r21 0.0055 0.0055 s−1 47 Table 1. Reactions and deterministic rates obtained from the physiological ranges. Table 1. Reactions and deterministic rates obtained from the physiological ranges. transcription and translation of PhoB and none on PhoA (Fig. S7). Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w www.nature.com/scientificreports/ Figure 3. The variations in the mRNA and active promoter levels due to the unbinding rate of the promoter and the active transcription factor. An unbinding rate of 100/sec (A) results in much less spread in the steady state distributions in comparison to unbinding rates of 1000/sec (B) and 5000/sec (C). The variations are quantified as the ratio of the standard deviation and the mean. Figure 3. The variations in the mRNA and active promoter levels due to the unbinding rate of the promoter and the active transcription factor. An unbinding rate of 100/sec (A) results in much less spread in the steady state distributions in comparison to unbinding rates of 1000/sec (B) and 5000/sec (C). The variations are quantified as the ratio of the standard deviation and the mean. To this end, we have experimented in stochastic simulations with different DNA unbinding rates, that is, r10r and r11r, from 100/s up to 5000/s. In accordance with the common practice, we have used stochasticity to quan- tify the noise that arises from the binding of a regulatory protein to a promoter21. The resulting amplification in oscillations in stochastic simulations, shown in Fig. 3 as well as Figs S9, S10 and S11, are due to the increase in the promoter unbinding rates. In Fig. 3, we have quantified the decrease in noise in the steady state distribution of mRNA and active promoter levels in terms of the ratio of the standard deviation over the mean. In this respect, the deterministic simulations display how DNA binding rates affect the mean behaviour, while the stochastic sim- ulations bring about the loss of coherence due to noise in gene expression. Figure 3B displays the simulations with the rates of the control model. In Fig. 3A, DNA unbinding rate is decreased by an order of magnitude, whereas in Fig. 3C, the effect of a higher DNA unbinding rate of 5000/s is depicted. These results indicate that lower unbind- ing rates, as observed in saturation conditions, are required for stable gene regulation that is not affected by noise. This also reflects how both genetics and noise due to environmental factors can affect the development of targeted pathway interventions for faster Pi accumulation.hf The results above show that our control model provides detailed predictions about the complex effects of pro- duction pathways of the Pi accumulation system. Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w Results Ph h Figure 2 as well as Figure S2 include a comparison of the deterministic and the stochastic simulations. The stochastic simulations are performed by applying the standard conversion to obtain molecule numbers from the concentrations. The grey fluctuating lines show the stochastic results at a single representative simulation; the simulation shows the expected fluctuations in the model species with smaller numbers such as mRNA molecules as well as the qualitative agreement between the stochastic and the deterministic simulations. Binding and unbinding of transcription regulators are a primary mechanism for gene regulation, whereby transcription factors operate at a fast time-scale. While the rate of binding of transcription regulators are known in many cells, little is known about how cells can modulate their unbinding for regulation20. The unbinding rate of an active transcription factor can thus vary over many orders of magnitude6,20. Therefore, in the initial analysis, we have fixed the transcription factor unbinding rates (r10r, r11r) to 1000 s−1 as shown in Table 1, and analysed the system behaviour with respect to variations. Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w 5 www.nature.com/scientificreports/ www.nature.com/scientificreports/ The simulations are in good agreement with the experimental data and the general concepts described in the literature for the functionality of Pho regulon2,3. The control model proposed can thus serve as a virtual lab, which can be used to test and justify the theoretical approaches on the Pi intake system. PhoR tunes the Pi intake both up and down. Besides the chemical properties of the proteins in the reg- ulatory system4, also the activity of the TCS proteins influence the Pi intake. Moreover, as displayed in Fig. 2C, the feedback mechanism due to the increased expression of sensor histidine kinase PhoR and the response regulator PhoB introduces a speed-up of an hour in comparison to the simulations, where this feedback mechanism is not taken into consideration. This indicates that a faster response in terms of Pi intake is delivered by an increase in the histidine kinase levels as well as the increase of its activity due to signalling. A notable feature of the Pi response system is that the sensor histidine kinase is bifunctional: it participates in both phosphorylation and dephosphorylation of its cognate response regulator. In this respect, the TCS autoreg- ulatory design is a distinct mechanism from the conventional positive feedback loops. The bifunctional PhoR component is an autokinase with concomitant opposing phosphatase activity22–24.h The dual role of PhoR is a mechanism that enhances signal robustness18,25. Moreover, it has also been shown that the phosphatase activity in TCS provides a rapid dephosphorylation mechanism that shuts off the system, and thereby restores it to the original state19. Such a dynamics can be triggered, for example, by an increase in the external Pi concentration and the consequent decrease in the autophosphorylation activity of PhoR. Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w 6 www.nature.com/scientificreports/ We have analysed the effect of the changes in autophosphorylation rates to the system behaviour. Because autophosphrylation becomes possible when the starvation signal prevents PhoU from inhibiting PhoR, the pro- pensity of autophosphorylation depends on the incoming signal, which is a function of the external Pi levels. By decreasing the autophosphorylation propensity by applying various fold changes, we can thus see the effect of an increase in external Pi concentration on the system as depicted in Figs 2D and S12. www.nature.com/scientificreports/ In these simulations, a decrease in PhoR activity due to increased external Pi concentration results in a proportional decrease in the active PhoB dimers, and a decrease in the promoter activity as well as the PhoR activity. p y y A complementary realisation of this mechanism is given by the association of PhoB to PhoR, that is, r8. Although the physiological range for this parameter is narrow, as a result of a hypothetical increase in the asso- ciation rate of PhoB and PhoR, the PhoB concentration stays low for a longer time period and the levels of active PhoB dimers decrease proportionally as displayed in the fold change experiments in Fig. S13. p p y p y g p g In our model, we assume that the growth in the cell culture within the considered time interval is negligible. However, due to cell cycle, which has a time scale in the order of an hour, the protein concentrations can be subject to dilution besides the active degradation of the molecules we have considered. To this end, Figs S14, S15 and S16 explore the effect of higher degradation rates due to dilution in growth conditions, (r17, r18, r19, r20, and r21), together with higher external Pi levels, given with a decrease in the rates r1 and r2. As it can be seen in Fig. S12, a decrease in the autophosphorylation rates does not only lower the steady state levels, but also slows down the activation of the transcription factor by preventing the formation of an initial peak in DiPhoBpp levels. A concomitant increase in the degradation and dilution rates delays reaching a steady state. However, this does not drastically alter the eventual active DiPhoBpp concentrations. Starvation response can be obtained with synthetic promoters at higher external Pi concen- trations. The simulation results in Figs 2D and S12 demonstrate the system’s adaptation to the stimuli due to Pi concentration, whereby the autophosphorylation propensity of PhoR acts as a proxy for the external Pi levels. These simulations predict how changes in the external Pi concentration affect the Pho regulon, and in particular, how the promoter activity decreases with an increase in the external Pi concentration. These results thus confirm that the adaptation of gene expression is clearly dependent on the Pi response stimuli2,12. www.nature.com/scientificreports/ Accumulation of ppGpp triggers the stringent response and a radical decrease in ribosome and protein synthesis, even leading to growth arrest31. When the level of RpoS is higher, E. coli is more resistant to stress but grows more slowly under a variety of conditions32,33.f We have further assessed our analysis to factor for the possible effects of stress response in Pi starvation con- ditions. As depicted in Fig. S17, Pi limitation induces the general stress response regulated by RpoS, sigma fac- tor29,30. Concomitantly, Pi limitation increases the intracellular level of guanosine tetraphosphate (ppGpp), which is known as stringent stress response12,30,31. RpoS and ppGpp are thought to maintain a balance between vegetative d / i f E li Th G l id i d R S l i 32 Th R S d and stress/starvation states of E. coli. The ppGpp nucleotide induces RpoS accumulation32. The RpoS responds to stress and shifts transcription away from vegetative growth and towards stress resistance31–34. Accumulation of ppGpp triggers the stringent response and a radical decrease in ribosome and protein synthesis, even leading to growth arrest31. When the level of RpoS is higher, E. coli is more resistant to stress but grows more slowly under a variety of conditions32,33.f y It is known that RpoS and ppGpp direct RNA polymerase to promoters, and negatively affect the expression of several PHO genes with varying effects18,35,36. We have assessed the effect of the RpoS and SpoT activity on the system by applying sensitivity analysis on the transcription rate of PhoB, as depicted in Fig. S3 for r14. In the light of this analysis, we have investigated the effect of stress response on the results depicted in Fig. 4, whereby the model is calibrated under varying stress response conditions due to Pi starvation. By repeating the analysis in Fig. 4 under these conditions, we have tested the activity of various promoter designs (Fig. S18). These results confirm the previous observations, and indicate that stress response can have a modulating effect, which can however be factored for by promoter design.h y p g These results indicate that the steady state promoter activity and the PhoA yield are highly correlated in all the regimes and for all the promoter binding and unbinding rates. Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w www.nature.com/scientificreports/ Moreover, the model provides a mechanistic explanation for the interplay between the system components under the conditions of varying external Pi concentrations, which result in variations in the promoter activity.li It is well established that the protein production rate is greatly influenced by the specific nucleotide sequence of the promoter26–28. In this respect, synthetic biology and genetic engineering methods aim at synthesising pro- moters with the desired strength. To this end, in order to observe the possible variations in gene expression due to variations in promoter strength, we have performed a class of simulations. The results of these simulations in Fig. 4 display measurements of the steady state levels of PhoA promoter activity (pPhoAa) as well as the PhoA yield of the system as the resulting product in terms of the area under the curve (PhoA AUC). In these simulations, we have scanned regimes with varying external Pi concentrations simulated by applying fold changes to the rates r1 and r2 as in the simulations in Figs 2D and S12. We have applied a fold change of 1 for the control regime with 0 μM external Pi, and 0.5, 0.2 and 0.1 for increasing external Pi levels. For each external Pi regime, we have scanned 100 different promoter designs by means of simulations that apply 10 fold change values from 0 to 2.5 with steps of 0.25 to the promoter binding rates r10 and r11 as well as 10 such fold change values to the promoter unbinding rates r10r and r11r. The heatmaps resulting from these 100 simulations are depicted in Fig. 4, where the control values for 1 fold change for binding and unbinding rates are marked by dashed lines.f g g g g y We have further assessed our analysis to factor for the possible effects of stress response in Pi starvation con- ditions. As depicted in Fig. S17, Pi limitation induces the general stress response regulated by RpoS, sigma fac- tor29,30. Concomitantly, Pi limitation increases the intracellular level of guanosine tetraphosphate (ppGpp), which is known as stringent stress response12,30,31. RpoS and ppGpp are thought to maintain a balance between vegetative and stress/starvation states of E. coli. The ppGpp nucleotide induces RpoS accumulation32. The RpoS responds to stress and shifts transcription away from vegetative growth and towards stress resistance31–34. www.nature.com/scientificreports/ As expected, when the control system’s output in Pi starvation condition is compared with the output in regimes with increased external Pi, we observe a decrease in PhoA yield. Moreover, these results predict that in order to obtain the starvation response in the conditions with higher external Pi concentration, promoter binding rates need to be increased and unbinding decreased. Promoters that provide the required strengths can be obtained by modifying the nucleotide sequences, for exam- ple, as in28,37,38. p We have tested the predictions of the model on experimental data obtained with the synthetic promoter under starvation conditions with 0 μM external Pi concentration. Moreover, we have used the model to explore the effect of such synthetic promoters under various external Pi concentration conditions. The experimental data and 7 www.nature.com/scientificreports/ Figure 4. Heatmaps for the activity of various promoter designs for pPhoA and pPhoB, and the resulting PhoA expression (bottom-row) under different external Pi concentration conditions. The heatmaps are ordered decreasingly from left to right according to the external Pi concentration given by the fold changes applied to the PhoR autphosphorylation reactions r1 and r2. The left most column with 1 as the fold change value is the starvation condition with 0 μM external Pi. Each heatmap scans 100 simulations by applying 10 different fold change values to the promoter binding rates r10 and r11 as well as 10 different fold change values to the promoter unbinding rates r10r and r11r. The upper row displays the resulting steady state levels of the active promoter pPhoAa, whereas the lower row displays the yield of PhoA gene expression measured as the area under the curve (AUC). The intersection of the dashed lines in the left column delivers the experimentally observed regime observed in Fig. 2. The levels of this regime, that display the starvation response, are highlighted in all the heatmaps. Figure 4. Heatmaps for the activity of various promoter designs for pPhoA and pPhoB, and the resulting PhoA expression (bottom-row) under different external Pi concentration conditions. The heatmaps are ordered decreasingly from left to right according to the external Pi concentration given by the fold changes applied to the PhoR autphosphorylation reactions r1 and r2. The left most column with 1 as the fold change value is the starvation condition with 0 μM external Pi. www.nature.com/scientificreports/ Each heatmap scans 100 simulations by applying 10 different fold change values to the promoter binding rates r10 and r11 as well as 10 different fold change values to the promoter unbinding rates r10r and r11r. The upper row displays the resulting steady state levels of the active promoter pPhoAa, whereas the lower row displays the yield of PhoA gene expression measured as the area under the curve (AUC). The intersection of the dashed lines in the left column delivers the experimentally observed regime observed in Fig. 2. The levels of this regime, that display the starvation response, are highlighted in all the heatmaps. Figure 5. Comparison of the experimental data on PhoA expression with the synthetic promoter together with the simulation results with varying external Pi concentrations and promoter parameters that model synthetic designs. The experimental data on the starvation response with the synthetic promoter is represented as hollow circles. As in Fig. 4, the plots are ordered decreasingly from left to right according to the external Pi concentration given by the fold changes applied to the PhoR autphosphorylation reactions r1 and r2. The left most column with 1 as the fold change value is the starvation condition with 0 μM external Pi. Each plot displays four simulations with varying fold change values applied to promoter binding and unbinding rates that model various promoter designs. A modified promoter (blue curve) can reproduce the starvation response in low as well as high external Pi concentration, and reproduce the experimental data under the synthetic promoter starvation conditions. Figure 5. Comparison of the experimental data on PhoA expression with the synthetic promoter together with the simulation results with varying external Pi concentrations and promoter parameters that model synthetic designs. The experimental data on the starvation response with the synthetic promoter is represented as hollow circles. As in Fig. 4, the plots are ordered decreasingly from left to right according to the external Pi concentration given by the fold changes applied to the PhoR autphosphorylation reactions r1 and r2. The left most column with 1 as the fold change value is the starvation condition with 0 μM external Pi. Each plot displays four simulations with varying fold change values applied to promoter binding and unbinding rates that model various promoter designs. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 6. Heatmap displaying the results of the sensitivity analysis by considering the physiological interval in Table 1. For each parameter, the maximum and minimum values within its physiological range are considered for simulation, and the area under the curve (AUC) for each species is computed. The difference of the AUC for the maximum and minimum parameter values are then normalised with the AUC of the control model. Red represents the decreasing effect and green represents the increasing effect. Figure 6. Heatmap displaying the results of the sensitivity analysis by considering the physiological interval in Table 1. For each parameter, the maximum and minimum values within its physiological range are considered for simulation, and the area under the curve (AUC) for each species is computed. The difference of the AUC for the maximum and minimum parameter values are then normalised with the AUC of the control model. Red represents the decreasing effect and green represents the increasing effect. the simulation results with our model are depicted in Fig. 5. The selected synthetic promoter verifies the model predictions as it has a similar behaviour as the control model for the pPhoA promoter in starvation conditions. Moreover, the model further predicts the synthetic promoters with increased strength, given with higher bind- ing rates and lower unbinding rates, deliver responses similar to the starvation response, also in the presence of higher external Pi concentrations. Within a modular framework, the simulation results mechanistically quantify how changes in the genetic components affect the behaviour of the circuit. the simulation results with our model are depicted in Fig. 5. The selected synthetic promoter verifies the model predictions as it has a similar behaviour as the control model for the pPhoA promoter in starvation conditions. Moreover, the model further predicts the synthetic promoters with increased strength, given with higher bind- ing rates and lower unbinding rates, deliver responses similar to the starvation response, also in the presence of higher external Pi concentrations. Within a modular framework, the simulation results mechanistically quantify how changes in the genetic components affect the behaviour of the circuit. Sensitivity Analysis. To assess the sensitivity of our model to the parameters, we have performed a two-step analysis. In the first step, we have considered the physiological interval of the parameters given in Table 1. www.nature.com/scientificreports/ For this analysis, we have only included the rate parameters that have been taken from literature and have been estimated within a given range. For each parameter, we have run simulations by instantiating the model with the maximum and minimum values of its physiological range, and, for each species, we have computed the yield of the system in terms of the area under the curve (AUC). Figure 6 displays the results obtained by taking the difference of the AUC for the maximum and minimum parameter values, normalised with the AUC of the control model. The resulting heatmap quantifies the impact of each parameter on the system dynamics with respect to plausible var- iations within its physiological range. The results demonstrate that the changes in the translation parameters are more pronounced than in the others. Moreover, in accordance with the results above, the autophosphorylation rate of PhoR impacts the active transcription factor levels and the transient species that lead to it. p p p In the second step, we have performed a sweeping analysis by considering all the model reactions and spe- cies within a range of 3 orders of magnitude both up and down. That is, each reaction rate is multiplied with a fold-change factor within a spectrum of 6 orders of magnitude, that is, from 10−3 to 103. We have then computed the AUC for each species and normalised the outcome with the AUC of the control model. The heatmap depicted in Fig. S19 quantifies the impact of these changes and predicts the system behaviour under hypothetical condi- tions simulated by such variations in parameters. www.nature.com/scientificreports/ A modified promoter (blue curve) can reproduce the starvation response in low as well as high external Pi concentration, and reproduce the experimental data under the synthetic promoter starvation conditions. Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w 8 Discussion We have presented a recipe for the design process of such an application, which integrates gene expression data from E. coli into a computer model. p Our model, which includes 29 reactions that describe the dynamic behaviour of the key regulatory network components, provides a quantification of the phosphate starvation response by means of both deterministic and stochastic simulations. The deterministic simulations allowed us to estimate the missing parameters of the model and analyse the mean behaviour of the system dynamics. In particular, we have exposed the phosphorylation cycle in TCS signalling and its role in the positive feedback mechanism in determining the network yield in terms of PhoA. Moreover, the steady state properties of the system could be displayed by sensitivity analysis on deterministic simulations.f In accordance with the common practice, we have employed stochastic simulations to highlight the effect of noise in the system39. As expected, the stochastic simulations are consistent with the deterministic simulations, which display the mean behaviour. Stochastic simulations, on the other hand, capture the noise due to small species numbers and concomitant fast and slow reactions. As a result of this, stochastic simulations with our model display the fluctuations observed in experimental observations, thereby exposing the fluctuations due to binding and unbinding of the transcription factors with the promoters, which operate at a much faster time scale in comparison to the preceding signalling cascade40–42. In these simulations, higher unbinding rates result in greater fluctuations that correlate with the decrease in binding saturation, a requirement for a robust signal. While confirming the notion that the complex networks of interacting molecules within cells should be robust43,44, these results highlight the additional role of the transcription factor unbinding rate in tuning the protein synthesis. Our stochastic simulation results, together with the experimental data on PhoB concentration in wild-type, suggest that the noise at the level of promoters controls the phenotypic variability in the mean behaviour. Large levels of noise at the level of PhoA and PhoB promoters are results of DNA unbinding rates. Moreover, our model shows that the smaller DNA unbinding rates also reduce the response time.i g p Our results confirm that the dynamics of the TCS and its responsiveness to both genetic and environmental perturbations play a key role in tuning the E. coli Pi response. Discussion We have presented a computational model and its experimental validation for quantifying dynamic mechanisms of auto-regulation in Escherichia coli in response to external Pi levels. Our model provides a mechanistic expla- nation of the interplay between transcriptional regulatory network, given by a two-component system (TCS) and Pho regulon, and promoter efficiency under variations in external Pi concentrations. Being parameterised within the physiological ranges of its components, the output of the model in terms of gene regulation delivers the expected system dynamics. The results provided by the model are in good agreement with the theory and the general concepts described in the literature for the functionality of Pho regulon2,3. The model provides pre- dictions for the complex effects of TCS activity and consequent dynamics, including synthetic promoters with varying affinities to their transcription factor. A direct validation of the predictions is provided by the good fit of the experimental data. Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w 9 www.nature.com/scientificreports/ With these insights, this work offers a mechanistic understanding of inorganic phosphate intake, realised by Pho regulon in a way that connects signalling with the genetic level. It provides a quantitative descrip- tion of how different proteins interact to form a biological control system. It also describes the control of the phosphate-starvation response at the genetic level. Our work provides measurements of protein, phosphoryla- tion, and promoter activity levels that are fundamental to define features of TCS circuits. One of the major out- comes is that our results do not only explain the observed experimental data, but also provide predictions on the physiology of the Pho regulon and insights for the synthetic promoter design. Moreover, our work has implica- tions for applications in artificial life and for others in biotechnology that exploit such mechanisms. As the model architecture is intrinsically open to integrate supplementary layers, together with experimental findings, it should provide insights in investigations on engineering new dynamic sensors and regulators for living technologies. p g g g g y g g g Our model describes the responsive structure of the TCS system, which can be used to synthesize bacteria that detect changes in their environment and respond by modulating the synthesis and intake. Applications of such an engineering approach for bacteria should provide a basis for a new generation of bio-materials. Discussion In this respect, with parameter values obtained by fitting the experimental data on PhoA and PhoB promoters, we have investigated and identified the response dynamics of individual system species. Moreover, we have analysed synthetic promoter affinities that reproduce the wild type response to Pi starvation. For this purpose, we have tested and verified the model with experimental data for the response regulator binding affinity to infer the functional relation between the fraction of bound response regulator and the transcriptional activity. We have then quantified the effect of each protein in the path- way, through sensitivity analysis, singling out the main regulator mechanisms of the TCS. Our results indicate the extent to which synthetic promoters can be tuned for various TCS detection thresh- olds such that the organism adapts to various environmental conditions. In this regard, because our model includes mechanisms for quantifying the information E. coli has about its phosphate environment, the model can be used to perform further experiments on the Pi intake system. Moreover, it can be used to explore and test various promoter designs, for example, in biotechnology applications such as sensors for wastewater treatment or detecting environmental pollutants to relocate towards them. g p In conclusion, our study reveals how the tight interplay between theory and wet-lab experiments greatly helps to improve our understanding of bacterial sensing and signalling pathways. In particular, the TCS component of our model, which relays the signal on environmental changes to the genetic components for tuning protein expression, belongs to one of the largest and most diverse families of sensory components in biology. In this respect, the TCS features examined in this work provide a template for the models of similar systems that regulate the response for various external signals. An improved and quantitative understanding of such systems by formal mechanistic models will likely contribute to our understanding of the engineering of biosensors for diverse syn- thetic biology applications. www.nature.com/scientificreports/ www.nature.com/scientificreports/ When the external Pi concentration is limited, PhoR is free to bind ATP. This allows PhoR to autophosphoryl- ate itself. PhoR is stable as a dimer, which is denoted by DiPhoR. Therefore, it is doubly phosphorylated. ↔ DiPhoR DiPhoRp (1) ↔ iPhoRp DiPhoRpp (2) (1) ↔ DiPhoR DiPhoRp ↔ DiPhoRp DiPhoRpp (2) PhoR is essential for the control of the activity of PhoB2,8,45. It phosphorylates PhoB through an autokinase/ phosphotransferase activity2,8. After autophosphorylating, PhoR relays the signal to the transcription factor PhoB. The bidirectional reactions 3 and 5 below model the association of phosphorylated PhoR dimer and PhoB, and the unidirectional reactions 4 and 6 model the phosphotransferase. PhoB has been reported to exist primarily as monomers and phosphorylation greatly enhances dimerisation of PhoB (DiPhoBpp), modelled by reaction 710. + ↔ − DiPhoRpp PhoB DiPhoRpp PhoB (3) − → + DiPhoRpp PhoB DiPhoRp PhoBp (4) + ↔ − DiPhoRp PhoB DiPhoRp PhoB (5) − → + DiPhoRp PhoB DiPhoR PhoBp (6) + ↔ PhoBp PhoBp DiPhoBpp (7) + ↔ − DiPhoRpp PhoB DiPhoRpp PhoB (3) − → + DiPhoRpp PhoB DiPhoRp PhoBp (4) + ↔ − DiPhoRp PhoB DiPhoRp PhoB (5) − → + DiPhoRp PhoB DiPhoR PhoBp (6) + ↔ PhoBp PhoBp DiPhoBpp (7) + ↔ − DiPhoRpp PhoB DiPhoRpp PhoB (3) + ↔ − DiPhoRpp PhoB DiPhoRpp PhoB (3) (6) + ↔ PhoBp PhoBp DiPhoBpp + ↔ PhoBp PhoBp DiPhoBpp (7) (7) In E. coli, the sensor histidine kinase PhoR is a bifunctional enzyme that paradoxically performs two opposed tasks: in one direction, it catalyzes the phosphorylation of response regulator PhoB, and in the other, it also per- forms the dephosphorylation of phosphorylated PhoB, which is PhoBp18,25. The association of PhoR dimers with phosphorylated PhoB is modelled by the bidirectional reaction 8, whereas the phosphatase activity is given by reaction 9. + ↔ − DiPhoR PhoBp DiPhoR PhoBp (8) − → + DiPhoR PhoBp DiPhoR PhoB (9) + ↔ − DiPhoR PhoBp DiPhoR PhoBp (8) + ↔ − DiPhoR PhoBp DiPhoR PhoBp (8) − → + DiPhoR PhoBp DiPhoR PhoB (9) − → + DiPhoR PhoBp DiPhoR PhoB (9) Phosphorylated dimer structure PhoB (DiPhoBpp) is enabled for activating Pho regulon by binding to a con- sensus promoter region. PhoB and PhoR in Pho regulon are encoded by the same operon, that is, the phoBR operon. Methodsh The model is based on a mechanistic description of the system dynamics within a chemical reaction network rep- resentation with respect to mass action kinetics. To build the simulation model, we have selected the part of the system from the TCS to gene regulation, and used the promoter activity as well as the levels of active transcription factor as indicators for quantifying the response to external Pi levels. q y g In the following, to better illustrate the response of Pho regulon and TCS signalling to the Pi starvation, we describe the network in terms of the interactions of system components as a chemical reaction network. Figure 2B provides an alternative representation of the regulatory system interactions given by the chemical reaction network. Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w 10 Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w →∅ mRNAb →∅ mRNAb We have implemented the deterministic ordinary differential equation (ODE) model in Matlab by using the standard translation from the chemical reaction network above, based on stoichiometries. The ODEs and detailed descriptions of each system variable are listed in Fig. S1 and Table S1. We have also implemented a version of the model for stochastic simulation, again based on mass action kinetics. The stochastic simulations capture fluctuations due to small molecule numbers, which are not captured by the deterministic simulations. By using the standard conversion factors for mass action kinetics, we could use the same rates for the deterministic and stochastic simulations.h The initial concentrations of the model variables have been derived from the literature or obtained from our experiments, described below. The control model has been calibrated for an initial culture containing 0 μM exter- nal Pi. Prior to Pi starvation, the concentrations of proteins PhoR and PhoB are approximately 0.22 μM. The concentrations of active PhoR and active PhoB are μ ⋅ − M 4 10 8 and μ ⋅ − M 6 10 8 , as determined by Keasling et al.3. With a single plasmid, average mRNA number is 2–3 in E. coli47. Therefore, the initial states of mRNAa and mRNAb are set to 0.00166 μM by taking E. coli volume as 1 μm3, and the initial promoters numbers are set to 10 for each. We assume that the ATP concentration stays constant throughout the considered time intervals.h The rates of chemical reactions are obtained in accordance with the variability of physiological ranges given in the literature5,6,47–50. The model includes 29 reactions, including the reverse reactions. The control model has been used to reproduce the data and the unknown parameters have been estimated by least square inference within the plausible physiological ranges. When possible, parameter values are fixed or estimated by using experimental measurements found in the literature. The parameter values taken from the literature and their physiological ranges for the rates, if applicable, are listed in Table 1. The parameters without a range are fitted to the experimen- tal data by using the deterministic model to reproduce the response curves. The parameter estimation procedure has been carried out by using a multi-start approach. www.nature.com/scientificreports/ Thus, the synthesis of the regulatory proteins PhoB and PhoR is under Pho regulon control2,8,46. h Based on experimental data, we consider the PhoA and PhoB promoters (pPhoA, pPhoB), whereby the PhoB promoter provides feedback to the system as this results in the expression of both PhoB and PhoR. PhoR expres- sion during Pi limitation is dependent on the upstream pPhoB; the operon structure indicates that PhoR gene function requires expression from the pPhoB46. For this, phosphorylated PhoB dimers (DiPhoBpp) bind to the promoter as active transcription factors. + ↔ DiPhoBpp pPhoA pPhoAa (10) + ↔ DiPhoBpp pPhoB pPhoBa (11) + ↔ DiPhoBpp pPhoA pPhoAa (10) + ↔ DiPhoBpp pPhoB pPhoBa (11) + ↔ DiPhoBpp pPhoA pPhoAa (10) (10) + ↔ DiPhoBpp pPhoB pPhoBa + ↔ DiPhoBpp pPhoB pPhoBa (11) (11) Active promoters pPhoAa and pPhoBa lead to the transcription of mRNA, which carry the information for the subsequent translation, resulting in protein synthesis, which are PhoA, PhoB and DiPhoR2,8. → + pPhoAa pPhoAa mRNAa (12) → + mRNAa PhoA mRNAa (13) → + pPhoBa pPhoBa mRNAb (14) → + mRNAb PhoB mRNAb (15) → + mRNAb DiPhoR mRNAb (16) With the inclusion of the degradation/dilution terms, we obtain: →∅ PhoA (17) →∅ PhoB (18) →∅ DiPhoR (19) → + pPhoAa pPhoAa mRNAa (12) → + mRNAa PhoA mRNAa (13) → + pPhoBa pPhoBa mRNAb (14) → + mRNAb PhoB mRNAb (15) → + mRNAb DiPhoR mRNAb (16) dation/dilution terms, we obtain: →∅ PhoA (17) →∅ PhoB (18) →∅ DiPhoR (19) → + pPhoAa pPhoAa mRNAa → + mRNAb DiPhoR mRNAb With the inclusion of the degradation/dilution terms, we obtain: Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w 11 www.nature.com/scientificreports/ (20) →∅ mRNAa →∅ mRNAa (21) →∅ mRNAb The rate values have been selected with respect to the best fit to the physiological ranges, also listed in Table 1, and the dynamics in accordance with the experimental findings in order to avoid discontinuities or states with unrealistic values. Th d f h h d h h b b d l fi d f l The data for the PhoA and PhoB expression have been obtained using PCR amplified DNA from E. coli MG1655 genome and transcriptionally fused to the translational coupler BCD251 and the fluorescent ms-fgfp gene. Subsequently the PphoA-BCD2-msfgpf and PphoB-BCD2-msfgfp fragments were cloned using the PacI/ HindIII restrictions sites in pSEVA234 plasmid (http://seva.cnb.csic.es/), generating the pSEVA237PphoA and pSEVA237PphoB vectors as depicted in Fig. 2A.hi The synthetic promoter Pliar was obtained as in52,53 with modifications. Activities of the PphoA and PphoB promoters have been determined as follows. Cells were maintained on LB rich medium. For Pi assays, an over- night preculture of E. coli DH10B carrying pSEVA237PphoA, pSEVA237PphoB or pSEVA237PLiar00117 plas- mids harbouring PhoA, PhoB or PLiar00117 promoters driving the expression of MsfGFP, respectively were grown in 10 ml MOPS medium (pH = 7.2), supplemented with 0.02% casamino acids and 5 mM KH2PO4 at 37 °C under constant shaking at 250 rpm. The activity of PhoB and PhoA promoters were analysed in resting cells, both in presence or absence of 50 μg/ml antibiotic kanamycin, which did not have any impact on the promoter activity as it can be seen in the Supplementary Figs S2 and S20. The bacterial precultures were used to inoculate 50 ml of the same medium reaching an OD600 of 0.1 and grown until mild-late exponential phase (OD600 of 0.6–0.9). At this point, the bacterial cells were pelleted at 1500 × g for 10 min at room temperature, and finally washed twice in MOPS medium without − PO4 3 . Subsequently, the cells were suspended in 250 μl of MOPS to reach a final OD600 of 2 with increasing concentrations of − PO4 3 (from 0 to 50 mM). The bacterial cell suspensions were loaded in 96-well plates and the expression of MsfGFP (fluorescence) was measured at different times in a SpectraMax i3x (Molecular Device) at 30 °C. The excitation wavelength was set to 485 nm and the fluorescence emission was measured at different times at 525 nm. Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w www.nature.com/scientificreports/ Biological insights from structures of two-component proteins. Annu Rev Microbiol. 63 (2009).h 46. Warner, B. & Chang, B. The phoBR operon in Escherichia coli K-12. Bacteriol 169, 5569–5574 (1987). h 47. OpenWetWare. Parameter estimation in E. coli. Available at http://www.openwetware.org/wiki/Computational_Biology/Gene_ Expression_modeling (2007). g 48. Dexter, J. & Gunawardena, J. 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The constancy of global regulation across a species: the concentrations o ppGpp and RpoS are strain-specific in Escherichia coli. BMC Microbiology 11, 62 (2011). 2 Spira B Hu X & Ferenci T Strain variation in ppGpp concentration and RpoS levels in laboratory strains of Escherichia coli K 12 i 2. Spira, B., Hu, X. & Ferenci, T. Strain variation in ppGpp concentration and RpoS levels in laboratory strains of Escherichia coli K-12 Microbiology 154, 2887–2895 (2008). gy . Ferenci, B. Maintaining a healthy SPANC balance through regula gy 3. Ferenci, B. Maintaining a healthy SPANC balance through regulatory and mutational adaptation. Mol. Author Contributions C.U. and O.K. designed the model and performed the simulations and the sensitivity analysis. J.T.-B., C.U., J.L.G. and J.N. designed and performed the experiments. C.U., O.K. and M.H. wrote and edited the manuscript. All the authors analysed the results and reviewed the manuscript. Acknowledgements Th k h b ll g This work has been partially funded by the European Union’s Horizon 2020 research and innovation programme under the grant agreement No 686585 - LIAR, Living Architecture. This work has been partially funded by the European Union’s Horizon 2020 research and innovation programme under the grant agreement No 686585 - LIAR, Living Architecture. Scientific Reports | (2019) 9:2076 | https://doi.org/10.1038/s41598-018-38223-w 13 www.nature.com/scientificreports/ Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-38223-w. Competing Interests: The authors declare no competing interests. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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How to Construct Effective Consultation System for S&amp;T Decision-making
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1 Introduction The consultation system of S&T decision-making is the inevitable result of scientific and technological progress and social development, and also the inherent requirement for government decision-making. As a result of the new round of scientific and technological revolution and industrial transformation, the complexity of science and technology strategy and policy making is increasing, and the comprehensive participation of scientific, technological, economic, educational and other social factors and various think tanks is needed. China also attaches great importance to the construction of the consultation system of S&T decision-making. In January 2015, The Opinions on Strengthening the Construction of a New Think Tank with Chinese Characteristics, issued by the Office of the CPC Central Committee and General Office of State Council, emphasized that "the consultation system of the decision-making is an important part of the construction of socialist democratic politics in China, and the new think tank with Chinese characteristics is an important content of the modernization of the national governance system and also an important part of the country's soft power "[1]. In September 2015, the Office of the CPC Central Committee and General Office of State Council issued The Implementation Plan for Deepening the Reform of the Scientific and Technological System, which called for "the establishment of a national consultation mechanism of innovation and S&T decision- making, the development of a good scientific and technological community and think tank to support innovation and S&T decision-making, the establishment of a national advisory committee of S&T decision-making” [2].On the May 30, 2016, General Secretary Xi Jinping How to Construct Effective Consultation System for S&T Decision-making Zijun Tang1,a 1Office of Scientific Research Management Jishou University Jishou Hunan China Abstract. With the development of a new round of science and technology (S&T) revolution and industrial transformation in the world, the complexity of S&T decision-making is increasing. It not only needs the comprehensive participation of scientific, technological, economic, educational and other social factors but also an effective consultation system to guarantee. To build an effective consultation system for S&T decision-making, a lot of measures can be taken, such as establishing the system of the policy and regulation, perfecting the multi-mode operation system, constructing the support system of talent team, developing of multi-channel financing system, optimizing the system of social public participation and strengthening the system of interactive international cooperation. stressed in his important speech Striving to Build a World Science and Technology Power, "We should speed up the establishment of mechanism of S&T decision-making to support administrative decision-making, strengthen the consultation system of S&T decision-making, and build a high level science and technology think tank to accelerate the institutionalization of major scientific and technological decisions "[3] February 2017, the CPC Central Committee passed the Plan of the Construction Program of National Consultation System of S&T Decision-making, pointed out to build a national consultation system and a think tank at the highest level of S&T decision-making. The Plan is a re-promotion of China's current consultation mechanism of S&T decision- making, marking the China’s consultation of S&T decision-making is on the process of institutionalization and systematization. There are a lot of measures can be taken to construct the effective consultation system for S&T decision-making in China. a Corresponding author: zijuntang@163.com © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). ITM Web of Conferences 25, 03007 (2019) ICICCI 2018 ITM Web of Conferences 25, 03007 (2019) ICICCI 2018 https://doi.org/10.1051/itmconf/20192503007 3 Perfecting the multi-mode operation system It is a common direction for the development of consultation system of S&T decision-making in various countries to construct different and complementary consultation systems at different levels and of various types. It is a common practice in the international community to set up a committee with members spanning all fields, controlling the number of members and highlighting their representativeness. China has already established the construction plan of the national science and technology advisory committee, and it is necessary to further improve the operation mode of the institution. At the horizontal level, to promote the coordinated development of official advisory bodies and parastatal and civil advisory bodies, encourage the Government to purchase consultation services of S&T decision-making, support the development of market-oriented decision- making advisory bodies through corresponding policy measures, strengthen their financial support, and enhance the status and influence of civil decision-making advisory bodies, finally form a useful supplement to the official advisory body, make China's consultation of S&T decision-making to the direction of specialization. At the vertical level, there is a need for a multi-tiered and complementary advisory body layout, based on the hierarchical distribution of existing decision-making advisory bodies, from the national level, the sectoral level to the grass-roots level and social institutions. For example, in the case of high-level consultation on major political and diplomatic decisions, the national advisory committee on science and technology may make advisory recommendations on such decisions. When it comes to decision-making in the social and public fields, appropriate decision-making consultation bodies may be set up according to the local characteristics and actual conditions 4 Constructing the support system of talent team The high-quality S&T decision-making consultation needs the novelty, reliability and policy relevance of knowledge, which will inevitably put forward special requirements for the construction of talent team in decision-making consultation bodies. According to the present situation of decision-making consultation in China, it should be firstly focused on cultivating interdisciplinary knowledge talents and strengthening the ability of strategic research. In order to better cope with increasingly complex decision-making problems and enhance the effectiveness of decision- making suggestions, it is necessary to cultivate research talents with multi-disciplinary complementarity and cross- disciplinary advantages, and pay attention to the selection and training of talents in terms of professional depth, disciplinary breadth, scientific height and social perspective. Secondly, the evaluation and incentive mechanism of S&T decision consultation talents should be constructed through targeted measures. It will be formulated that unified and standardized evaluation standards for the employment, professional title assessment, moral character and ability and social contribution of talents, and take the social impact of the consultation recommendations as the core assessment indicators. At the same time, by encouraging original ideas, respect for intellectual property rights, the establishment of talent program projects and other methods, to stimulate the healthy development of consulting talent team, to provide sustainable talent support for decision-making consultation, enhance the capacity and level of S&T decision-making advisory bodies. Thirdly, it should be pay more attention to cultivating and promoting the professional ethics of decision-making consultation talents. In the selection of advisory members and experts, social responsibility and academic ethics should be the basic standards, and the professionalism of responsible consultation should be actively promoted so that S&T decision-making consultation can truly win the affirmation and trust of the society. 2 Improving the policy and regulation guarantee system The smooth development of consultation of S&T decision- making is guaranteed by sound policies and regulations. Developed countries all regard the construction of perfect legal system as the key to promote the consultation of decision-making. The supporting policies and regulations of consultation of S&T decision-making in China are in the initial stage and need to be strengthened and improved in the following four aspects. The first is to incorporate consultation into the legal procedures for major decision- making of government departments, protect the legal status ITM Web of Conferences 25, 03007 (2019) ITM Web of Conferences 25, 03007 (2019) ICICCI 2018 https://doi.org/10.1051/itmconf/20192503007 of consulting bodies through relevant laws and regulations, and specify their power and responsibility relationship, expert selection, organizational structure and operation mode; Secondly, the status and role of different types of consultation bodies in decision-making should be clarified through legislation. In particular, the consultation procedures should be standardized and designed through specific legal provisions to ensure the relative independence of consultation bodies. Meanwhile, corresponding incentive measures should be formulated according to different consultation situations; Thirdly, establish the application and feedback system of important S&T decision-making consultation achievements, and strengthen the legal regulation of the formation, release and use of consultation reports. Fourthly, establish a supervision system and restriction mechanism for S&T decision-making consultation, and a public disclosure and hearing system for major decision-making matters, ensure the openness of all links and procedures of decision- making consultation, protect the right of supervision and information of the public, and promote the openness, transparency and fairness of decision-making consultation activities. of various departments, institutions and regions. The perfect operation mechanism is the premise to ensure the decision-making level to grasp the relevant policy information accurately, timely and systematically, provide objective and reasonable scientific support for decision- making, and is also the key to test its effectiveness. References 1. The Executive Office of the CPC Central Committee and General Office. On strengthening Chinese characteristics Views on the construction of a new think tank. [2016-09-30]. http://www.gov.cn/Xinwen/201501/20/content_2807 126.htm 2. The Executive Office of the CPC Central Committee and General Office. Deepening the implementation of scientific and technological system reform case. [2016-09-30]. http://www.gov.cn/guowuyuan/2015/09/24/Content_ 2938314.htm 5 Developing multi-channel financing system Of course, whether it is from the public finances or corporate sponsorship and social donations, there should be transparent and open management of financial intervention in order to prevent the possible existence of "undercover operation" through financial support, affecting or shaping the advisory opinions. Then the advisory bodies are not disturbed by some kind of "external" factors and can objectively provide independent advice on a fair attitude. 5 Developing multi-channel financing system A certain amount of financial support is a necessary condition for the normal operation of decision-making advisory bodies. The advisory bodies of developed countries are mainly funded by government grants, corporate grants, private donations and dues. At present, China mainly supports the operation of decision-making advisory bodies through the way of government financial allocation, while the proportion of enterprises and private 2 2 ITM Web of Conferences 25, 03007 (2019) ITM Web of Conferences 25, 03007 (2019) ICICCI 2018 https://doi.org/10.1051/itmconf/20192503007 international research, operation and exchange in order to obtain a voice on international issues. In order to meet the requirements of the new era, China should further promote the international development of S&T decision-making consultation, and promote the competitive platform of intellectual products to the international market. Firstly, carry out cooperative research and exchange dialogue with foreign S&T decision-making advisory bodies, draw on their organizational methods, operating models and discourse paradigms, and selectively integrate them into the construction of our own advisory bodies in accordance with the actual situation in China. Secondly, expand the voice of domestic decision-making advisory bodies in the international arena, strive to "lead" in some global issues and China issues that arouse world concern, and build a group of decision-making advisory bodies with international influence. Thirdly, cultivate a team of consulting talents with critical spirit and innovative consciousness, international discourse ability and competitiveness, launch original research achievements with the value of the times and global consciousness, and improve the academic reputation in the international arena. The last is to actively promote the establishment of S&T decision-making advisory branches abroad, as a means of establishing effective international cooperative relations for the exchange platform, and to expand the international recognition and influence of advisory bodies through advisory activities on China's foreign policy. financial support needs to be further improved. Therefore, the management system of decision-making consultation funds should be reformed, change the homogenization mode of financing, encourage enterprises to invest in the establishment of advisory bodies and promote their market-oriented operation. At the same time, civil and individual donations, etc., should also be given some recognition and support, in order to further enrich the funding sources of decision-making advisory bodies. Acknowledgement This paper was financially supported by the project of Science and Technology Plan of Xiangxi Autonomous Prefecture, "The Research on the Mechanism of University -affiliated think tanks participating in Local Science and Technology Innovation Decision-making consultation" (No.2018SF5015) 6 Optimizing the system of public participation in society. Many decision-making problems in social development are often closely related to the public interest, and actively promoting public participation is of great significance to improve the sound consultation system of S&T decision- making. Firstly, through the systematic formulation of system rules, introducing orderly and effectively public participation in the progress of decision-making advisory activities can help to jointly promote the formation of decision-making solutions, and also can make the decision-making advisory bodies widely accept public supervision, better promote the openness and transparency of the consultation process; Secondly, the Government should strengthen the guiding role of public participation in decision-making, speed up the institutionalization of public participation, create a good institutional environment, make public participation an integral part of the decision-making consultation process, so that the public really enjoy the right to participate in decision- making consultation, and have a practical role and influence in the process of decision-making consultation; Thirdly, a multi-subject decision-making consultation platform including government, S&T decision-making advisory bodies and the public should be further built, so as to smooth out the information communication channels between the government and the society. 7 Strengthening the system of interactive international cooperation In the context of globalization, international influence is one of the important goals pursued by S&T decision- making advisory bodies, for which most national decision- making advisory bodies adopt open policies and conduct 3. Xi Jinping. Strive to build a world science and technology power. People's Daily, 6(2016). 3
https://openalex.org/W4393149662
https://link.springer.com/content/pdf/10.1007/s00383-024-05683-3.pdf
English
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A systematic review of Sandifer syndrome in children with severe gastroesophageal reflux
Pediatric surgery international
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cc-by
4,266
Abstract Abstract Purpose  Sandifer syndrome (SS), which combines gastroesophageal reflux (GER) and a neurological or psychiatric disorder, is an uncommon condition that often takes a long time to diagnosis. We aimed to systematically review available papers regarding SS. Methods  After presenting our two cases of SS, we systematically reviewed articles published in MEDILINE/PubMed, Cochrane Library, and Web of Science. Results  The meta-analysis included 54 reported cases and 2 of our own cases. Our results showed that all cases achieved symptom improvement with appropriate treatment for GER. Notably, 19 of the 56 cases exhibited anatomical anomalies, such as hiatal hernia and malrotation. Significantly more patients with than without anatomical anomalies required surgery (p < 0.001). However, 23 of the 29 patients without anatomical anomalies (79%) achieved symptom improvement without surgery. Patients who did not undergo surgery had a median (interquartile range) duration to symptom resolution of 1 (1–1) month. Conclusion  The primary care providers should keep SS in the differential diagnosis of patients presenting with abnormal posturing and no apparent neuromuscular disorders. Fundoplication may be effective especially for patients with anatomical anomalies or those whose symptoms do not improve after more than 1 month with nonsurgical treatment. Keywords  Sandifer syndrome · Gastroesophageal reflux · Fundoplication · Laparoscopic surgery Pediatric Surgery International (2024) 40:91 https://doi.org/10.1007/s00383-024-05683-3 Pediatric Surgery International (2024) 40:91 https://doi.org/10.1007/s00383-024-05683-3 ORIGINAL ARTICLE ORIGINAL ARTICLE A systematic review of Sandifer syndrome in children with severe gastroesophageal reflux Daiki Kato1 · Hiroo Uchida1 · Hizuru Amano1 · Kazuki Yokota2 · Chiyoe Shirota1 · Takahisa Tainaka1 · Wataru Sumida1 · Satoshi Makita1 · Akihiro Yasui1 · Yousuke Gohda1 · Takuya Maeda1 · Akinari Hinoki3 Daiki Kato1 · Hiroo Uchida1 · Hizuru Amano1 · Kazuki Yokota2 · Chiyoe Shirota1 · Takahisa Tainaka1 · Wataru Sumida1 · Satoshi Makita1 · Akihiro Yasui1 · Yousuke Gohda1 · Takuya Maeda1 · Akinari Hinoki3 Accepted: 19 March 2024 © The Author(s) 2024 * Hiroo Uchida hiro2013@med.nagoya-u.ac.jp 3 Department of Rare/Intractable Cancer Analysis Research, Nagoya University Graduate School of Medicine, 65 Tsurumai‑cho, Showa‑ku, Nagoya 466‑8550, Japan 1 Department of Pediatric Surgery, Nagoya University Graduate School of Medicine, 65 Tsurumai‑cho, Showa‑ku, Nagoya 466‑8550, Japan Fig. 1   a Upper gastrointestinal series showing gastroesophageal reflux. b Esophagogastroduo- denoscopy showing reflux esophagitis (Los Angeles clas- sification: Grade D) and a hiatal hernia Introduction abnormal posture and involuntary movements that disturb clinicians or parents given that they can mimic seizures [2]. SS is often misdiagnosed as a neurological or musculoskel- etal condition. The difficulty in accurately diagnosing this clinical manifestation is that there are often no obvious gas- trointestinal symptoms such as abdominal pain or vomit- ing. This can lead to unnecessary and expensive neurologic examinations such as MRI, EEG, and electromyography. These exams may lead to a missed and delayed diagnosis, and mismanagement. When a patient has abnormal posture or movement without neuromuscular disease, SS should be one of the differential diagnoses. Fortunately, symptoms of SS improve with GER treatments, such as medication and surgery. Sandifer syndrome (SS) is an uncommon condition charac- terized by a combination of gastroesophageal reflux (GER) or hiatal hernia and a neurological or psychiatric disorder [1]. SS is associated with abnormal posture and move- ments of the neck and trunk. Patients with SS present with 1 Department of Pediatric Surgery, Nagoya University Graduate School of Medicine, 65 Tsurumai‑cho, Showa‑ku, Nagoya 466‑8550, Japan 1 Department of Pediatric Surgery, Nagoya University Graduate School of Medicine, 65 Tsurumai‑cho, Showa‑ku, Nagoya 466‑8550, Japan Recognizing SS and treating GER will quickly resolve this disease. However, reports on SS have been infrequent, with the diagnosis of SS often taking a long time. Moreo- ver, to the best of our knowledge, only a few systematic review and meta-analysis have been published on this 2 Department of Pediatric Surgery, Aichi Developmental Disability Center Central Hospital, 713‑8 Kagiya‑cho, Kasugai 480‑0392, Japan 2 Department of Pediatric Surgery, Aichi Developmental Disability Center Central Hospital, 713‑8 Kagiya‑cho, Kasugai 480‑0392, Japan (0123 3456789) Pediatric Surgery International (2024) 40:91 Page 2 of 7 Pediatric Surgery International 91 A 9‑year‑old boy This case involved a patient who was suffering from vomit- ing and an abnormal left-leaning posture that started 4 years prior to presentation. He initially sought consultation from a pediatric neurologists. However, when no abnormalities were found on blood examination and head magnetic reso- nance imaging, he was placed under observation. Unfortu- nately, his symptoms did not improve, prompting referral to a pediatric psychiatrists 2 years prior to presentation. Elec- troencephalography and development examinations showed no abnormalities. As such, SS was suspected based on his- tory, for which upper gastrointestinal examinations were per- formed. Upper gastrointestinal series (UGI) showed GER (Fig. 1a), whereas esophagogastroduodenoscopy (EGD) showed reflux esophagitis (Los Angeles classification: Grade D) and a hiatal hernia (Fig. 1b). Low esophageal pH (< 4) accounted for 19% of the 24-h recording cycle. These find- ings confirmed that SS was the correct diagnosis. Postural abnormalities temporarily disappeared after initi- ating PPI treatment 1 month prior to presentation. However, we opted to perform laparoscopic Toupet fundoplication given the recurrence of symptoms despite PPI treatment. At 2 weeks after the surgery, his symptoms improved as shown by the disappearance of postural abnormalities and cessation of voluble speech. No symptom recurrence had been noted for 9 months. Systematic review and meta‑analysis We subsequently reviewed articles published in MEDIL- INE/PubMed, Cochrane Library, and Web of Science using the following combinations of search terms: “Sandifer syn- drome” and “gastroesophageal reflux.” A 7‑year‑old boy subject. Therefore, the current study first aimed to present our experience with two cases of SS. Second, a systematic review was conducted to evaluate all published studies on patients with SS. Our article on the diagnosis and treatment of patients with SS aimed to help consider SS as an early differential diagnosis in children with these disorders. This case involved a patient who was suffering from abnor- mal posture with backward bending of the neck, violent speech, chronic cough, and vomiting 2 months prior to presentation (Fig. 2a), which prompted him to initially seek consultation from pediatricians. Computed tomography of the head performed in the emergency department of the pre- vious hospital showed no abnormality. The patient was then transferred to our hospital. UGI showed GER (Fig. 2b), and EGD showed reflux esophagitis (Los Angeles classification: Grade D) and hiatal hernia (Fig. 2c). We could not perform low esophageal pH 24-h recording due to his symptom of abnormal posture. Results In total, 201 articles were identified via a comprehensive review. After removing 116 duplicates and 61 studies that satisfied the exclusion criteria, 24 articles and 54 cases were ultimately identified (Table 1) [1–24]. Figure 3 shows the flow diagram for study selection. Selection criteria i Given the lack of improvement with proton pump inhibi- tor (PPI) treatment for 1 month, laparoscopic Toupet fun- doplication was performed, which improved the patients symptoms 1 month after surgery. The left-leaning posture disappeared, and the patient did not develop any symptom recurrence for 2 years. Original articles and case reports reporting details regard- ing patients with SS were included in the analysis. Two reviewers independently scanned the titles and abstracts of the identified articles. The exclusion criteria were as fol- lows: (1) articles not written in English, (2) non-original Fig. 1   a Upper gastrointestinal series showing gastroesophageal reflux. b Esophagogastroduo- denoscopy showing reflux esophagitis (Los Angeles clas- sification: Grade D) and a hiatal hernia Page 3 of 7  91 Pediatric Surgery International (2024) 40:91 Page 3 of 7  91 Pediatric Surgery International ) Fig. 2   a Abnormal posture with backward bending of the neck was observed. b Upper gastrointestinal series show- ing gastroesophageal reflux. c Esophagogastroduodenoscopy showing reflux esophagitis (Los Angeles classification: Grade D) and a hiatal hernia articles or case reports (meeting abstracts, reviews), (3) studies that did not focus on patients with SS, and (4) studies with insufficient data on patients with SS. This systematic review was conducted based on the PRISMA principles. Data extraction l g y All 54 published cases and both of our own were evalu- ated via meta-analysis (Table 1). In all cases, symptoms improved with appropriate GER treatment. In terms of ana- tomical anomaly, there were 18 cases of hiatal hernia, 1 case of malrotation, and 8 cases of no description. Notably, 17/19 (89%) cases with anatomical anomalies required surgery. Patients with anatomical anomalies required significantly more surgeries than did those without anatomical anomalies (p < 0.001). However, 23 of the 29 patients without anatomi- cal anomalies (79%) showed symptom improvement with- out surgery. In both of our cases, surgery promoted early improvement in symptoms of hiatal hernia. Many patients were diagnosed with GER by UGI, EGD, and low esopha- geal pH 24-h recording. The pH measurement has increased frequency over time and the diagnostic criteria for GER in all cases. The median (interquartile range) age at diagnosis Two reviewers separately collected the following data from the selected articles: the first author, year of publication, study design, sample size, patient characteristics, treatment course, and outcomes. Both reviewers reached a consensus at each stage of the data extraction process. Fig. 2   a Abnormal posture with backward bending of the neck was observed. b Upper gastrointestinal series show- ing gastroesophageal reflux. c Esophagogastroduodenoscopy showing reflux esophagitis (Los Angeles classification: Grade D) and a hiatal hernia Statistical analysis Continuous variables were presented as medians and inter- quartile ranges, whereas categorical variables were pre- sented as frequencies and percentages. Fisher’s exact test was used to evaluate categorical variables. p values < 0.05 were considered statistically significant. Statistical analysis 3   The flow diagram for study inclusion based on the PRISMA 2020 statement Fig. 3   The flow diagram for study inclusion based on the PRISMA 2020 statement was 2 (0–5) years. The median (interquartile range) dura- tion to diagnosis was 6 (2–28) months. The duration from nonsurgical treatment to surgery was more than 1 month, although only a few articles described this. In patients who did not undergo surgery, the median (interquartile range) duration to resolution of symptom was 1 (1–1) month. various symptoms such as torticollis, dystonia, and seizures [25, 26]. While the actual incidence of SS remains unknown, estimates place it at probably < 1% of children with GER [27, 28]. Given its lack of recognition, SS is often mistaken for neuromuscular or neuropsychiatric disorders due to lim- ited regarding the same. Available articles have shown that SS takes several months or more to diagnose (Table 1), often resulting in the repetition of unnecessary tests that further delay diagnosis [29]. Statistical analysis Pediatric Surgery International (2024) 40:91 Pediatric Surgery International Page 4 of 7 91 91 Table 1   Data collected from reported and our cases Author Year Age Anatomical anomaly Diagnostic examination Definitive treatment Duration to diagnosis (M) Duration to surgery (M) Duration to resolution (M) Shrestha 2021 4y None UGI Drug N/A – 1 Sharif 2020 3y None UGI, EGD, pH Drug N/A – N/A Bamji 2015 3m None Clinical history Drug 2 – 1 2m None UGI Drug 1 – 1 Nalbantoglu 2008 9m None EGD, pH Diet 2 – 1 Tokuhara 2008 8y None UGI, EGD, pH Surgery 36 3 3 Lehwald 2007 9y Hiatal hernia UGI, EGD Surgery 60 N/A 3 Firat 2007 2y Malrotation UGI, pH Surgery 21 N/A 1 Kabakus 2006 2m None Scintigraphy Drug 5 – N/A 4m None Scintigraphy Drug 2 – N/A 6m None Scintigraphy Drug 3 – N/A 1y None Scintigraphy Drug 8 – N/A Corrado 2006 1y None UGI, pH Drug 2 – 1 Frankel 2006 4y None UGI, EGD, pH Surgery 2 30 1 Corrado 2000 15d None UGI, pH Diet 14 – N/A Ybarrondo 2000 5y None UGI Surgery 0 12 1 Olguner 1999 5y None UGI, pH Surgery 12 N/A 2 Deskin 1995 2y None UGI Surgery 12 6 3 Gorrotxategi 1995 N/A N/A UGI, EGD, pH Drug N/A – N/A N/A N/A UGI, EGD, pH Drug N/A – N/A N/A N/A UGI, EGD, pH Drug N/A – N/A N/A N/A UGI, EGD, pH Surgery N/A N/A N/A N/A N/A UGI, EGD, pH Surgery N/A N/A N/A N/A N/A UGI, EGD, pH Surgery N/A N/A N/A N/A N/A UGI, EGD, pH Surgery N/A N/A N/A N/A N/A UGI, EGD, pH Surgery N/A N/A N/A Senocak 1993 N/A Hiatal hernia N/A Surgery N/A N/A N/A Puntis 1989 5y Hiatal hernia UGI, pH Surgery 3 2 3 Nanayakarra 1985 2y None UGI, pH, scintigraphy, Drug 19 – N/A 2y None UGI, pH Drug 18 – 1 2y None UGI, pH Drug 19 – 1 Hadari 1984 13y Hiatal hernia UGI Surgery N/A N/A N/A Werlin 1980 2w None UGI, pH Drug 0 – N/A 2w None UGI, pH Drug 0 – N/A 8m None UGI, pH Drug 2 – 1 3m None UGI, pH Drug N/A – 1 2w None UGI, pH Drug 0 – N/A Murphy 1977 8m Hiatal hernia UGI Drug 6 – 1 Bray 1977 3m None UGI Drug 2 – 4 3m None UGI Drug 2 – 2 2m Hiatal hernia UGI Drug 3 – 2 4y Hiatal hernia UGI Surgery 3 N/A N/A 5y Hiatal hernia UGI Surgery 30 N/A 1 5y None UGI Surgery 54 N/A N/A 7m None UGI Drug 6 – N/A 2m None UGI Drug 1 – N/A Sutcliffe 1969 10y Hiatal hernia UGI Surgery N/A N/A N/A 6y Hiatal hernia UGI Surgery N/A N/A N/A Table 1   Data collected from reported and our cases Pediatric Surgery International (2024) 40:91 Page 5 of 7  91 Pediatric Surgery International 91 UGI upper gastrointestinal series, EGD esophagogastroduodenoscopy, pH low esophageal pH 24-h recording Table 1   (continued) Author Year Age Anatomical anomaly Diagnostic examination Definitive treatment Duration to diagnosis (M) Duration to surgery (M) Duration to resolution (M) 4y Hiatal hernia UGI Surgery N/A N/A N/A Kinsbourne 1964 1y Hiatal hernia UGI Surgery 52 N/A N/A 7y Hiatal hernia UGI Surgery 36 N/A N/A 4y Hiatal hernia UGI Surgery 47 N/A N/A 9y Hiatal hernia UGI Surgery 72 N/A N/A 14y Hiatal hernia UGI Surgery 108 N/A N/A Kato 2023 9y Hiatal hernia UGI, EGD, pH Surgery 54 1 1 7 Hiatal hernia UGI, EGD Surgery 3 1 1 UGI upper gastrointestinal series, EGD esophagogastroduodenoscopy, pH low esophageal pH 24-h recording Table 1   (continued) Author Year Age Anatomical anomaly Diagnostic examination Definitive treatment Duration to diagnosis (M) Duration to surgery (M) Duration to resolution (M) 4y Hiatal hernia UGI Surgery N/A N/A N/A Kinsbourne 1964 1y Hiatal hernia UGI Surgery 52 N/A N/A 7y Hiatal hernia UGI Surgery 36 N/A N/A 4y Hiatal hernia UGI Surgery 47 N/A N/A 9y Hiatal hernia UGI Surgery 72 N/A N/A 14y Hiatal hernia UGI Surgery 108 N/A N/A Kato 2023 9y Hiatal hernia UGI, EGD, pH Surgery 54 1 1 7 Hiatal hernia UGI, EGD Surgery 3 1 1 Fig. Discussion To date, the etiology of the muscle dystonia in SS remains unclear. It may be related with the diaphragm and neck shar- ing common innervation. Some authors have postulated the movements and abnormal postures were a learned behav- ior to relieve abdominal discomfort and improve esopha- geal motility. It was proved the direct relationship between SS, which is named after neurologist Paul Sandifer, had first been reported by Kinsbourne in 1964 after recognizing a dysfunction in the upper gastrointestinal tract with neuro- logical manifestations occurring in children and adolescents [24]. SS consists of an unusual combination of GER and Pediatric Surgery International (2024) 40:91 Page 6 of 7 91 dystonic movements and low pH, and it revealed the pH in a patient with SS. During 83 episodes of posturing, none of these episodes occurred during a period of pH > 5 for ≥ 30 s[10]. SS could be caused by gastroesophageal reflux. Suc- cessful treatment of the underlying GERD led to a com- plete resolution of the symptoms. Although GER episodes in patients with SS had initially been considered to be induced by postural abnormalities, radiography during the torsion episode suggested that GER was actually worsening [24]. Previous study reported that neurological manifestations were the result of the vagal reflex [10]. However, it does not explain why these postures cannot adopt this position dur- ing sleep. The possible pathophysiologic relationship is that GER episodes cause postural abnormalities. Several articles have reported that esophageal motility improved during head tilting as evidence by the increase in esophageal contraction pressure from 47 to 74 mmHg and propagation velocity from 2.5 to 4 cm/s [13, 17]. The symptoms observed in SS are thought to clear gastric acid, suggesting that GER causes postural abnormalities, as supported by the disappearance of symptoms after GER treatment in our cases and in the reported literature. gastrointestinal examination. Fundoplication may be effec- tive especially for patients with anatomical anomalies or those whose symptoms fail to improve for over 1 month with nonsurgical treatment. Quick detection of subtle symptoms can facilitate early diagnosis and treatment and omission of unnecessary examinations in patients with SS. Our study has several limitations. First, all eligible stud- ies on patients with SS were case reports and non-rand- omized in nature. A prospective study may provide addi- tional insights into the diagnosis and outcomes of patients with SS. Conclusion It is important that GER be resolved when treating SS. Should appropriate nonsurgical treatment fail to improve symptoms, surgery may be necessary. Fundoplication, the primary surgical procedure considered safe for the treat- ment of GER [30], should always be considered as a pos- sible treatment option for patients with SS. The current study found that significantly more patients with anatomi- cal anomalies required surgery than did those without the same (Table 1). Fundoplication may be more effective than drug or diet therapy in controlling SS should gross anatomi- cal anomalies, such as hiatal hernia and malrotation, be the cause of GER. Moreover, most patients treated with drug or diet improved within 1 month (Table 1). Fundoplication may need to be considered if symptoms do not improve for more than 1 month with nonsurgical treatment. The primary care providers should keep SS in the dif- ferential diagnosis of patients presenting with abnormal posturing and no apparent neuromuscular disorders. Prompt treatment of GER may lead to early symptomatic improvement and omission of unnecessary examinations. Fundoplication leads to early symptomatic improvement and may be effective especially for patients with anatomi- cal anomalies or those whose symptoms fail to improve for over 1 month with nonsurgical treatment. Author contributions  D.K. had the idea for the article, and wrote the manuscript. D.K. and H.A. performed the literature search and data analysis. H.U. drafted and critically revised the work. All authors approved the final draft of this manuscript. The primary care providers should keep SS in the dif- ferential diagnosis of patients presenting with abnormal posturing and no apparent neuromuscular disorders. Rec- ognition and treatment of GER in patients with SS is key to medical management. The important first step is to sus- pect SS and obtain a detailed medical history. If symptoms appear immediately after feeding, SS may be present. As the diagnosis of GER is most reliably made by assess- ing the presence or absence of reflux, pH measurement is recommended in principle. In reported articles, the pH measurement has increased frequency over time (Table 1). The pH measurement is useful as it provides an objective assessment of reflux and clinicians should always con- sider performing this examination. Treatment should be initiated immediately after confirming GER through upper Funding  Open Access funding provided by Nagoya University. Funding  Open Access funding provided by Nagoya University. Discussion Second, the sample size was small, suggesting the need for large, high-quality randomized controlled trials in the future. Third, the duration of conservative therapy was unknown although we reviewed articles. If conserva- tive treatment is effective, it may be acceptable to continue treatment as is for long time. How long the effect of con- servative treatment lasts is a major issue to be addressed in the future. Conclusion Data availability  No datasets were generated or analysed during the current study. References 19. Hadari A, Azizi E, Lernau O, Nissan S (1984) Sandifer’s syn- drome–a rare complication of hiatal hernia. A case report. Z Kinderchir 39:202–203. https://​doi.​org/​10.​1055/s-​2008-​10442​08 1. Shrestha AB, Rijal P, Sapkota UH et al (2021) Sandifer syndrome: a case report. JNMA J Nepal Med Assoc 59:1066–1068. https://​ doi.​org/​10.​31729/​jnma.​6472 20. Werlin SL, D’Souza BJ, Hogan WJ et al (1980) Sandifer syn- drome: an unappreciated clinical entity. Dev Med Child Neurol 22:374–378. https://​doi.​org/​10.​1111/j.​1469-​8749.​1980.​tb037​19.x g j 2. Sharif A, Carr L, Saliakellis E, Chakraborty H (2020) Paroxys- mal head drops with ataxia-like symptoms presenting as Sandifer syndrome in a 3-year old girl. BMJ Case Rep 13:e230989. https://​ doi.​org/​10.​1136/​bcr-​2019-​230989 21. Murphy WJ, Gellis SS (1977) Torticollis with hiatus hernia in infancy. Sandifer syndrome. Am J Dis Child 131:564–565. https://​ doi.​org/​10.​1001/​archp​edi.​1977.​02120​18007​8015 22. Bray PF, Herbst JJ, Johnson DG et al (1977) Childhood gastroe- sophageal reflux: neurologic and psychiatric syndromes mim- icked. JAMA 237:1342–1345. https://​doi.​org/​10.​1001/​jama.​1977.​ 03270​40004​6017 g 3. Bamji N, Berezin S, Bostwick H, Medow MS (2015) Treatment of Sandifer syndrome with an amino-acid–based formula. AJP Rep 5:e51–e52. https://​doi.​org/​10.​1055/s-​0035-​15456​72 p g 4. Nalbantoglu B, Metin DM, Nalbantoglu A (2013) Sandifer’s syn- drome: a misdiagnosed and mysterious disorder. Iran J Pediatr 23:715–716 23. Sutcliffe J (1969) Torsion spasms and abnormal postures in childen with hiatus hernia, Sandifer’s syndrome. Prog Pediatr Radiol 2:190–197 5. Tokuhara D, Yamano T, Okano Y (2008) Letters to the editor. J Paediatr Child Health 44:311–312. https://​doi.​org/​10.​1111/j.​1440-​ 1754.​2008.​01304-​01305.x 24. Kinsbourne M (1964) Hiatus hernia with contortions of the neck. Lancet 283:1058–1061. https://​doi.​org/​10.​1016/​S0140-​6736(64)​ 91264-4 6. Lehwald N, Krausch M, Franke C et al (2007) Sandifer syndrome– a multidisciplinary diagnostic and therapeutic challenge. Eur J Pediatr Surg 17:203–206. https://​doi.​org/​10.​1055/s-​2007-​965145 25. Gellis SS, Feingold M (1971) Syndrome of hiatus hernia with torsion spasms and abnormal posturing (Sandifer’s syndrome). Am J Dis Child 121:53–54 g g 7. Firat AK, Karakas HM, Firat Y, Yakinci C (2007) Unusual symp- tom of intestinal malrotation: episodic cervical dystonia due to Sandifer syndrome. Pediatr Int 49:519–521. https://​doi.​org/​10.​ 1111/j.​1442-​200x.​2007.​02409.x 26. Torticollis with hiatus hernia in infancy. Sandifer syndrome - Pub- Med. https://​pubmed.​ncbi.​nlm.​nih.​gov/​855842/. Accessed 15 Sep 2023 27. Narchi H (2004) A Rare Under-Recognized Cause of Pseudo- Seizures. Int Pediatr 19:39–41 8. Kabakuş N, Kurt A (2006) Sandifer Syndrome: a continuing prob- lem of misdiagnosis. Pediatr Int 48:622–625. https://​doi.​org/​10.​ 1111/j.​1442-​200X.​2006.​02280.x 28. Sommer A (1993) Occurrence of the Sandifer complex in the Brachmann-de Lange syndrome. Am J Med Genet 47:1026–1028. https://​doi.​org/​10.​1002/​ajmg.​13204​70719 9. Declarations Conflict of interest  The authors declare no competing interests that are relevant to the content of this article. Ethical approval  All procedures performed in studies involving human participants were in accordance with the ethical standards of the insti- tutional or national research committee and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. Informed consent  Informed consent to participate in the study was obtained from the parents of all individual participants by disclosing Page 7 of 7 91 Pediatric Surgery International (2024) 40:9 Pediatric Surgery International the study information and giving those who did not wish to participate an opportunity to request it (opt-out). the study information and giving those who did not wish to participate an opportunity to request it (opt-out). syndrome. Pediatr Int 41:321–322. https://​doi.​org/​10.​1046/j.​1442-​ 200x.​1999.​01051.x 14. Deskin RW (1995) Sandifer syndrome: a cause of torticollis in infancy. Int J Pediatr Otorhinolaryngol 32:183–185. https://​doi.​ org/​10.​1016/​0165-​5876(95)​01130-4 Open Access  This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. g 15. Gorrotxategi P, Reguilon MJ, Arana J et al (1995) Gastroesopha- geal reflux in association with the Sandifer syndrome. Eur J Pedi- atr Surg 5:203–205. https://​doi.​org/​10.​1055/s-​2008-​10662​05 16. Senocak ME, Arda IS, Büyükpamukçu N (1993) Torticollis with hiatus hernia in children. Sandifer syndrome. Turk J Pediatr 35:209–213f 17. Puntis JW, Smith HL, Buick RG, Booth IW (1989) Effect of dys- tonic movements on oesophageal peristalsis in Sandifer’s syn- drome. Arch Dis Child 64:1311–1313. https://​doi.​org/​10.​1136/​ adc.​64.9.​1311 18. Nanayakkara CS, Paton JY (1985) Sandifer syndrome: an over- looked diagnosis? Dev Med Child Neurol 27:816–819. https://​doi.​ org/​10.​1111/j.​1469-​8749.​1985.​tb038​08.x References Corrado G, Fossati C, Turchetti A et al (2006) Irritable oesopha- gus: a new cause of Sandifer’s syndrome. Acta Paediatr 95:1509– 1510. https://​doi.​org/​10.​1080/​08035​25060​06432​51 29. Mandel H, Tirosh E, Berant M (1989) Sandifer syndrome recon- sidered. Acta Paediatr Scand 78:797–799. https://​doi.​org/​10.​ 1111/j.​1651-​2227.​1989.​tb111​50.x p g 10. Frankel EA, Shalaby TM, Orenstein SR (2006) Sandifer syndrome posturing: relation to abdominal wall contractions, gastroesopha- geal reflux, and fundoplication. Dig Dis Sci 51:635–640. https://​ doi.​org/​10.​1007/​s10620-​006-​3184-1 30. Gordon N (2015) Sandifer’s syndrome: investigations and treat- ment. J Pediatr Neurol 05:275–278. https://​doi.​org/​10.​1055/s-​ 0035-​15574​13 11. Corrado G, Cavaliere M, D’Eufemia P et al (2000) Sandifer’s syn- drome in a breast-fed infant. Am J Perinatol 17:147–150. https://​ doi.​org/​10.​1055/s-​2000-​9285 Publisher's Note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 12. de Ybarrondo L, Mazur JL (2000) Sandifer’s syndrome in a child with asthma and cerebral palsy. South Med J 93:1019–1021 13. Olguner M, Akgür FM, Hakgüder G, Aktuğ T (1999) Gastroe- sophageal reflux associated with dystonic movements: Sandifer’s
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Engendrando desenvolvimento e etnicidade nas terras baixas do Pacífico colombiano
Revista Estudos Feministas
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cc-by
7,516
Copyright  2004 by Revista Estudos Feministas * Excepcionalmente neste artigo, as notas estão editadas ao final do texto. Kiran Asher Engendrando desenvolvimento e Engendrando desenvolvimento e Engendrando desenvolvimento e Engendrando desenvolvimento e Engendrando desenvolvimento e etnicidade nas terras baixas do etnicidade nas terras baixas do etnicidade nas terras baixas do etnicidade nas terras baixas do etnicidade nas terras baixas do PPPPPacífico colombiano acífico colombiano acífico colombiano acífico colombiano acífico colombiano Clark University Kiran Asher Clark University Estudos Feministas, Florianópolis, 12(1): 360, janeiro-abril/2004 15 Engendrando desenvolvimento e Engendrando desenvolvimento e Engendrando desenvolvimento e Engendrando desenvolvimento e Engendrando desenvolvimento e etnicidade nas terras baixas do etnicidade nas terras baixas do etnicidade nas terras baixas do etnicidade nas terras baixas do etnicidade nas terras baixas do PPPPPacífico colombiano acífico colombiano acífico colombiano acífico colombiano acífico colombiano RRRRResumo esumo esumo esumo esumo: Neste ensaio, exploro como as organizações e redes de mulheres afro-colombianas moldam e são moldadas por iniciativas do Estado para desenvolver e modernizar a região do Pacífico. Argumento que, agindo assim, elas mobilizam e vão além da retórica desenvolvimentista do Estado e do discurso de uma etnia negra ‘gendrada’ e da tradição das organizações políticas negras da região. PPPPPalavras-chave alavras-chave alavras-chave alavras-chave alavras-chave: mulheres afro-colombianas, estratégias de desenvolvimento, políticas étnicas e de gênero. O Litoral Pacífico da Colômbia, ou a região Choco, é uma área rica em recursos naturais, que se estende do extremo sul do Panamá ao extremo norte do Equador, ao longo da costa do Pacífico. Em 1991, a Colômbia adotou uma nova Constituição que inclui leis e políticas com implicações contraditórias para a região do Pacífico e seus/ suas habitantes. Por um lado, a Lei 70, de 1993, baseada no Artigo Provisório 55 da Constituição de 1991, reconhece os/as afro-colombianos/as (que compõem 90% da população da região) como um grupo étnico separado, com direitos a possuir terras coletivamente e utilizá-las dentro dos moldes tradicionais.1 Por outro lado, as reformas econômicas neoliberais esboçadas no ‘Charter’ de 1991 identificam áreas ‘marginais’, como as da região do Pacífico, para vários projetos de macrodesenvolvimento, com vistas a extrair seus recursos naturais e modernizá-las. Além disso, em consonância com os apelos da Constituição pela proteção do meio ambiente colombiano, as Terras Baixas 15 Estudos Feministas, Florianópolis, 12(1): 360, janeiro-abril/2004 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... elas se mobilizam e vão além da retórica desenvolvimentista do Estado e do discurso de uma etnia gendrada e da tradição dos movimentos sociais negros na região. Concluo discutindo como o ativismo das afro-colombianas complica a nossa compreensão de como operam as intervenções para o desenvolvimento. O ensaio mostra também como as subjetividades e o agenciamento das mulheres negras são moldados diferenciadamente, desigualmente e discursivamente pelas, e contra as, intervenções políticas e culturais na região. Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 17 Plan P Plan P Plan P Plan P Plan Pacífico acífico acífico acífico acífico: da “inércia do : da “inércia do : da “inércia do : da “inércia do : da “inércia do subdesenvolvimento” para o subdesenvolvimento” para o subdesenvolvimento” para o subdesenvolvimento” para o subdesenvolvimento” para o “desenvolvimento sem sujeitos”? “desenvolvimento sem sujeitos”? “desenvolvimento sem sujeitos”? “desenvolvimento sem sujeitos”? “desenvolvimento sem sujeitos”? Um tráfico incessante de mercadorias e pessoas fluía ao longo do rio Atrato, em Quibdó (a capital do estado de Choco). Os prédios de gesso e cimento, delineando o malécon, rachavam e mofavam sob o intenso calor, com sua pintura, outrora brilhante, empolando e descascando na umidade do ar. Ao passar pelos vendedores, que compravam peças artesanais de ouro de mulheres e homens chocoanos, lembrei-me da vista aérea do rio Atrato enquanto voava para Quibdó. Contaminado com o mercúrio usado na mineração comercial, o rio Atrato parecia uma corda grossa e verde insinuando-se lentamente pela paisagem. Enquanto a mineração do ouro e de outros metais preciosos (juntamente com a extração da madeira e o comércio do marfim vegetal) tem sido a base da economia extrativa da região desde os tempos coloniais, a pesca, caça e a agropecuária são as principais atividades de subsistência das comunidades negras dos quatro estados do Litoral do Pacífico. Refletindo sobre a situação da região durante uma visita a Quibdó nos anos 1980, Thomas Sanders, um ex- professor de Religião e membro do Universities Field Staff International, observou que “... o Choco continua a simbolizar um tipo de pobreza e de subdesenvolvimento em que cultura e raça têm influência”.4 De acordo com Sanders, a região do Choco continuou a sofrer da mesma “inércia do subdesenvolvimento” que ele havia testemunhado durante sua primeira visita em 1970, apesar do aumento do preço do ouro no mundo. Para Sanders, o futuro da região residia no exercício do poder político e da responsabilidade econômica para desenvolver os ricos recursos naturais e humanos e melhorar o padrão de vida.5 Mais de uma década depois, um relatório feito pelo Departamento Nacional de Planejamento (DNP) reflete o diagnóstico de Sanders, observando que “a pobreza é a Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 17 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 19 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... coligados a uma política partidária formal, opõem-se a tais medidas modernizadoras. Janeth Rojas, uma feminista mestiça de Cali, com experiência em projetos de desenvolvimento a longo prazo na região do Pacífico, resume sucintamente as reações de muitos/as afro- colombianos/as: Essa visão de desenvolvimento com respeito às comunidades negras baseia-se fundamentalmente em [a noção de] “ter” (TENER) mais do que na possibilidade de “ser” (SER). Com isso subentende-se que a melhoria de qualidade de vida é medida mais através da satisfação das necessidades básicas, melhores salários e aumento do consumo, do que do fortalecimento do direito de ser diferente.11 Talvez os mais notáveis críticos das iniciativas do Estado colombiano para o desenvolvimento sejam os membros de um grupo de intelectuais negros/as e ativistas que uniram forças coletivamente sob o nome de Processo de Comunidades Negras (PCN).12 Carlos Rosero, um antropólogo de Buenaventura e membro da liderança do PCN, rejeita a representação que o Estado faz da região e do seu povo como “marginal e pobre”.13 Ele argumenta que o Plan Pacífico foi concebido nos escritórios de planejamento do governo e é uma forma de “desenvolvimento sem sujeitos”, porque ignora as percepções, necessidades e agenciamento dos/as afro- colombianos/as.14 Rosero e outros/as citam como evidência o fato de que as primeiras versões do Plan Pacífico enfocavam primordialmente planos de macrodesenvolvimento e investimento a fim de promover a atividade econômica na região.15 Eles/as corretamente mencionam que as questões sócio-culturais e ecológicas foram introduzidas na agenda desse plano inicial após muito lobby de grupos étnicos e de ONGs ambientalistas. y g p Refletindo a respeito da onda anterior de desenvolvimento no Pacífico, os/as críticos/as observam como tais projetos – planejados na capital andina e incompatíveis com as realidades sócio-culturais da região – foram malsucedidos e fizeram surgir ondas de migração em direção a cidades do Pacífico e além.16 A população rural deslocada, inclusive um grande número de mulheres, foi incorporada à economia monetária como mão-de-obra barata na agroindústria e na cultura aquática, o que levou a um colapso dos sistemas de subsistência locais e, geralmente, deixou as comunidades mais empobrecidas que antes.17 Betty Ruth Lozano18 observa que a intensificação dos sistemas de acumulação de capital levou a uma duplicação ou triplicação da carga de trabalho das mulheres e reforçou a subordinação de gênero das mulheres 19 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 21 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... articula essa preocupação em termos coloquiais: Pra que serve água potável, eletricidade, escolas, clínicas, aeroportos, reservas florestais, grandes empresas e dinheiro se temos vergonha de sermos negros, se denunciamos nossas fazendas e vendemos nossas terras, se nos acanhamos de comer o que tiramos da floresta, ou as nossas comidas tradicionais, e se nos esquecemos de como fazer uma catanga [uma armadilha para lagostas], se abandonamos o território que tem sido a nossa vida, se todo esse desenvolvimento traz violência e pobreza? Nossa busca principal agora é pelo direito a “ser” [SER] sem negar a importância de “ter” [TENER] condições materiais para gozar a vida como camponeses dignos, aqui nos nossos rios.25 Em reuniões públicas, em negociações com o Estado, assim como em entrevistas e conversas, os/as ativistas do PCN chamam a atenção para as várias práticas de subsistência das comunidades negras – agricultura, pesca artesanal e caça, catação de mariscos, extração de vários produtos renováveis, como também extração de madeira e mineração em pequena escala. Observam que os/as afro- colombianos/as vivem na região desde o período colonial e desenvolveram esses sistemas de produção econômica ecologicamente sustentáveis baseados/as nas tradições africanas e ameríndias. Para os/as ativistas do PCN, são essas práticas ‘ancestrais’ e as relações sociais associadas a elas, baseadas no parentesco e nos laços de família, que representam o locus da etnia e da diferença dos negros, que devem ser o fulcro do “desenvolvimento culturalmente apropriado” na região. Leyla Arroyo, um outro membro ativo no PCN de Buenaventura, observa o seguinte: Acredito que a identidade étnica está no centro do processo organizativo do povo negro. O reconhecimento das comunidades negras como um grupo étnico é uma de nossas maiores vitórias. Trabalhamos para que o nosso povo sinta orgulho de ser negro; reafirmamos quem nós somos, conquistamos auto-estima. [...] Acredito que podemos desenvolver uma visão de vida alternativa, um modelo econômico diferente, baseado nas nossas tradições, que seja justo e que inclua todos os aspectos da sociedade.26 Contudo, os/as afro-colombianos são marcados/as por muitas distinções regionais, ideológicas, de classe e de gênero. Dada essa heterogeneidade, os debates em torno do que significa ser ‘negro/a’ ou ‘afro-colombiano/a’ e o estágio incipiente das organizações negras em nível nacional, não existe uma visão comum ou politicamente viável de um “desenvolvimento culturalmente apropriado”. Dentro deste contexto, os movimentos sociais de negros na 21 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 23 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... observa que os primeiros planos de desenvolvimento para a região surgiram nas décadas de 1950 e 1960, durante o período de desenvolvimento do Terceiro Mundo pós- Segunda Guerra Mundial. Uma série de desastres naturais na região (inclusive um incêndio em Quibdó em 1966 e um maremoto em Tumaco em 1979) também abriram espaço para grandes projetos de reconstrução apoiados pelo Estado. Nas décadas seguintes, vários eventos, instituições e práticas – evangelização, concessões para florestamento, estudos para avaliar o potencial agroindustrial, desenvolvimento de culturas aquáticas, projetos para a construção de portos e canais – levaram o desenvolvimento e integração da região ao interior do país.28 No final da década de 1960, projetos de desenvolvimento como o PLADEICOP (Projeto de Desenvolvimento Integrado para o Pacífico Colombiano), do presidente Belisario Betancur, tinham as mulheres como alvos de projetos de bem-estar social, contra a pobreza e de saúde rural. Essas iniciativas continuaram na forma de programas de assistência como o El Plan de Hogares de Bienestar (Plano para Casas de Bem-Estar), que era parte do plano sócio-econômico do presidente Virgilio Barcos para a região do Pacífico.29 Programas do convênio entre a Corporación Autônoma Regional Del Valle Del Cauca e a Holanda introduziram novas tecnologias e técnicas para a intensificação da produção agrícola em pequena escala na região. Embora esses programas produtivos não visassem às mulheres, como Lozano e Rojas demonstram,30 eles incorporaram com sucesso as mulheres negras ao processo de desenvolvimento. Por exemplo, na região de Tumaco, no Pacífico Sul, mulheres, em grande número, foram empregadas em pequenas tarefas em fazendas de camarão e em plantações de palmeiras para a extração de óleo, e integradas ao setor agroindustrial. Em 1988, um PLADEICOP mais abrangente lançou um projeto direcionado às mulheres para facilitar a contribuição delas ao setor produtivo.31 Esses projetos de desenvolvimento correspondiam à tendência internacional das abordagens Mulheres-no-Desenvolvimento (Women-in-Development/ WID), que defendia a necessidade de integrar as mulheres ao processo de desenvolvimento e demonstrava que, além da contribuição ao bem-estar da família, as mulheres são uma parte importante do setor econômico produtivo e devem ter acesso igual aos seus benefícios.32 Como a pequena descrição no início desta seção indica, as mulheres negras são uma parte ativa e visível da economia da região. Na década de 1980, mulheres padeiras, costureiras, peixeiras, vendedoras de frutas em Guapi e Tumaco formavam pequenos grupos em torno de 23 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 2 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 25 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... princípio fundamental da rede era “criar organizações de mulheres autônomas que manifestassem e refletissem nosso desenvolvimento, interesses e identidade étnica e cultural”.40 CoopMujeres, Ser Mujer e Fundemujer estavam entre as muitas organizações ligadas à Red. princípio fundamental da rede era “criar organizações de mulheres autônomas que manifestassem e refletissem nosso desenvolvimento, interesses e identidade étnica e cultural”.40 CoopMujeres, Ser Mujer e Fundemujer estavam entre as muitas organizações ligadas à Red. Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 27 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... Desarollo de la Mujer de Buenaventura (FUNDEMUJER), era a maior das três cooperativas, consistindo de 25 grupos de mulheres, com um total de 800 membros. Com uma história organizacional e institucional estabelecida, as mulheres negras começaram a questionar, nas cooperativas, sobre suas identidades étnicas e de gênero, e procuraram ir além das atividades produtivas.36 Em abril de 1995, houve um encontro entre o CoopMujeres de Guapi com um novo programa para mulheres negras fundado pelo governo do Canadá e administrado pela Fundación FES, uma fundação privada colombiana. Os executores desse fundo colombiano- canadense encontraram-se com membros das cooperativas para discutir suas propostas e planos para os anos seguintes. No encontro, Sylveria Rodríguez, a diretora do CoopMujeres, disse que os membros queriam direcionar novos fundos para o treinamento técnico das mulheres. Além disso, ela e membros da cooperativa queriam realizar oficinas sobre mecanismos de participação política, cidadania, relações interpessoais e familiares. Quando perguntada a respeito de oficinas sobre os direitos das mulheres, a resposta de Sylveria Rodríguez foi: Já conhecemos os nossos direitos; agora queremos aprender como obter nossos direitos; ensinar a outras pessoas sobre os nossos direitos. Precisamos educar os nossos homens sobre os direitos das mulheres. No ano passado, comemoramos o Dia dos Pais no Coop Mujeres. Este ano, estamos tentando fazer com que cada membro “conquiste” o seu parceiro e o traga para a oficina. No ano anterior, uma oficina sobre cozinha regional foi realizada paralelamente à comemoração do Dia dos Pais. Tal evento combinou atividades produtivas (cozinhar) com o resgate de tradições culinárias locais e exibiu as atividades dos membros das cooperativas para os pais e esposos presentes. No dia seguinte ao encontro, Cipriana Diuza, uma professora e membro recente do CoopMujeres, contou-me que se engajou na cooperativa porque era “organizada, fundamentada, e ajudava ativamente mulheres solteiras, mães solteiras, mulheres pobres e chefes de famílias”. Mas confessou ter curiosidade a respeito de “toda essa confusão em torno de ser mulher negra”. Ela sentia que “nós somos mulheres negras, alegres, mas ainda escravizadas, ainda temerosas. Ainda precisamos aprender a valorizar o nosso dialeto, a nossa religião, as nossas danças”. Mulheres nas cooperativas de Buenaventura, Guapi e Tumaco expressaram sentimentos semelhantes. Elas queriam manter o enfoque nas atividades produtivas geradoras de renda, 25 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... número um, o amigo-homem” tratava-se de uma alusão não apenas aos homens em casa, como também aos homens inseridos nas lutas mais amplas dos/as negro/as na região. Os membros do Fundemujer afirmam: Queremos ver as lutas étnico-políticas com os olhos de mulheres negras. Mas o PCN não quer as duas lutas juntas. A opinião deles é que a luta de gênero enfraquece a luta étnica.46 Apontando exemplos das muitas mulheres negras que estavam envolvidas no processo de mobilização para a ratificação da AT 55, membros do Fundemujer ecoaram a observação de Teofila de que as mulheres negras e suas organizações desempenharam um papel crucial na luta pela Lei 70. Mercedes, Patrícia, Dora e Myrna observaram também que as mulheres negras ofereceram um apoio logístico importante (cozinhando para o encontro, administrando o escritório, controlando as finanças) dentro do PCN. No entanto, como Teofila, elas sentiram que os membros do PCN não reconheceram nem valorizaram o trabalho e a importância das mulheres negras enquanto mulheres.47 De acordo com elas, até as mulheres que têm um papel importante no PCN não possuem uma “consciência de gênero”. Patricia Moreno continua: Armando [do PCN] disse que eles tinham medo que nós falássemos sobre gênero e esquecêssemos a luta étnica. Eles acham que questões de gênero vindas do “interior” [referindo-se às capitais andinas da região e do país] irão diluir a luta étnica. Isso os apavora. Eles acreditam que nós não sentimos a nossa própria opressão. São as forças externas que nos “pesquisam”. Como se não sentíssemos nossa subordinação sexual e física! Os ativistas do PCN contestam tal caracterização de seu posicionamento em relação às mulheres e às questões de gênero. Eles enfatizam que não apenas reconhecem o papel central desempenhado pelas mulheres negras na vida social, cultural e política do Pacífico, como também admitem a necessidade de se abordar questões de gênero dentro do PCN.48 Entretanto, reiteram seu posicionamento firme em relação às intervenções do Estado na região, inclusive “a nova política de gênero”. Os membros do PCN afirmam que as relações de gênero são relações sociais fundamentais, e que a dinâmica entre homens e mulheres negras são mais bem abordadas através de lutas para se conquistar direitos étnicos e não através de programas estatais direcionados às mulheres. VVVVVendo através dos olhos das mulheres endo através dos olhos das mulheres endo através dos olhos das mulheres endo através dos olhos das mulheres endo através dos olhos das mulheres negras: as redes afro negras: as redes afro negras: as redes afro negras: as redes afro negras: as redes afro-colombianas -colombianas -colombianas -colombianas -colombianas Teofila Betancur é uma guapireña e membro ativo da Red de Mujeres Negras. Durante uma conversa com ela em Cali, em 1995, ela me disse: “As mulheres negras ajudaram a Lei 70 a partir da sua condição de mulher (ser mujer). Contudo, elas têm pouca ou nenhuma participação pública ou política enquanto mulheres negras. Isso também é verdade nas cooperativas de mulheres negras. As mulheres negras são discriminadas triplamente – enquanto pobres, enquanto mulheres e enquanto negras. Isso as leva a se subvalorizar. As mulheres negras na costa assim como nas cidades estão alisando, pintando ou fazendo permanentes em seus cabelos para ficarem ‘brancas’ e adotam hábitos da alta sociedade colombiana em uma tentativa de conseguir aceitação social. Isso representa uma perda de valores e de beleza. Então, membros da Rede organizaram oficinas de cabelos e concursos de penteados entre as mulheres negras de Guapi. Agora, mais de metade das guapireñas usam trancinhas, tranças e outros penteados afro-colombianos tradicionais. Precisamos trabalhar a partir da ‘cabeça’ para recuperar nossa identidade externa como mulheres negras, para refletirmos a respeito e afirmarmo-nos como negras”.41 Teofila Betancur e suas companheiras acreditam que o enfoque nas identidades e na auto-estima das mulheres negras representa um verdadeiro primeiro passo em direção aos objetivos mais amplos da Red de trabalhar para a melhoria das mulheres negras e suas comunidades.42 Quando encontrei Teofila novamente em 1999, ela me disse que a seção da Rede em Guapi agora incluía 74 grupos locais, ribeirinhos e regionais de mulheres, no estado de Cauca. Cada grupo está envolvido em vários tipos de atividades, inclusive projetos ‘produtivos’ como o cultivo e a recuperação de grãos alimentícios e plantas medicinais nativas, assim como projetos de ‘conservação’ com foco nas práticas agrícolas e silvícolas para conservar o meio ambiente e a biodiversidade.43 De acordo com Teofila, as diversas atividades e projetos empreendidos por grupos dentro da Rede surgem de “suas necessidades, percepções e experiências como mulheres negras, e representam a sua força coletiva”. Para simbolizar essa força, a Rede de Guapi adotou o nome Red de Organizaciones Femeninas del 27 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 29 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... étnicas e de gênero, para contestar as forças hegemônicas do desenvolvimento apoiado pelo Estado. Abaixo, ofereço uma leitura diferente do ativismo das mulheres negras. Argumento que as subjetividades e atividades das mulheres negras são moldadas através e contra as intervenções políticas e culturais na região. Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 31 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... A posição do PCN é a de que as organizações e redes de mulheres negras funcionando de maneira independente dos movimentos étnicos mais amplos da região terminariam sendo 29 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... Através de Através de Através de Através de Através de ser y tener ser y tener ser y tener ser y tener ser y tener: constituindo etnia, : constituindo etnia, : constituindo etnia, : constituindo etnia, : constituindo etnia, gênero e desenvolvimento no P gênero e desenvolvimento no P gênero e desenvolvimento no P gênero e desenvolvimento no P gênero e desenvolvimento no Pacífico acífico acífico acífico acífico Ponderando sobre uma política de desenvolvimento para e por mulheres no Pacífico colombiano, Maria Del Rosario Minaescreve: Temos um interesse básico: trabalhar conjuntamente entre nós mesmas e com outras para propor uma política de desenvolvimento para as mulheres afrodescendentes da região do Pacífico que corresponda à nossa existência básica (SER), a partir da qual seja possível identificar as condições e necessidades materiais e sociais (TENER) que garantam essa existência.49 Para Mina, um enfoque nas necessidades materiais não se choca com a recuperação e o orgulho da identidade. Pelo contrário, na sua formulação da “política de desenvolvimento para mulheres negras do Pacífico”, ‘ser’ (existir) e ‘tener’ (ter as necessidades que garantam essa existência) estão dialeticamente ligados. Para as mulheres negras do Pacífico, participar dos projetos de modernização do Estado não significa apenas abandonar suas identidades e tradições para conquistar aquilo que o desenvolvimento promete. Pelo contrário, para as mulheres negras, engajar- se no desenvolvimento significa engajar-se na luta pelo poder de definir o que se entende por ‘necessidades’ e de criar meios de atendê-las. Isso requer que as mulheres se engajem estrategicamente com o poder estatal e com suas intervenções desenvolvimentistas, assim como com as lutas étnicas. Também requer que recorram ao seu conhecimento das práticas tradicionais e culturais de sobrevivência no Pacífico. Minha discussão sobre o ativismo das mulheres afro- colombianas refere-se a pelo menos duas questões sobre o debate em torno do desenvolvimento: primeiro, as lutas dos grupos locais contra o Estado e a modernização; e, segundo, os problemas do ativismo político e ‘agenciamento’ das mulheres do Terceiro Mundo.50 A década de 1980 presenciou muitos países latino- americanos enfrentando graves crises financeiras, tumultos políticos e levantes sociais. Estudiosos/as dos novos movimentos sociais (NMSs) na América Latina afirmam que coalizões pouco organizadas de trabalhadores/as de 31 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 3 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... estados do sudoeste do Pacífico), assim como de representantes de organizações da Costa Atlântica e da capital Bogotá. Grupos do estado de Choco, que fica ao noroeste, preferiram se organizar separadamente. estados do sudoeste do Pacífico), assim como de representantes de organizações da Costa Atlântica e da capital Bogotá. Grupos do estado de Choco, que fica ao noroeste, preferiram se organizar separadamente. p Minhas discussões sobre o posicionamento e estratégias do PCN baseiam-se em vários encontros e conversas com membros de organizações do PCN durante trabalho de campo na região. Líderes do PCN, como Carlos Rosero, Libia Grueso, Hernan Cortés e Leyla Andréa Arroyo também expressam seus pontos de vista em seminários, conferências e fóruns com vistas a discutir os direitos dos/as negros/as, que são amplamente publicados em minutas ou anais das conferências. Baseio-me nesses pareceres coletados durante o meu trabalho de campo e forneço citações paralelas de fontes publicadas sempre que possível. 13 Tais críticas ao desenvolvimento são ecoadas largamente por acadêmicos/as e ativistas, no norte e no sul. Por exemplo, os/as vários/ as colaboradores/as do Dicionário do desenvolvimento: um guia para o conhecimento como poder (Wolfgang SACHS, 1992) analisam certos conceitos-chave do desenvolvimento – tais como Planejamento, Pobreza, Produção, Progresso, Padrão de Vida, etc. – em um esforço de expor os limites do pensamento acerca do desenvolvimento e de imaginar uma “era pós-desenvolvimento”. Esses/as críticos/as argumentam que, através de alegações enganosas de superioridade dos sistemas econômico e político do Ocidente, os discursos sobre o desenvolvimento constroem um Terceiro Mundo ‘subdesenvolvido’ e impõem uma ‘racionalidade’ e conhecimento ocidental a esses espaços. p ç 14 ROSERO, 1996, p. 182. 15 Alguns projetos propostos no Plan Pacífico pelo Conselho Nacional de Política Econômica e Social (CONPES) e pelo Departamento Nacional de Planejamento (DNP) da Colômbia incluem: a construção de estradas ligando centros regionais-chave com centros de troca e comércio andinos, construindo oleodutos e usinas hidroelétricas, e construindo e modernizando vários portos. Ver em Diego PIEDRAHÍTA e María Estella PINEDA, 1993, uma lista detalhada e uma descrição das propostas desses projetos. Ver também www.dnp.gov.co para uma visão geral das agendas de desenvolvimento da Colômbia. g 16 Gustavo DE ROUX, 1990-1991; Alvaro PEDROSA, 1996; ROJAS, 1994. ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... alternativas para o desenvolvimento e as literaturas dos NMSs histórica e analiticamente, a fim de argumentar que o desenvolvimento e os movimentos contra ele estão implicados uns nos outros. Afirma que as buscas que vemos hoje por alternativas às forças destrutivas do capitalismo precisam começar a examinar como o moderno e o não- moderno produzem um ao outro em inúmeros locais. p Minha discussão do ativismo das mulheres negras no Pacífico mostra que gênero desempenha um papel crucial nessa produção e contestação do desenvolvimento. Enquanto os primeiros estudos acerca de gênero e desenvolvimento retratavam as mulheres como vítimas e recipientes passivas das políticas de bem-estar social, estudos feministas recentes revelam a multiplicidade de localidades e de questões em torno das quais uma diversidade de mulheres age.59 Feministas pós-coloniais como Chandra Mohanty, Aihwa Ong e Gayatri Spivak60 elucidam como estruturas do capitalismo, discursos de desenvolvimento e de gênero, patriarcados locais e noções de cultura constituem as subjetividades das mulheres de maneiras específicas e são utilizados para reforçar certos significados e práticas, e para evitar outros. Essas feministas chamam a atenção para a natureza dinâmica e oposicional da dominação e da resistência das mulheres dentro de relações desiguais de poder. Por exemplo, em Women Workers and Capitalist Scripts, Mohanty61 enfoca não apenas a exploração de mulheres trabalhadoras do Terceiro Mundo pela atual expansão capitalista, com também “seu agenciamento enquanto trabalhadoras, em nome dos interesses comuns das mulheres trabalhadoras, baseado na compreensão de localidades e necessidades compartilhadas, e nas estratégias/práticas de organização que estão ancoradas na, e levam à, transformação das vidas cotidianas das mulheres trabalhadoras”.62 Similarmente, no seu estudo sobre as operárias de fábricas na Malásia, Ong63 discute como essas mulheres camponesas que se tornaram assalariadas são moldadas pelas, e resistem às, relações industriais capitalistas. O exemplo das cooperativas de mulheres negras e da Red de Mujeres Negras destaca como as subjetividades e as organizações afro-colombianas são moldadas pela linguagem e pelas práticas de instituições para o desenvolvimento e suas políticas de gênero atuais. Por sua vez, as mulheres negras utilizam-se dos termos e recursos das iniciativas desenvolvimentistas e dos discursos culturais predominantes na região para abordar as suas necessidades e subverter as agendas tanto do Estado como das lutas étnicas. Através dessa constituição mútua, elas ajudam a institucionalizar as intervenções do Estado e, simultaneamente, 33 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 35 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... manuscritos publicados e não-publicados sobre e pela rede de mulheres, e de um videoteipe do segundo encontro da rede de mulheres da região de Cauca e Nariño, realizado em Timbiqui, Cauca, no período de 1 a 4 de maio de 1997. manuscritos publicados e não-publicados sobre e pela rede de mulheres, e de um videoteipe do segundo encontro da rede de mulheres da região de Cauca e Nariño, realizado em Timbiqui, Cauca, no período de 1 a 4 de maio de 1997. 43 Ver ASHER, 2002, para uma análise mais detalhada sobre o envolvimento de mulheres negras (inclusive do grupo de Teofila) em projetos de desenvolvimento sustentável e de preservação da biodiversidade, no estado de Cauca, no sudoeste colombiano. Ver Juana CAMACHO, 1999 e 2001, para uma discussão sobre o papel central das mulheres na administração de recursos agrícolas e da biodiversidade, e na manutenção de redes sociais nas áreas costeiras do estado de Choco, no noroeste. 44 Observação participativa no seminário e na marcha organizada pela Red de Mujeres Chocoanas, no Dia Internacional da Mulher, 8 de março de 1995, e em entrevistas com as organizadoras do evento, Adriana Eliza Fox Porro e Nimia Teresa Cuesta. 45 Talvez a explicação por que as mulheres da OBAPO resolveram se organizar sob os auspícios de uma organização negra resida no fato de que a maior parte das posições de liderança na OBAPO é de mulheres. 46 Isso me traz à mente as reflexões de Audre Lorde sobre o movimento negro nos Estados Unidos: “Dentro das comunidades negras onde o racismo é uma realidade viva, as diferenças entre nós geralmente parecem suspeitas e perigosas. A necessidade de unidade é normalmente mal interpretada como uma necessidade de homogeneidade, e o olhar de uma feminista negra, confundido com traição dos nossos interesses comuns enquanto um povo” (LORDE, 1992, p. 51). p ( p ) 47 Ver ASHER, 1997, para uma discussão sobre a questão contraditória da presença de mulheres negras como líderes em movimentos sociais negros e a sua invisibilidade enquanto mulheres negras nessas organizações. 48 OCN, 1996, p. 256-260. 49 MINA, 1995. ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... 17 Por exemplo, a pesquisa de Carmen Diana DEERE e Magdalena LEÓN (1981), em três regiões andinas que se desenvolvem com maior rapidez na Colômbia e no Peru, mostrou que o maior envolvimento de mulheres no trabalho agrícola caminha lado a lado com o empobrecimento geral de grandes setores rurais. Ver também seu volume editado Rural Women and State Policy: Feminist Perspectives on Latin American Agricultural Development (DEERE e LEÓN, 1987) para discussões sobre como as políticas agrícolas do Estado afetem as mulheres em várias partes da América Latina e da bacia caribenha. 19 GRUESO, 1998; GRUESO et al., 1998. 20 OCN, 1996; ROSERO, 1995 e 1996. 21 Julio CARRIZOSA UMANÃ, 1993; Arturo ESCOBAR, 1996; ROJAS, 1994; Juan Pablo RUIZ, 1993. 21 Julio CARRIZOSA UMANÃ, 1993; Arturo ESCOBAR, 1996; ROJAS, 1994; Juan Pablo RUIZ, 1993. 23 Como era de se esperar, há debates intensos na Colômbia, inclusive entre negros/as, sobre o que constitui a etnia e a diferença ‘afro- colombiana’. Muitas pesquisas têm enfocado a forma como as diferenças culturais e de identidade dos/as negros/as ou afro- colombianos/as são entendidas e construídas pelos movimentos sociais negros depois da Lei 70. Ver ASHER, 1998; Eduardo RESTREPO, 1998 e 2001; e Peter WADE, 1996, para algumas discussões importantes. Ver 35 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 37 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... Ourselves as Women, Rescuing our Black Identity: Ethnicity and Gender in the Pacific Lowlands.” Current World Leaders, v. 40, n. 6, 1997. p. 106-127. Leaders, v. 40, n. 6, 1997. p. 106-127. ____. “Culture/Politics and Black Ethnicities in the Pacific Coast of Colombia.” Paper presented at XXI International Conference of the Latin American Studies Association, Chicago, September 24-26, 1998. ____. “Mobilizing the Discourses of Sustainable Economic Development and Biodiversity Conservation in the Pacific Lowlands of Colombia.” Strategies: A Journal of Theory, Culture and Politics, v. 13, n. 1, 2000. p. 111-125. ____. “Texts in Context: Afro-Colombian Women’s Activism in the Pacific Lowlands of Colombia.” Feminist Review, 2002. Under review. Í ___. “Culture/Politics and Black Ethnicities in the Pacific Coast of Colombia.” Paper presented at XXI International C f f th L ti A i St di A i ti Culture and Politics, v. 13, n. 1, 2000. p. 111 125. ____. “Texts in Context: Afro-Colombian Women’s Activism in the Pacific Lowlands of Colombia.” Feminist Review, 2002. Under review. BALANTA, Olivia; RODRÍGUEZ, Betty; SINISTERRA, Sonia; Quiñonez, Piedad; ARROYO, Leila; EQUIPO DINAMIZADOR. “Red de Mujeres Negras del Pacífico: tejiendo procesos organizativos autonomos”. Esteros, n. 9, p. 37-42, feb. 1997. BRAIDOTTI, Rosi, CHARKIEWICZ, Ewa, HÄUSLER, Sabine, and WIERINGA, Saskia (eds.). Women, the Environment and Sustainable Development: Towards a Theoretical Synthesis. London: Zed Books, 1994. CAMACHO, Juana. “‘Todos tenemos derecho a su parte’: derechos de herencia, acceso y control de bienes en comundiades negras de la costa Pacífica chocoana”. In: CAMACHO, Juana; RESTREPO, Eduardo (Coords.). De montes, ríos, y ciudades: territorios e identidades de la gente negra en Colombia. Santa Fe de Bogotá: Fundación Natura, Ecofondo, Instituto Colombiano de Antropología, 1999. p. 107-130. _____. “Mujeres, zoteas y hormigas arrieras: prácticas de manejo de flora en la costa Pacífica chocoana”. In: ARROYO, Jesús; CAMACHO, Juana; LEYTON, Mireya; GONZÁLEZ, Maribel (Coords.). Zoteas: biodiversidad y relaciones culturales en el Chocó biogeográfico colombiano. Bogotá: Instituto de Investigaciones Ambientales del Pacífico-IIAP, Fundación Natura, Fundación Swissaid-Colombia, 2001. p. 35-58. CAMACHO, Juana. “‘Todos tenemos derecho a su parte’: derechos de herencia, acceso y control de bienes en comundiades negras de la costa Pacífica chocoana”. In: CAMACHO, Juana; RESTREPO, Eduardo (Coords.). De montes, ríos, y ciudades: territorios e identidades de la gente negra en Colombia. Santa Fe de Bogotá: Fundación Natura, Ecofondo, Instituto Colombiano de Antropología, 1999. p. 107-130. p g , p _____. ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... 50 O ativismo das mulheres afro-colombianas baseado na ligação entre ‘ser’ e ‘tener’ também problematiza a caracterização da luta das mulheres do Terceiro Mundo como sendo ou lutas ‘femininas’ para atender às ‘necessidades práticas’ ou lutas ‘feministas’ pelo poder de redefinir as relações de gênero e contestar as posturas políticas e econômicas do Estado. Ver Marianne MARCHAND, 1995, para uma discussão sobre como essas visões sobre as lutas das mulheres são aplicadas para uma melhor compreensão do ativismo das mulheres. 51 Susan ECKSTEIN, 1989; David SLATER, 1985; e Arturo ESCOBAR e Sonia ALVAREZ, 1992. 52 A literatura dos NMSs é influenciada em vários níveis pelos debates pós-estruturalistas nas ciências sociais e humanas e textos ‘anti- desenvolvimento’ que surgiram nas décadas de 1980 e 1990. Muitos/as estudiosos/as e ativistas do Terceiro Mundo (ESCOBAR, 1992; SACHS, 1992; e Vandana SHIVA, 1988) denunciam os projetos desenvolvimentistas como um subgrupo da modernidade hegemônica, eurocêntrica. Baseando- se em trabalhos recentes em historiografia pós-colonial, eles chamam a atenção para a necessidade de se reconhecer racionalidades ‘não- ocidentais’. De acordo com esses/as críticos/as, alternativas às tendências homogeneizantes e excludentes do capitalismo global são encontradas nas práticas tradicionais dos grupos locais. p g p 53 ESCOBAR e PEDROSA, 1993 e 1996. 54 Ou para usar a frase de Verónica Schild, questiono a “separação tão definida entre Estado e sociedade civil” (SCHILD, 1998, p. 111), que está implícita em muito da literatura dos NMSs sobre as lutas locais contra o desenvolvimento. Escrevendo sobre o projeto de democratização e 37 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... _____.“Viejas y nuevas formas de capital y los dilemas de la biodiversidad”. In: ESCOBAR, Arturo and PEDROSA, Alvaro (eds.). Pacífico ¿Desarrollo o Diversidad?: Estado, capital y movimientos sociales en el Pacífico colombiano. Bogotá: CEREC y Ecofondo 1996 p 109 131 _____.“Viejas y nuevas formas de capital y los dilemas de la biodiversidad”. In: ESCOBAR, Arturo and PEDROSA, Alvaro (eds.). Pacífico ¿Desarrollo o Diversidad?: Estado, capital y movimientos sociales en el Pacífico colombiano. Bogotá: CEREC y Ecofondo 1996 p 109 131 _____.“Viejas y nuevas formas de capital y los dilemas de la biodiversidad”. In: ESCOBAR, Arturo and PEDROSA, Alvaro (eds.). Pacífico ¿Desarrollo o Diversidad?: Estado, capital y movimientos sociales en el Pacífico colombiano. B tá CEREC E f d 1996 109 131 _____.“Cultural Politics and Biological Diversity: State, Capital, and Social Movements in the Pacific Coast of Colombia.” In: LOWE, Lisa, and LLOYD, David (eds.). The Politics of Culture in the Shadow of Capital. Durham, NC: Duke University Press, 1997. p. 201-225. ESCOBAR, Arturo, and ALVAREZ, Sonia. The Making of Social Movements in Latin America: Identity, Strategy, and Democracy. Boulder, CO: Westview Press, 1992. ESCOBAR, Arturo; PEDROSA, Alvaro. “¿Laboratorio para el postdesarrollo?” Revista Universidad del Valle, n. 5, p. 34-45, 1993. ESCOBAR, Arturo; PEDROSA, Alvaro. “Camaroneras comunitarias: ¿Alternativas al capital? Entrevista con Harold Moreno y Relato de Doña Ester Caicedo”. In: ESCOBAR, Arturo, and PEDROSA, Alvaro (Coords.). Pacífico ¿Desarrollo o Diversidad?: Estado, capital y movimientos sociales en el Pacífico colombiano. Bogotá: CEREC y Ecofondo, 1996. p. 144-175. ESTEROS. Mujeres negras e indigenas definiendo futuro, v. 5, n. 9, feb. 1997. Número especial. FEMPRESS: Red de comunicación alternativa de la mujer. Mujeres negras, Latinoamerica, 1995. Número especial. FEMPRESS: Red de comunicación alternativa de la mujer. Mujeres negras, Latinoamerica, 1995. Número especial. FERGUSON, James. The Anti-Politics Machine: “Development”, Depoliticization, and Bureaucratic Power in Lesotho. Cambridge, UK: Cambridge University Press, 1990. GROS, Christian. Colombia indigena: identidad cultural y cambio social. Bogotá, Colombia: CEREC, 1991. Cambridge, UK: Cambridge University Press, 1990. GROS, Christian. Colombia indigena: identidad cultural y cambio social. Bogotá, Colombia: CEREC, 1991. GRUESO, Jesus Alberto; ESCOBAR, Arturo. “Las cooperativas agrarias y la modernización de los agricultores”. In: ESCOBAR, Arturo, and PEDROSA, Alvaro (eds.). Pacífico ¿Desarrollo o Diversidad?: Estado, capital y movimientos sociales en el Pacífico colombiano. Bogotá: CEREC y Ecofondo. 1996. p. 90-108. GRUESO, Libia Rosario. ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... “Mujeres, zoteas y hormigas arrieras: prácticas de manejo de flora en la costa Pacífica chocoana”. In: ARROYO, Jesús; CAMACHO, Juana; LEYTON, Mireya; GONZÁLEZ, Maribel (Coords.). Zoteas: biodiversidad y relaciones culturales en el Chocó biogeográfico colombiano. Bogotá: Instituto de Investigaciones Ambientales del Pacífico-IIAP, Fundación Natura, Fundación Swissaid-Colombia 2001 p 35-58 CAMACHO, Juana; RESTREPO, Eduardo (Coords.). De montes, ríos, y ciudades: territorios e identidades de la gente negra en Colombia. Santa Fe de Bogotá: Fundación Natura, ECOFONDO, Instituto Colombiano de Antropología, 1999. CARRIZOSA UMAÑA, Julio. “El Chocó y el resto del mundo.” EcoLogica: Politica, Medio Ambiente, Cultura, v. 4, n. 15- 16, p. 12-17, 1993. 39 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... “El ejericio del derecho al territorio en la comunidad negra del Pacífico Sur: un reto en doble vía”. Buenaventura, Colombia, 1998. Manuscrito. GRUESO, Libia, ROSERO, Carlos, and ESCOBAR, Arturo. “The Process of Black Community Organizing in the Southern Pacific Coast Region of Colombia.” In: ALVAREZ, Sonia, DAGNINO, Evelina and ESCOBAR, Arturo (eds.). Cultures of Politics/Politics of Cultures: Re-visioning Latin American Social Movements. Boulder, CO: Westview Press, 1998. p. 196-219. 41 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... TINKER, Irene (ed.). Persistent Inequalities: Women and World Development. Oxford: Oxford University Press, 1990. TINKER, Irene (ed.). Persistent Inequalities: Women and World Development. Oxford: Oxford University Press, 1990. VILLA, William. “El Pacífico colombiano: políticas de desarrollo”. Esteros, n. 1, p. 15-19, enero/marzo, 1993. _____.“El plan de desarrollo de comunidades negras en la ley 70 de 1993”. Esteros, n. 7, p. 36-40, agosto 1995. VILLA, William. “El Pacífico colombiano: políticas de desarrollo”. Esteros, n. 1, p. 15-19, enero/marzo, 1993. _____.“El plan de desarrollo de comunidades negras en la ley 70 de 1993”. Esteros, n. 7, p. 36-40, agosto 1995. , , p , / , _____.“El plan de desarrollo de comunidades negras en la ley 70 de 1993”. Esteros, n. 7, p. 36-40, agosto 1995. WADE, Peter. “The Language of Race, Place and Nation in Colombia.” America Negra, n. 2, 1991. p. 41-68. _____. Blackness and Race Mixture: The Dynamics of Racial Identity in Colombia. Baltimore and London: The Johns Hopkins Press, 1993. _____. “The Cultural Politics of Blackness in Colombia.” American Ethnologist, n. 22, v. 2, 1995. p. :341-357. _____. “Identidad y etnicidad”. In: ESCOBAR, Arturo; PEDROSA, Al (C d ) P ífi ¿D ll Di id d? WADE, Peter. “The Language of Race, Place and Nation in Colombia.” America Negra, n. 2, 1991. p. 41-68. WADE, Peter. “The Language of Race, Place and Nation in Colombia.” America Negra, n. 2, 1991. p. 41-68. _____. Blackness and Race Mixture: The Dynamics of Racial Identity in Colombia. Baltimore and London: The Johns Hopkins Press, 1993. _____. “The Cultural Politics of Blackness in Colombia.” American Ethnologist, n. 22, v. 2, 1995. p. :341-357. _____. “Identidad y etnicidad”. In: ESCOBAR, Arturo; PEDROSA, Alvaro (Coords.). Pacífico ¿Desarrollo o Diversidad?: Estado, capital y movimientos sociales en el Pacífico colombiano. Bogotá: CEREC y Ecofondo, 1996. p. 283- 298. WADE, Peter. “The Language of Race, Place and Nation in Colombia.” America Negra, n. 2, 1991. p. 41-68. _____. Blackness and Race Mixture: The Dynamics of Racial Identity in Colombia. Baltimore and London: The Johns Hopkins Press, 1993. _____. Blackness and Race Mixture: The Dynamics of Racial Identity in Colombia. Baltimore and London: The Johns Hopkins Press, 1993. Hopkins Press, 1993. _____. “The Cultural Politics of Blackness in Colombia.” American Ethnologist, n. 22, v. 2, 1995. p. :341-357. American Ethnologist, n. 22, v. 2, 1995. p. :341 357. _____. “Identidad y etnicidad”. In: ESCOBAR, Arturo; PEDROSA, Alvaro (Coords.). ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... OCN (Organización de Comunidades Negras). “Movimiento negro, identidad y territorio: entrevista con la Organización de Comunidades Negras de Buenaventura”. In: ESCOBAR, Arturo; PEDROSA, Alvaro (Coords.). Pacífico ¿Desarrollo o Diversidad?: Estado, capital y movimientos sociales en el Pacífico colombiano. Bogotá: CEREC y Ecofondo, 1996. p. 245- 265. ONG, Aihwa. Spirits of Resistance and Capitalist Discipline: Factory Women in Malaysia. Albany, NY: State University of New York Press, 1987. _____.“Colonialism and Modernity: Feminist Re-presentations of Women in Non-Western Societies.” Inscriptions, v. 3-4, n. 2, 1988. p. 79-93. PARDO, Mauricio (Coord.). Acción colectiva, Estado y etnicidad en el Pacífico colombiano. Bogotá: ICANH, Colciencias, 2001. PARDO, Mauricio; ALVAREZ, Manuela. “Estado y movimiento negro en el Pacífico colombiano”. In: PARDO, Mauricio (Coord.). Acción colectiva, Estado y etnicidad en el Pacífico colombiano. Bogotá: ICANH, Colciencias, 2001. p. 229-258. PCN (Proceso de Comunidades Negras). “El Proceso de Comunidades Negras en el centro-sur del Pacífico y el problema del ‘desarrollo’”. Buenaventura, Colombia, n.d. Manuscrito. PEDROSA, Alvaro. “La institucionalizacion del desarrollo”. In: ESCOBAR, Arturo; PEDROSA, Alvaro (Coords.). Pacífico ¿Desarrollo o Diversidad?: Estado, capital y movimientos sociales en el Pacífico colombiano. Bogotá: CEREC y Ecofondo, 1996. p. 66-89. PEET, Richard, and WATTS, Michael (eds.). Liberation Ecologies: Environment, Development, Social Movements. London and New York: Routledge, 1996. Í PIEDRAHÍTA, Diego; PINEDA, María Estella. “En vías de desarrollo: lista de proyectos de desarrollo para el Pacífico”. EcoLogica: Politica, Medio Ambiente, Cultura, v. 4, n. 15-16, 1993. p. 83-86. p RESTREPO, Eduardo. “La construcción de la etnicidad: comunidades negras en Colombia”. In: SOTOMAYOR, Maria Lucia (Coord.). Modernidad, identidad y desarrollo: construcción de sociedad y re-creación cultural en contextos de modernización. Bogotá: ICAN, 1998. p. 341-358. _____. “Imaginando comunidad negra: etnografía de la etnización de poblaciones negras en el Pacífico sur colombiano”. In: PARDO, Mauricio (Coord.). Acción colectiva, Estado y etnicidad en el Pacífico colombiano. Bogotá: ICANH, Colciencias, 2001. p. 41-70. RESTREPO, Eduardo. “La construcción de la etnicidad: comunidades negras en Colombia”. In: SOTOMAYOR, Maria Lucia (Coord.). Modernidad, identidad y desarrollo: construcción de sociedad y re-creación cultural en contextos de modernización. Bogotá: ICAN, 1998. p. 341-358. _____. “Imaginando comunidad negra: etnografía de la etnización de poblaciones negras en el Pacífico sur colombiano”. In: PARDO, Mauricio (Coord.). Acción colectiva, Estado y etnicidad en el Pacífico colombiano. Bogotá: ICANH, Colciencias, 2001. p. 41-70. 43 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 45 ENGENDRANDO DESENVOLVIMENTO E ETNICIDADE... Pacífico ¿Desarrollo o Diversidad?: Estado, capital y movimientos sociales en el Pacífico colombiano. Bogotá: CEREC y Ecofondo, 1996. p. 283- 298. WATTS, Michael. “‘A New Deal in Emotions’: Theory and Practice and the Crisis of Development.” In: CRUSH, Jonathan (ed.). Power of Development. London and NY: Routledge, 1995. p. 44-62. WESTWOOD, Sallie, and RADCLIFFE, Sarah A. “Gender, Racism and the Politics of Identities in Latin America.” In: RADCLIFFE, Sallie A., and WESTWOOD, Sarah (eds.). ‘Viva’: Women and Popular Protest in Latin America. London and New York: Routledge, 1993. p. 1-29. Ser y T Ser y T Ser y T Ser y T Ser y Tener ener ener ener ener: Engendering Development and Ethnicity in the P : Engendering Development and Ethnicity in the P : Engendering Development and Ethnicity in the P : Engendering Development and Ethnicity in the P : Engendering Development and Ethnicity in the Pacific L acific L acific L acific L acific Lowlands of Colômbia owlands of Colômbia owlands of Colômbia owlands of Colômbia owlands of Colômbia Abstract Abstract Abstract Abstract Abstract: In this paper I explore how Afro-Colombian women’s organizations and networks shape and are shaped by state initiatives to develop and modernize the Pacific region. I argue that in doing so these mobilize and go beyond the developmentalist rhetoric of the state and the discourse of gendered black ethnicity and tradition of black political organizations in the region. Key words Key words Key words Key words Key words: Afro-Colombian women, development strategies, gendered ethnicity in politics. Tradução de Ana Cecília Acioli Revisão da tradução: Susana Bornéo Funck Tradução de Ana Cecília Acioli Revisão da tradução: Susana Bornéo Funck Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 45 Estudos Feministas, Florianópolis, 12(1): 15-45, janeiro-abril/2004 45
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https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0233640&type=printable
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Computational model of tranexamic acid on urokinase mediated fibrinolysis
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PLOS ONE RESEARCH ARTICLE Tie Bo WuID1*, Thomas Orfeo2, Hunter B. Moore3, Joshua J. Sumislawski3, Mitchell J. Cohen3, Linda R. PetzoldID1 1 Department of Mechanical Engineering, University of California Santa Barbara, Santa Barbara, California, United States of America, 2 Department of Biochemistry, University of Vermont, Burlington, Vermont, United States of America, 3 Department of Surgery, Denver Health and Hospital Authority, Denver, Colorado, United States of America * tiebo@ucsb.edu * tiebo@ucsb.edu * tiebo@ucsb.edu a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Wu TB, Orfeo T, Moore HB, Sumislawski JJ, Cohen MJ, Petzold LR (2020) Computational model of tranexamic acid on urokinase mediated fibrinolysis. PLoS ONE 15(5): e0233640. https:// doi.org/10.1371/journal.pone.0233640 Citation: Wu TB, Orfeo T, Moore HB, Sumislawski JJ, Cohen MJ, Petzold LR (2020) Computational model of tranexamic acid on urokinase mediated fibrinolysis. PLoS ONE 15(5): e0233640. https:// doi.org/10.1371/journal.pone.0233640 Editor: Raghavan Raju, Medical College of Georgia, Augusta, UNITED STATES Received: February 6, 2020 Accepted: May 9, 2020 Published: May 26, 2020 Received: February 6, 2020 Accepted: May 9, 2020 Published: May 26, 2020 Abstract Understanding the coagulation process is critical to developing treatments for trauma and coagulopathies. Clinical studies on tranexamic acid (TXA) have resulted in mixed reports on its efficacy in improving outcomes in trauma patients. The largest study, CRASH-2, reported that TXA improved outcomes in patients who received treatment prior to 3 hours after the injury, but worsened outcomes in patients who received treatment after 3 hours. No consen- sus has been reached about the mechanism behind the duality of these results. In this paper we use a computational model for coagulation and fibrinolysis to propose that defi- ciencies or depletions of key anti-fibrinolytic proteins, specifically antiplasmin, a1-antitrypsin and a2-macroglobulin, can lead to worsened outcomes through urokinase-mediated hyperfibrinolysis. Computational model of tranexamic acid on urokinase mediated fibrinolysis Tie Bo WuID1*, Thomas Orfeo2, Hunter B. Moore3, Joshua J. Sumislawski3, Mitchell J. Cohen3, Linda R. PetzoldID1 PLOS ONE PLOS ONE Introduction Copyright: © 2020 Wu 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. Severe trauma often induces a coagulopathic state known as Acute Traumatic Coagulopathy (ATC) that manifests in increased bleeding and resultant mortality [1]. The mechanisms underlying ATC are not yet fully understood, making treatment difficult [2]. One treatment in particular, tranexamic acid (TXA), has been found to be often effective [3, 4]. However, there are unknown circumstances in which the treatment has been reported to worsen the condi- tion, with increased bleeding and mortality. A meta-analysis of the CRASH-2 study found that the relative risk of death due to bleeding in patients who received TXA compared to placebo was 0.68 for patients who arrived within 1 hour after injury, 0.79 for patients who arrived between 1 and 3 hours after injury, but 1.44 for patients who arrived more than 3 hours after injury [5, 6]. The risk of worsening the situation is one of the main reasons why many health care organizations are hesitant to incorporate TXA into their trauma protocol. Developing a mechanistic understanding of how TXA can produce such contrasting results is necessary for widespread adoption of the treatment. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This material is based upon work supported by the US Army Contracting Command, Aberdeen Proving Ground, Natick Contracting Division under Contract No. W911QY-15-C-0026. Funding was granted to Linda Petzold (LP). The funders had no role in study design, analysis, decision to publish, or preparation of the manuscript. Tranexamic acid is an antifibrinolytic amino acid derivative that prevents the binding of plasmin(ogen) to fibrin. This binding inhibits fibrinolysis in two ways (Fig 1). First, it slows 1 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0233640 May 26, 2020 PLOS ONE Computational model of TXA on uPA mediated fibrinolysis Competing interests: The authors have declared that no competing interests exist. down tissue-plasminogen-activator(tPA) mediated conversion of plasminogen to plasmin, as fibrin acts as a substrate that increases the catalytic efficiency of the reaction 500-fold. Even after activation, TXA-bound plasmin cannot bind to fibrin, directly preventing the digestion of fibrin [7]. Competing interests: The authors have declared that no competing interests exist. Introduction The mechanisms behind the anti-fibrinolytic properties of TXA are well under- stood, but the mechanism responsible for the possible increased bleeding and mortality have yet to be determined. Competing interests: The authors have declared that no competing interests exist. A possible explanation for the varying effectiveness of TXA treatment is the interaction between TXA and urokinase plasminogen activator (uPA or urokinase) [8]. Studies have shown that uPA levels are elevated in many instances in traumatic injury [9, 10], and since uPA-mediated plasmin activation occurs in solution, the inhibition of fibrin binding by TXA is inconsequential to this process. Furthermore, there is another binding site for TXA on plas- minogen that induces a conformation change that speeds up plasmin activation approximately 3-fold [11]. However this explanation is incomplete, as multiple studies have shown that despite the increase of plasmin generation, TXA still inhibits fibrinolysis in uPA-mediated sys- tems [12, 13]. This suggests that other conditions must be met to change the behavior of TXA from anti-fibrinolytic to pro-fibrinolytic. To explore the possible conditions under which TXA can increase fibrinolysis through uPA-mediated plasmin generation, we constructed a differ- ential equation based computational model. This model builds on previous ODE models [14– 18] of the coagulation and fibrinolysis system, but focuses specifically on investigating the interaction between uPA and TXA, which are not present in these models. Fig 1. TXA interaction with plasminogen. Tranexamic Acid(TXA) inhibits fibrinolysis through its binding to plasminogen (Pg). This binding prevents plasminogen from binding to fibrin, which inhibits activation through tPA. https://doi.org/10.1371/journal.pone.0233640.g001 Fig 1. TXA interaction with plasminogen. Tranexamic Acid(TXA) inhibits fibrinolysis through its binding to plasminogen (Pg). This binding prevents plasminogen from binding to fibrin, which inhibits activation through tPA. Fig 1. TXA interaction with plasminogen. Tranexamic Acid(TXA) inhibits fibrinolysis through its binding to plasminogen (Pg). This binding prevents plasminogen from binding to fibrin, which inhibits activation through tPA. https://doi.org/10.1371/journal.pone.0233640.g001 https://doi.org/10.1371/journal.pone.0233640.g001 https://doi.org/10.1371/journal.pone.0233640.g001 PLOS ONE | https://doi.org/10.1371/journal.pone.0233640 May 26, 2020 2 / 11 PLOS ONE Computational model of TXA on uPA mediated fibrinolysis Fig 2. Simplified diagram of reaction network. This network diagram shows the important reactions in our model. Tissue factor (TF) initiates a chain of reactions in the coagulation cascade resulting in thrombin (IIa). Thrombin converts fibrinogen into fibrin, forming the clot. Introduction The clot is broken down by plasmin (Pn) which is activated from plasminogen (Pg) in one of two ways, by tissue-plasminogen activator (tPA) or urokinase-plasminogen activator (uPA). This diagram illustrates how tranexamic acid (TXA) has properties that both inhibit and promote fibrinolysis. The purpose of this paper is to explore which conditions cause the net effect to change from anti-fibrinolytic to pro-fibrinolytic. Additional abbreviation: Fibrin Degradation Productions (FDP). Fig 2. Simplified diagram of reaction network. This network diagram shows the important reactions in our model. Tissue factor (TF) initiates a chain of reactions in the coagulation cascade resulting in thrombin (IIa). Thrombin converts fibrinogen into fibrin, forming the clot. The clot is broken down by plasmin (Pn) which is activated from plasminogen (Pg) in one of two ways, by tissue-plasminogen activator (tPA) or urokinase-plasminogen activator (uPA). This diagram illustrates how tranexamic acid (TXA) has properties that both inhibit and promote fibrinolysis. The purpose of this paper is to explore which conditions cause the net effect to change from anti-fibrinolytic to pro-fibrinolytic. Additional abbreviation: Fibrin Degradation Productions (FDP). https://doi.org/10.1371/journal.pone.0233640.g002 https://doi.org/10.1371/journal.pone.0233640.g002 We found that additional plasmin inhibitors such as α1-antitrypsin(A1AT) and α2-macro- globulin(A2M), two proteins not often associated with fibrinolysis, play a critical role in pre- venting TXA-induced hyperfibrinolysis (Fig 2. If the other plasmin inhibitors are depleted, α2-antiplasmin (AP or antiplasmin) is not sufficient in preventing fibrinolysis, and the pres- ence of TXA acts to increase the rate of fibrinolysis. Computational model In this paper we constructed an ordinary differential equation (ODE) model of the coagulation process. This includes reactions for thrombin generation, fibrin formation and fibrin degrada- tion (Fig 2). Most of these reactions and their associated rate constants are taken from models based on in vitro experimental results, and most of them can be found in previously published ODE models [14–18]. In our model we also make the assumption that A2M and A1AT are not being consumed outside of the coagulation and fibrinolysis systems. Because of the difficulty 3 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0233640 May 26, 2020 PLOS ONE Computational model of TXA on uPA mediated fibrinolysis Fig 3. Diagram of different variants of TXA-bound plasminogen. Plasminogen has 2 binding sites that interact with TXA (labeled x and y in the model). One of the binding sites with Kd = 600 μM changes the conformation that speeds up activation by uPA. The other binding site with Kd = 1.1 μM prevents binding to fibrin, which prevents activation via tPA as well as fibrin degradation. UPA can activate all 4 of these variants, whereas tPA can activate only the variants with the second binding site free. Fig 3. Diagram of different variants of TXA-bound plasminogen. Plasminogen has 2 binding sites that interact with TXA (labeled x and y in the model). One of the binding sites with Kd = 600 μM changes the conformation that speeds up activation by uPA. The other binding site with Kd = 1.1 μM prevents binding to fibrin, which prevents activation via tPA as well as fibrin degradation. UPA can activate all 4 of these variants, whereas tPA can activate only the variants with the second binding site free. https://doi.org/10.1371/journal.pone.0233640.g003 of accurately modeling these additional systems and their low impact on the overall system, we took the conservative approach of excluding them. If these reactions were included, the rate of A2M and A1AT consumption would increase and the results would be amplified in the favor of our conclusions. Our model focuses on the interactions between TXA and fibrinolysis, so it includes a more detailed model of TXA-plasminogen binding (Fig 3). We model plasminogen with 2 binding sites for TXA (labeled x and y), resulting in 4 species of Pg, depending on whether TXA is bound to one or both of those sites (Pg, Pgx, Pgy, Pgxy). The x site controls the pro-fibrinolytic activity of TXA. PLOS ONE | https://doi.org/10.1371/journal.pone.0233640 May 26, 2020 Computational model If TXA is bound to this site (Kd = 600 μM), then its activation rate via uPA is increased 3-fold. The y site controls the anti-fibrinolytic activity of TXA and has a much higher binding affinity (Kd = 1.1 μM). This is the binding site that fibrin and plasmin inhibitors such as α2-antiplasmin bind to. If the y site is occupied by TXA, it cannot bind to fibrin, which pre- vents it from being activated by tPA and prevents it from degrading fibrin even after being converted to plasmin. While tPA cannot activate the plasminogen species with its y site bound (Pgy, Pgxy), uPA can activate all of these species of plasminogen. Therefore, in uPA-mediated systems, TXA will increase the amount of plasmin generated [11, 12]. 4 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0233640 May 26, 2020 PLOS ONE Computational model of TXA on uPA mediated fibrinolysis The reversibility of the TXA binding with plasmin(ogen) has an important consequence. It means that eventually TXA bound to the y site will be replaced by a irreversible plasmin inhibi- tor as long as there is some left in the system. This means that TXA is effective at inhibiting fibrinolysis only if there is a sufficient supply of plasmin inhibitors [18]. A complete descrip- tion of the model, a complete list of reactions, rate constants and initial conditions for our model can be found in S1 Appendix in S1 File. The full model can be found online at https:// github.com/taicheeze/coag_ode_julia. We initiated coagulation with 5 pM of tissue factor (TF), which is frequently used in other computational models [15]. We examined the rate of fibrinolysis initiated with 2.5 nM tPA or 5 nM uPA, and varied the amount of TXA in the system (0, 1, 3, 14, 54 and 3470 μM) to com- pare to experimental results to those of Longstaff 2019 [13]. PLOS ONE | https://doi.org/10.1371/journal.pone.0233640 May 26, 2020 UPA mediated fibrinolysis The interactions between TXA and uPA are more complex. Overall, fibrinolysis with uPA is slower than with tPA, which can be for a variety of reasons. UPA activates plasmin in solution rather than on the fibrin surface, making it easier to inhibit by plasmin inhibitors. In addition, uPA is inhibited by protein C inhibitor (PCI) [20, 21] which is present in the body at a higher concentration than the inhibitors of tPA. Since we are assuming that A2M and A1AT are not being consumed by reactions outside of the coagulation and fibrinolysis systems, this affects uPA-activated plasmin more than tPA activated plasmin. Focusing on the plasmin inhibition role of these proteins, we simulated the effects of consumption of these proteins by varying their initial concentrations. Under conditions simulated with high concentrations of plasmin inhibitors (Fig 5A), TXA behaves in an anti-fibrinolytic manner. Despite increasing the amount of plasmin in the system, the activated plasmin is quickly bound to TXA, preventing it from degrading fibrin. The TXA bound to the active plasmin is later replaced by one of the non-reversible plasmin inhibitors, which permanently inhibits it. Higher concentrations of TXA shift the equilibrium between TXA-bound plasmin and free plasmin, lowering the amount of free plasmin at any given time, resulting in quick inhibition by other plasmin inhibitors. When α1-antitrypsin is removed from the system (Fig 5B), the behavior of the system remains mostly unchanged. The effects of A1AT are not obvious until both A1AT and A2M are removed. In this system, A1AT appears to play a role redundant to A2M except much weaker, due to its much slower binding to plasmin [22]. When A2M is removed from the sys- tem (Fig 5C), uPA mediated fibrinolysis becomes much more apparent. However, TXA retains its anti-fibrinolytic behavior, with a small change in behavior in the 10-100 μM range, which has also been shown experimentally [12]. However, once both A2M and A1AT are removed from the system(Fig 5D), the behavior of TXA flips. In this system, the only plasmin inhibitor left is antiplasmin. Since the ratio of plas- minogen to antiplasmin is nominally 2:1, a large amount of plasmin activation can deplete antiplasmin. The presence of TXA increases the rate of plasmin activation, and since the bind- ing between TXA and plasmin is reversible, antiplasmin will eventually deplete, and any remaining activated plasmin will rapidly degrade fibrin, resulting in hyperfibrinolysis. UPA mediated fibrinolysis Addi- tional results of uPA mediated fibrinolysis with varying levels of plasmin inhibitors can be found in S1 Fig in the S1 File. TPA mediated fibrinolysis The effects of TXA on tPA mediated fibrinolysis in our computational model behave as expected and are confirmed by results of previous empirical experiments(Fig 4) [12, 13, 19]. As more TXA was added to the system, fibrinolysis slowed down in a dose-dependent manner. This behavior is consistent through different initial concentrations of plasmin inhibitors (not Fig 4. Effects of TXA on tPA mediated fibrinolysis. Modeling results show that Tranexamic Acid (TXA) affects tPA mediated fibrinolysis in a dose dependent fashion. As the concentration of TXA increases, the rate of fibrinolysis decreases. At the highest dose of TXA, fibrinolysis appears to be completely inhibited. These simulations are initiated with 5 pM tissue factor, and an initial tPA concentration of 2.5 nM. https://doi.org/10.1371/journal.pone.0233640.g004 Fig 4. Effects of TXA on tPA mediated fibrinolysis. Modeling results show that Tranexamic Acid (TXA) affects tPA mediated fibrinolysis in a dose dependent fashion. As the concentration of TXA increases, the rate of fibrinolysis decreases. At the highest dose of TXA, fibrinolysis appears to be completely inhibited. These simulations are initiated with 5 pM tissue factor, and an initial tPA concentration of 2.5 nM. https://doi.org/10.1371/journal.pone.0233640.g004 5 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0233640 May 26, 2020 PLOS ONE Computational model of TXA on uPA mediated fibrinolysis shown) as expected since the interactions between TXA and tPA are straightforward and well established. These results show that the bulk of the model, with the exception of the uPA path- way of fibrinolysis, is in agreement with empirical data. TPA and uPA mediated fibrinolysis https://doi org/10 1371/journal pone 0233640 g005 https://doi.org/10.1371/journal.pone.0233640.g005 https://doi.org/10.1371/journal.pone.0233640.g005 PLOS ONE | https://doi.org/10.1371/journal.pone.0233640 May 26, 2020 TPA and uPA mediated fibrinolysis The behavior of TXA also depends on the balance between tPA and uPA in the system. In sys- tems depleted of A1AT and A2M, TXA remains anti-fibrinolytic in the tPA pathway, but becomes pro-fibrinolytic in the uPA pathway. By modulating the ratio of tPA to uPA, the over- all effect of TXA can go from pro-fibrinolytic (Fig 6A and 6B), to no effect(Fig 6C) to anti- fibrinolytic(Fig 6D). We can see that across these concentrations of tPA, the curves for the highest TXA concentrations are more-or-less identical. This shows that at high concentrations of TXA, the tPA pathway is completely inhibited. 6 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0233640 May 26, 2020 PLOS ONE Computational model of TXA on uPA mediated fibrinolysis Fig 5. Effects of TXA on uPA mediated fibrinolysis. Modeling results reveal that the effect of TXA on uPA mediated fibrinolysis greatly depends on the presence or absence of plasmin inhibitors in the system. When A2M is in the system, with (A) or without (B) A1AT, fibrinolysis is inhibited and TXA behaves in an anti-fibrinolytic fashion. When A2M is removed from the system, but A1AT is still present (C), fibrinolysis can be observed. Under these circumstances, TXA exhibits anti-fibrinolytic behavior and acts to slow down fibrinolysis in a dose dependent manner. When both A2M and A1AT are removed from the system (D), TXA becomes pro-fibrinolytic, and speeds up the rate of fibrinolysis through increased plasmin generation and antiplasmin depletion. These simulations are initiated with 5 pM tissue factor, and an initial uPA concentration of 5 nM. https://doi.org/10.1371/journal.pone.0233640.g005 Fig 5. Effects of TXA on uPA mediated fibrinolysis. Modeling results reveal that the effect of TXA on uPA mediated fibrinolysis greatly depends on the presence or absence of plasmin inhibitors in the system. When A2M is in the system, with (A) or without (B) A1AT, fibrinolysis is inhibited and TXA behaves in an anti-fibrinolytic fashion. When A2M is removed from the system, but A1AT is still present (C), fibrinolysis can be observed. Under these circumstances, TXA exhibits anti-fibrinolytic behavior and acts to slow down fibrinolysis in a dose dependent manner. When both A2M and A1AT are removed from the system (D), TXA becomes pro-fibrinolytic, and speeds up the rate of fibrinolysis through increased plasmin generation and antiplasmin depletion. These simulations are initiated with 5 pM tissue factor, and an initial uPA concentration of 5 nM. Discussion In this paper we have proposed a mechanistic explanation for the variance of outcomes in patients treated with TXA. In situations with elevated uPA and depletion of plasmin inhibitors such as A1AT and A2M, TXA can speed up fibrinolysis, leading to increased bleeding. With- out A1AT and A2M, antiplasmin is quickly depleted and the generated plasmin rapidly breaks down any fibrin clots uninhibited. One possible mechanism for depletion of plasmin inhibi- tors during trauma may arise from the interactions between A1AT and A2M with activated Protein C (APC) [23]. Many studies have shown that high levels of protein C are correlated with trauma patients with worse outcomes [24, 25]. This is more likely to occur in patients that arrive late, as there is more time for the plasmin inhibitors to deplete. Furthermore, the likely concurrence of high tPA in the system during trauma can work in tandem to push a system 7 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0233640 May 26, 2020 PLOS ONE Computational model of TXA on uPA mediated fibrinolysis Fig 6. Effects of TXA on systems with both tPA and uPA. Modeling results show that the effect of TXA on systems with both tPA and uPA depend on the balance between the two pathways. The concentration of uPA is held constant at 5nM, as tPA is increased from 0.1nM to 2.5nM. When tPA is low compared tp uPA (A and B), TXA exhibits the pro-fibrinolytic behavior similar to Fig 5D. This behavior shifts toward anti-fibrinolytic as tPA increases. At 1nM tPA (C), the hyperfibrinolysis occurs at similar times regardless of the concentration of TXA. At 2.5nM tPA (D), TXA is anti-fibrinolytic and acts to slow down hyperfibrinolysis. These simulations are initiated with 5 pM tissue factor. https://doi.org/10.1371/journal.pone.0233640.g006 Fig 6. Effects of TXA on systems with both tPA and uPA. Modeling results show that the effect of TXA on systems with both tPA and uPA depend on the balance between the two pathways. The concentration of uPA is held constant at 5nM, as tPA is increased from 0.1nM to 2.5nM. When tPA is low compared tp uPA (A and B), TXA exhibits the pro-fibrinolytic behavior similar to Fig 5D. This behavior shifts toward anti-fibrinolytic as tPA increases. At 1nM tPA (C), the hyperfibrinolysis occurs at similar times regardless of the concentration of TXA. At 2.5nM tPA (D), TXA is anti-fibrinolytic and acts to slow down hyperfibrinolysis. PLOS ONE | https://doi.org/10.1371/journal.pone.0233640 May 26, 2020 Discussion These simulations are initiated with 5 pM tissue factor. https://doi.org/10.1371/journal.pone.0233640.g006 https://doi.org/10.1371/journal.pone.0233640.g006 toward hyperfibrinolysis [26]. Under these conditions, TXA increases the amount of plasmin produced by uPA in solution that rapidly consumes antiplasmin. Once antiplasmin has been reduced significantly, TXA loses its ability to prevent tPA-mediated fibrinolysis and any plas- min generated on fibrin surfaces will go uninhibited as A2M and A1AT cannot bind to fibrin- bound plasmin. This model also provides an explanation for empirical studies that show how TXA can increase plasmin generation in uPA systems, but at the same time slow down fibrinolysis. This model shows that TXA binding to plasmin alone cannot prevent antiplasmin from depleting, because the binding between TXA and plasmin is reversible. Regardless of the amount of TXA in the system, free plasmin will always be present, but higher concentrations of TXA decreases the amount of free plasmin available. This free plasmin will be consumed by plasmin inhibitors and shifts the equilibrium to generate more free plasmin. In healthy plasma, there is an ample 8 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0233640 May 26, 2020 PLOS ONE Computational model of TXA on uPA mediated fibrinolysis supply of plasmin inhibitors, and TXA serves to lower the amount of free plasmin at any given time, leading to quick inhibition. However, if A2M and A1AT are depleted, TXA can act to speed up the rate of antiplasmin depletion because the concentration of plasminogen is approximately twice the concentration of antiplasmin. This leaves the remaining free plasmin free to break down fibrin unimpeded. Because of this, the presence of additional irreversible inhibitors is necessary in a functional fibrinolytic system. Even in the absence of TXA, these additional inhibitors are necessary in healthy fibrinolysis. During an injury, tPA deposits in endothelial cells are quickly released in large amounts into the site of injury. During the time immediately after injury, plasmin activators greatly outnum- ber its inhibitors such as PAI-1 and can quickly activate large amounts of plasmin [18]. With- out additional inhibitors, antiplasmin would quickly deplete, resulting in hyperfibrinolysis. As TXA continues to be explored as treatment for different situations [27–29], a mechanis- tic understanding of this phenomena becomes increasingly important in preventing unfore- seen side effects of TXA treatment. Currently, the decision on whether to use TXA in treatment depends on whether the patient arrived before or after 3 hours post injury. Discussion Although this heuristic protocol can be helpful, it will undoubtedly lead to instances in which patients who can benefit from TXA treatment are not receiving it and patients who would suffer adversely from it will be given it. With an understanding of the conditions under which TXA helps or worsens a patient’s condition, we can identify patients that should receive TXA and ones that shouldn’t. In addition, TXA treatment can potentially be improved by using plasmin inhibitors in conjection with TXA to mitigate the risk of hyperfibrinolysis through the deple- tion of plasmin inhibitors. Conclusion This paper uses computational modeling to present a mechanistic explanation for the different behaviors of tranexamic acid. TXA usually has anti-fibrinolytic properties, but in systems with high levels of uPA and low levels of plasmin inhibitors, TXA can become pro-fibrinolytic, resulting in worse outcomes. This understanding can potentially be used to adjust treatment protocols and stratify patients according to TXA risk to ensure that treatment is beneficial to patients receiving it. As we have done with previous models [18], this model can also be used to test potential treatments such as the replenishment of key proteins such as α2-antiplasmin. Author Contributions Conceptualization: Thomas Orfeo, Hunter B. Moore, Joshua J. Sumislawski, Mitchell J. Cohen. References 1. Cohen MJ. Acute traumatic coagulopathy: Clinical characterization and mechanistic investigation. Thrombosis Research. 2014; 133(SUPPL. 1):S25–S27. https://doi.org/10.1016/j.thromres.2014.03.013 PMID: 24759135 2. Godier A, Susen S. Trauma-induced coagulopathy. 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Effects of tranexamic acid on death, vascular occlusive events, and blood transfusion in trauma patients with significant haemorrhage (CRASH-2): a randomised, placebo-controlled trial. The Lancet. 2010; 376(9734):23–32. https://doi. org/10.1016/S0140-6736(10)60835-5 7. P L M. Tranexamic Acid: A review of its use in the treatment of hyperfibrinolysis. Drugs. 2012; 72 (5):585–617. https://doi.org/10.2165/11209070-000000000-00000 8. Takada A, Takada Y. Conversion of glu-plasminogen to plasmin by urokinase in the presence of tra- nexamic acid. Thrombosis Research. 1981; 22(4):437–443. https://doi.org/10.1016/0049-3848(81) 90105-5 PMID: 6457415 9. Hijazi N, Abu Fanne R, Abramovitch R, Yarovoi S, Higazi M, Abdeen S, et al. Endogenous plasminogen activators mediate progressive intracerebral hemorrhage after traumatic brain injury in mice. Blood. 2015; 125(16):2558–2567. https://doi.org/10.1182/blood-2014-08-588442 PMID: 25673638 10. Saes JL, Schols SEM, van Heerde WL, Nijziel MR. Hemorrhagic disorders of fibrinolysis: a clinical review. Journal of Thrombosis and Haemostasis. 2018; 16(8):1498–1509. https://doi.org/10.1111/jth. 14160 11. Ellis V, Behrendt N, Dano K. Plasminogen activation by receptor-bound urokinase: A kinetic study with both cell-associated and isolated receptor. Journal of Biological Chemistry. 1991; 266(19):12752– 12758. PMID: 1829461 12. Hoylaerts M, Lijnen HR, Collen D. Studies on the mechanism of the antifibrinolytic action of tranexamic acid. BBA—General Subjects. 1981; 673(C):75–85. https://doi.org/10.1016/0304-4165(81)90312-3 PMID: 7193484 13. Longstaff C, Locke M. Increased urokinase and consumption of α2-antiplasmin as an explanation for the loss of benefit of tranexamic acid after treatment delay. Journal of Thrombosis and Haemostasis. 2018; p. jth.14338. 14. Hockin MF, Jones KC, Everse SJ, Mann KG. A model for the stoichiometric regulation of blood coagula- tion. J Biol Chem. 2002; 277(21):18322–18333. Formal analysis: Tie Bo Wu. Formal analysis: Tie Bo Wu. Investigation: Hunter B. Moore, Joshua J. Sumislawski. Methodology: Tie Bo Wu. Methodology: Tie Bo Wu. Project administration: Mitchell J. Cohen, Linda R. Petzold. Project administration: Mitchell J. Cohen, Linda R. Petzold. Resources: Thomas Orfeo, Hunter B. Moore. Resources: Thomas Orfeo, Hunter B. Moore. Supervision: Mitchell J. Cohen, Linda R. Petzold. 9 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0233640 May 26, 2020 PLOS ONE Computational model of TXA on uPA mediated fibrinolysis Visualization: Tie Bo Wu. Writing – original draft: Tie Bo Wu. Writing – review & editing: Tie Bo Wu, Thomas Orfeo, Linda R. Petzold. Writing – review & editing: Tie Bo Wu, Thomas Orfeo, Linda R. Petzold. References https://doi.org/10.1074/jbc.M201173200 PMID: 11893748 15. Brummel-Ziedins KE, Orfeo T, Callas PW, Gissel M, Mann KG, Bovill EG. The Prothrombotic Pheno- types in Familial Protein C Deficiency Are Differentiated by Computational Modeling of Thrombin Gen- eration. PLoS ONE. 2012; 7(9):e44378. https://doi.org/10.1371/journal.pone.0044378 PMID: 22984498 16. Mitrophanov AY, Wolberg AS, Reifman J. Kinetic model facilitates analysis of fibrin generation and its modulation by clotting factors: implications for hemostasis-enhancing therapies. Mol BioSyst. 2014; 10 (9):2347–2357. https://doi.org/10.1039/c4mb00263f PMID: 24958246 10 / 11 PLOS ONE | https://doi.org/10.1371/journal.pone.0233640 May 26, 2020 PLOS ONE Computational model of TXA on uPA mediated fibrinolysis 17. Wootton DM, Popel AS, Rita Alevriadou B. An experimental and theoretical study on the dissolution of mural fibrin clots by tissue-type plasminogen activator. Biotechnology and Bioengineering. 2002; 77 (4):405–419. https://doi.org/10.1002/bit.10127 PMID: 11787013 18. Wu TB, Wu S, Buoni M, Orfeo T, Brummel-Ziedins K, Cohen M, et al. Computational Model for Hyperfi- brinolytic Onset of Acute Traumatic Coagulopathy. Annals of Biomedical Engineering. 2018; 46 (8):1173–1182. https://doi.org/10.1007/s10439-018-2031-9 PMID: 29675813 19. SILVA MMCG, THELWELL C, WILLIAMS SC, LONGSTAFF C. Regulation of fibrinolysis by C-terminal lysines operates through plasminogen and plasmin but not tissue-type plasminogen activator. Journal of Thrombosis and Haemostasis. 2012; 10(11):2354–2360. https://doi.org/10.1111/j.1538-7836.2012. 04925.x PMID: 22974122 20. Stump DC, Thienpont M, Collen D. Purification and characterization of a novel inhibitor of urokinase from human urine. Quantitation and preliminary characterization in plasma. The Journal of biological chemistry. 1986; 261(27):12759–66. PMID: 3091604 21. Heeb MJ, España F, Geiger M, Collen D, Stump DC, Griffin JH. Immunological identity of heparin- dependent plasma and urinary protein C inhibitor and plasminogen activator inhibitor-3. The Journal of biological chemistry. 1987; 262(33):15813–6. PMID: 2824468 22. Kolev K, Le´ra´nt I, Tenekejiev K, Machovich R. Regulation of fibrinolytic activity of neutrophil leukocyte elastase, plasmin, and miniplasmin by plasma protease inhibitors. The Journal of biological chemistry. 1994; 269(25):17030–4. PMID: 7516329 23. Martos L, Ramo´n LA, Oto J, Ferna´ndez-Pardo A´ , Bonanad S, Cid AR, et al. α 2 -Macroglobulin Is a Sig- nificant in Vivo Inhibitor of Activated Protein C and Low APC:α 2 M Levels Are Associated with Venous Thromboembolism. Thrombosis and Haemostasis. 2018; 118(4):630–638. https://doi.org/10.1055/s- 0038-1629902 PMID: 29448296 24. Brohi K, Cohen MJ, Ganter MT, Schultz MJ, Levi M, Mackersie RC, et al. Acute coagulopathy of trauma: hypoperfusion induces systemic anticoagulation and hyperfibrinolysis. The Journal of trauma. 2008; 64 (5):1211–7; discussion 1217. https://doi.org/10.1097/TA.0b013e318169cd3c PMID: 18469643 25. PLOS ONE | https://doi.org/10.1371/journal.pone.0233640 May 26, 2020 References Howard BM, Kornblith LZ, Cheung CK, Kutcher ME, Miyazawa BY, Vilardi RF, et al. Inducing Acute Traumatic Coagulopathy In Vitro: The Effects of Activated Protein C on Healthy Human Whole Blood. PLOS ONE. 2016; 11(3):e0150930. https://doi.org/10.1371/journal.pone.0150930 PMID: 27008408 26. Chapman MP, Moore EE, Moore HB, Gonzalez E, Gamboni F, Chandler JG, et al. Overwhelming tPA release, not PAI-1 degradation, is responsible for hyperfibrinolysis in severely injured trauma patients. The journal of trauma and acute care surgery. 2016; 80(1):15–16. 27. Shakur H, Elbourne D, Gu¨lmezoglu M, Alfirevic Z, Ronsmans C, Allen E, et al. The WOMAN Trial (World Maternal Antifibrinolytic Trial): tranexamic acid for the treatment of postpartum haemorrhage: an international randomised, double blind placebo controlled trial. Trials. 2010; 11(1):40. https://doi.org/10. 1186/1745-6215-11-40 PMID: 20398351 28. The Crash-3 Trial CollaboratorsEffects of tranexamic acid on death, disability, vascular occlusive events and other morbidities in patients with acute traumatic brain injury (CRASH-3): a randomised, pla- cebo-controlled trial. The Lancet. 2019; 394(10210):1713–1723. 29. Ahmadzia HK, Phillips JM, Katler QS, James AH. Tranexamic Acid for Prevention and Treatment of Postpartum Hemorrhage. Obstetrical & Gynecological Survey. 2018; 73(10):587–594. 11 / 11
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The impact and challenges of digital marketing in the health care industry during the digital era and the COVID-19 pandemic
Frontiers in public health
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Introduction Pasaribu SB, Novitasari D, Goestjahjanti FS and Hendratono T (2022) The impact and challenges of digital marketing in the health care industry during the digital era and the COVID-19 pandemic. According to Arni and Laddha (1), the healthcare industry was under great pressure due to the emergence of COVID-19. The COVID-19 pandemic has had devastating effects on the global economy, industries, and organizations, affecting marketing and spending strategies. Due to the total lockdown in various regions, digital marketing is crucial because traditional marketing strategies are no longer working. Due to a large number of quarantine patients at home, as well as concerns about the possibility of contracting COVID-19, people have chosen not to visit health care facilities, both clinics and hospitals, resulting in a decrease in the volume and income from prescribing drugs from 2021 until now. Khan and Nawaz (2) stated that digital marketing strategies have increased over the years, and spending on such plans has also increased. The COVID-19 pandemic accelerates the growth rate of digital marketing in the health industry and is expected to increase the return of both direct visits and patient telemedicine to hospitals so that hospital revenues also increase. Front. Public Health 10:969523. doi: 10.3389/fpubh.2022.969523 COPYRIGHT © 2022 Pasaribu, Novitasari, Goestjahjanti and Hendratono. 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. The development of the Internet, the World Wide Web, and digital technologies such as technology platforms from desktops, laptops, smartphones, to tablet devices used by consumers has changed marketing. Patients, who are convinced that they are consumers of health services and products, are increasingly using the internet or other digital technologies to find the right information, as well as choose and buy those goods or services. For organizations like hospitals, especially during the COVID-19 pandemic conditions, digital media and new technology platforms provide opportunities to expand into new markets, offer new services, apply new online communication techniques, and compete on a more equal footing with larger businesses. According to Arni and Laddha (1) and Al-Weshah et al. TYPE Opinion PUBLISHED 28 July 2022 DOI 10.3389/fpubh.2022.969523 TYPE Opinion PUBLISHED 28 July 2022 DOI 10.3389/fpubh.2022.969523 The impact and challenges of digital marketing in the health care industry during the digital era and the COVID-19 pandemic OPEN ACCESS EDITED BY Mochammad Fahlevi, Binus University, Indonesia REVIEWED BY Simon Grima, University of Malta, Malta Magdalena Syrkiewicz-Switala, Medical University of Silesia, Poland *CORRESPONDENCE Sahala Benny Pasaribu bennypasaribu@trilogi.ac.id SPECIALTY SECTION This article was submitted to Health Economics, a section of the journal Frontiers in Public Health RECEIVED 15 June 2022 ACCEPTED 07 July 2022 PUBLISHED 28 July 2022 CITATION Pasaribu SB, Novitasari D, Goestjahjanti FS and Hendratono T (2022) The impact and challenges of digital marketing in the health care industry during the digital era and the COVID-19 pandemic. Front Public Health 10:969523 OPEN ACCESS EDITED BY Mochammad Fahlevi, Binus University, Indonesia REVIEWED BY Simon Grima, University of Malta, Malta Magdalena Syrkiewicz-Switala, Medical University of Silesia, Poland *CORRESPONDENCE Sahala Benny Pasaribu bennypasaribu@trilogi.ac.id SPECIALTY SECTION This article was submitted to Health Economics, a section of the journal Frontiers in Public Health RECEIVED 15 June 2022 Sahala Benny Pasaribu1*, Dewiana Novitasari2, Francisca Sestri Goestjahjanti2 and Tonny Hendratono3 1Faculty of Economics and Business, Trilogi University, South Jakarta, Indonesia, 2Economics and Management Department, Sekolah Tinggi Ilmu Ekonomi Insan Pembangunan, Tangerang, Indonesia, 3Tourism Department, Sekolah Tinggi Pariwisata Ambarrukmo, Yogjakarta, Indonesia digital marketing, health care industries, digital era, COVID-19, pandemic, hospital frontiersin.org Introduction (3), most consumers searched for information online about treatment options or more generally, to learn about health problems or health care providers. Digital marketing can simply be defined as achieving marketing goals through the application of technology and digital media. Hospital use of digital technology increased by 50% to reach healthcare consumers. According to de Ruyter et al. (4) and Khan and Nawaz (2), 48% of healthcare provider executives see revenue growth increased by 50% to reach healthcare consumers. According to de Ruyter et al. (4) and Khan and Nawaz (2), 48% of healthcare provider executives see revenue growth Frontiers in Public Health Frontiers in Public Health 01 frontiersin.org 10.3389/fpubh.2022.969523 10.3389/fpubh.2022.969523 Pasaribu et al. as a key benefit of digital investment. Today, consumers are looking for a stress-free and trustworthy user experience. With the advancement of technology, the demand for innovative healthcare applications has increased. Therefore, the implementation of health marketing strategies on digital platforms will enable the health sector to grow. for other people in the future. Many people go to hospitals or doctor websites for reviews, and use Google reviews to examine and share medical experiences. In addition, a large number of people use social media platforms to share their experiences. In previous research, we can see in Figure 1 that the opinion framework shows a shift in digital marketing in the current health industry. Content marketing has a positive impact on factors such as customer engagement, trust, and loyalty. In comparison, content marketing was found to be more effective at persuading customer loyalty compared to trust and loyalty. Furthermore, customer trust in a brand has a strong positive relationship with loyalty. A positive effect of customer engagement on trust has also been found. Digital marketing increases patient satisfaction, loyalty, and engagement with hospital services. According to Savitri et al. (7), Tancharoenwong (8), and Qian et al. (9), one of the benefits of digital marketing is that it can be closer to consumers. Hospitals should adopt a multi-channel content marketing approach to take full advantage. Discussion and opinion Digital methods can promote medical services in order to expand the business. The strategic way of thinking, in this case, implies attracting new patients and offering them quality health services, which ensures satisfaction and the possibility for them to recommend further health facilities. This is consistent with the data from Mishra that digital marketing increases satisfaction, loyalty, and patient engagement with hospital services. According to Wijaya et al. (5) and Wisetsri et al. (6), one of the benefits of digital marketing is to expand the brand/business online. The results of a study in Bangladesh stated that in order to collect information about doctors, people rely on hospital websites because it is the most accurate and up- to-date source of information, and collect information posted on social media such as Facebook groups. Before deciding to visit a health service, people research health care professionals and share reviews about their experiences so that they can be useful According to Nunan and Di Domenico (10), Purcarea (11), Purwanto (12), and Pourkarim et al. (13), digital marketing effects and barriers are something to manage during COVID-19. A digital marketing strategy is adopted to manage work online and persist with safety precautions. Technology brings both negative and positive impacts to the society. However, during the pandemic, digital marketing tools and management are still FIGURE 1 Opinion framework. 02 Frontiers in Public Health frontiersin.org Pasaribu et al. 10.3389/fpubh.2022.969523 effective and helpful in moving normal life into the new norm. Digital marketing has gained a lot of advantages in terms of online businesses, small, or large entrepreneurs, excelling in the advertising market well. and promoting the services to patients’ relatives and family. The selection of methods or channels for digital marketing depends on the goals or targets to be achieved by the hospital. After determining the goals of digital marketing, the hospital determines the choice of methods or digital media channels that will be used, then determines the goals, targets, and objectives to be achieved from each of the selected media, analyzes the situation and audience/market share, and determines the budget and marketing frequency. Furthermore, for its implementation, it is necessary to determine who will carry out the marketing strategy that has been chosen. Moreover, monitoring and evaluation of each digital marketing strategy that has been implemented must be carried out so that the hospital can achieve its goals or targets effectively and efficiently. Author contributions All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Discussion and opinion According to Savitri et al. (7), Tancharoenwong (8), Qian et al. (9), Wijayaa et al. (5), and Wisetsri et al. (6), several challenges and barriers to the use of social media in hospital digital marketing, which make health services lag behind the industry in digital marketing, include security issues, patient privacy, regulatory issues, lack of guidance on how to use digital platforms properly, lack of staffinterest to use social media or the right infrastructure to respond to complaints, and unclear responsibilities for various internet marketing activities. As a result, management commitment and consistency are needed because the use of digital technology requires adequate resources in terms of finance, infrastructure, and manpower to be more effective. Electronic communications should be conducted through an experimental approach rather than a planned approach to avoid poor integration of online and offline marketing communications. Conflict of interest Digital technology marketing has a tremendous impact, including increasing engagement on social media and marketing, serving as a key performance indicator for analyzing organizational values, increasing demand for digital, growing product searches among users, and increasing demand for content platforms. All of them have been demonstrated in many companies and organizations. During the COVID-19 pandemic, digital marketing has hit its growth charts and has made technological advances all over the world. Digital marketing in hospitals during this pandemic is a marketing strategy that has many benefits, including attracting new patients, expanding business, increasing customer/patient trust, strengthening customer/patient loyalty, increasing brand awareness, encouraging patients to use hospital services, 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. 6. Wisetsri W, Soni N, Singh RK, Chaurasia PK, Gupta SK. The healthcare sector: a development of digital marketing methods. Linguist Antverpiensia. (2021) 3:2602–21. 7. Savitri C, Hurriyati R, Wibowo L, Hendrayati H. The role of social media marketing and brand image on smartphone purchase intention. Int J Data Network Sci. (2022) 6:185–92. doi: 10.5267/j.ijdns.2021. 9.009 8. Tancharoenwong AS. Facebook as a digital marketing tool: Facebook’s engagement strategy for maternal and child health page. UTCC Int J Bus Econ. (2018) 10:3–17. interaction and word-of-mouth on purchase intention: an empirical study from Indonesian smartphone consumers. Int J Data Network Sci. (2021) 5:231– 8. doi: 10.5267/j.ijdns.2021.6.011 interaction and word-of-mouth on purchase intention: an empirical study from Indonesian smartphone consumers. Int J Data Network Sci. (2021) 5:231– 8. doi: 10.5267/j.ijdns.2021.6.011 2. Khan RZ, Nawaz H. Impact and challenges of digital marketing during Covid-19 pandemic. Gorteria J. (2021) 34:31–9. 11. Purcarea EVL. The impact of marketing strategies in healthcare systems. J Med Life. (2019) 12:93–9. 9. Qian ZW, Wan GS, Du XL, Shi YF, Huang G. Performance evaluation of digital marketing in health care industry with the application of data envelopment analysis. Revista De Cercetare Si Interventie Sociala. (2018) 60:39–50. 10. Nunan D, Di Domenico M. Older consumers, digital marketing, and public policy: a review and research agenda. J Public Policy Mark. (2019) 38:469– 83. doi: 10.1177/0743915619858939 Pasaribu et al. 13. Pourkarim M, Nayebzadeh S, Alavian SM, Hataminasab SH. Digital Marketing: a unique multidisciplinary approach towards the elimination of viral hepatitis. Pathogens. (2022) 11:626. doi: 10.3390/pathogens 11060626 9. Qian ZW, Wan GS, Du XL, Shi YF, Huang G. Performance evaluation of digital marketing in health care industry with the application of data envelopment analysis. Revista De Cercetare Si Interventie Sociala. (2018) 60:39–50. 10. Nunan D, Di Domenico M. Older consumers, digital marketing, and public policy: a review and research agenda. J Public Policy Mark. (2019) 38:469– 83. doi: 10.1177/0743915619858939 11. Purcarea EVL. The impact of marketing strategies in healthcare systems. J Med Life. (2019) 12:93–9. 10.3389/fpubh.2022.969523 References 1. Arni P, Laddha S. Adoption of digital marketing in health industry. SIES J Manage. (2017) 13:3–10. 1. Arni P, Laddha S. Adoption of digital marketing in health industry. SIES J Manage. (2017) 13:3–10. interaction and word-of-mouth on purchase intention: an empirical study from Indonesian smartphone consumers. Int J Data Network Sci. (2021) 5:231– 8. doi: 10.5267/j.ijdns.2021.6.011 2. Khan RZ, Nawaz H. Impact and challenges of digital marketing during Covid-19 pandemic. Gorteria J. (2021) 34:31–9. 3. Al-Weshah GA, Kakeesh DF, Al-Ma’aitah NA. Digital marketing strategies and international patients’ satisfaction: an empirical study in Jordanian health service industry. Stud Appl. Econ. (2021) 39:90–99 doi: 10.25115/eea. v39i7.4811 4. de Ruyter K, Keeling DI, Ngo LV. When nothing is what it seems: a digital marketing research agenda. Aust Mark J. (2018) 26:199–203. doi: 10.1016/j.ausmj.2018.07.003 4. de Ruyter K, Keeling DI, Ngo LV. When nothing is what it seems: a digital marketing research agenda. Aust Mark J. (2018) 26:199–203. doi: 10.1016/j.ausmj.2018.07.003 8. Tancharoenwong AS. Facebook as a digital marketing tool: Facebook’s engagement strategy for maternal and child health page. UTCC Int J Bus Econ. (2018) 10:3–17. 5. Wijayaa O, Sulistiyanib S, Pudjowatic J, Kurniasih N, Purwanto A. The role of social media marketing, entertainment, customization, trendiness, Frontiers in Public Health frontiersin.org 03 Pasaribu et al. 10.3389/fpubh.2022.969523 12. Purwanto A. How the role of digital marketing and brand image on food product purchase decisions? An empirical study on Indonesian SMEs in the digital era. J Indus Eng Manage Res. (2022) 3: 34–41. 13. Pourkarim M, Nayebzadeh S, Alavian SM, Hataminasab SH. Digital Marketing: a unique multidisciplinary approach towards the elimination of viral hepatitis. Pathogens. (2022) 11:626. doi: 10.3390/pathogens 11060626 Frontiers in Public Health 04 Frontiers in Public Health frontiersin.org
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Assessment of Salivary Gland Dysfunction after Radioiodine Therapy for Thyroid Carcinoma Using Non-Contrast-Enhanced CT: The Significance of Changes in Volume and Attenuation of the Glands
American journal of neuroradiology
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ORIGINAL RESEARCH B. Nabaa K. Takahashi T. Sasaki A. Okizaki T. Aburano B. Nabaa K. Takahashi T. Sasaki A. Okizaki T. Aburano BACKGROUND AND PURPOSE: Although radiation induced damage to the salivary gland is a known complication of radioactive iodine (131I) therapy for thyroid carcinoma, prediction of the severity and reversibility of sialoadenitis is difficult. Our aim was to correlate the extent of salivary dysfunction assessed by salivary gland scintigraphy with changes in the volume and attenuation of salivary glands on nonenhanced CT in postoperative patients with thyroid cancer treated with RIT. MATERIALS AND METHODS: Forty patients with thyroid carcinoma, 13 men (age range, 21–80 years) and 27 women (age range, 28–75 years) who underwent a total thyroidectomy and were treated with RIT were assessed retrospectively. On CT, the percentage of volume reduction and the difference in attenuation of the parotid and submandibular glands after RIT were determined and correlated with the extent of radiation-induced salivary dysfunction on scintigraphy. RESULTS: The salivary gland volume significantly decreased with an increase in the dysfunction grade on scintigraphy for both the parotid and submandibular glands (P  .001). The attenuation significantly increased with an increase in the dysfunction grade on scintigraphy for the parotid gland (P  .001), but not for the submandibular gland. The cutoff value of volume reduction to diagnose severe gland dysfunction was 19.5% (sensitivity, 86.0%; specificity, 100%) for the parotid gland and 31.0% (sensitivity, 100%; specificity, 97.0%) for the submandibular gland, and that of the attenuation change was 9.8 HU (sensitivity, 81.0%; specificity, 95%) for the parotid gland. CONCLUSIONS: The reduction in volume of the parotid and submandibular glands and the increase in attenuation of the parotid gland on nonenhanced CT can be indicators of the grade of RIT-induced salivary dysfunction. ABBREVIATIONS: HU  hounsfield unit; RIT  radioiodine therapy; ROC  receiver operating characteristic analysis ABBREVIATIONS: HU  hounsfield unit; RIT  radioiodine therapy; ROC  receiver operating characteristic analysis obtained during the follow-up period after RIT. These changes are likely due to radiation-induced sialoadenitis and could be a predictor of salivary gland dysfunction. A few studies have reported dose-dependent volume loss of the salivary gland in patients undergoing external radiation for head and neck cancers,10-13 but no previous studies have as- sessed the effect of RIT on gland volume or attenuation, to our knowledge. Received October 26, 2011; accepted after revision January 24, 2012. From the Department of Radiology, Asahikawa Medical University, Asahikawa, Japan. Please address correspondence to Koji Takahashi, MD, PhD, Department of Radiology, Asahikawa Medical University, 2-1-1-1, Midorigaoka-higashi, Asahikawa, Japan 078-8510; e-mail: taka1019@asahikawa-med.ac.jp Indicates article with supplemental on-line figures. http://dx.doi.org/10.3174/ajnr.A3063 Aburano B. Nabaa, K. Takahashi, T. Sasaki, A. Okizaki and T. Aburano B. Nabaa, K. Takahashi, T. Sasaki, A. Okizaki and T. of October 23, 2024. This information is current as of October 23, 2024. This information is current as http://www.ajnr.org/content/33/10/1964 https://doi.org/10.3174/ajnr.A3063 doi: 2012, 33 (10) 1964-1970 AJNR Am J Neuroradiol http://www.ajnr.org/content/33/10/1964 https://doi.org/10.3174/ajnr.A3063 doi: 2012, 33 (10) 1964-1970 AJNR Am J Neuroradiol ORIGINAL RESEARCH T he use of RIT for ablation of residual thyroid tissue after surgery has been established for the management of differ- entiated thyroid cancer.1 Due to simultaneous accumulation of 131I in the salivary glands, radiation damage to the salivary gland is a known immediate and long-term complication of RIT.2-4 Patients with RIT-induced salivary dysfunction have a variety of symptoms, including swelling and pain in the pa- rotid region, dryness of the mouth, altered taste, and difficulty swallowing. With RIT, however, calculation of the actual ab- sorbed dose in the salivary glands and prediction of the sever- ity and reversibility of associated sialoadenitis are difficult. Generally, qualitative and quantitative salivary gland scintig- raphy has been used for the assessment and follow-up of salivary gland dysfunction after RIT.5-9 T In this study, we examine the relationship between the ex- tent of salivary gland dysfunction evaluated on scintigraphy and changes in volume and attenuation of the salivary glands on CT in postoperative patients with thyroid carcinoma treated with therapeutic doses of radioiodine. CT of the neck and chest is usually performed in patients who have undergone RIT for evaluation of the recurrence and metastasis of thyroid carcinoma. We have noticed that the salivary glands, especially the parotid glands, show some shrinkage and an increase in attenuation on CT scans ORIGINAL RESEARCH ORIGINAL RESEARCH All CT studies were assessed by 2 radiologists (T.S. and B.N., with 9 and 3 years’ experience in head and neck imaging, respec- tively), who were blinded to the results of scintigraphy. The volume and attenuation of the bilateral parotid and submandibular glands were retrospectively measured in each patient on both CT images obtained before the initial RIT and after the last RIT. The volume of the salivary gland was measured by summation of products of an area of the salivary gland on each section and section thickness. The area of the salivary gland was obtained by a manually contoured region-of- interest measurement. The percentage of volume reduction of each gland after treatment was calculated with the following formula: (Pretreatment Volume  Posttreatment Volume)/Pretreatment Vol- ume  100. The attenuation of each gland was determined by a re- gion-of-interest measurement (region-of-interest size, 40–60 mm2) on CT scans obtained before and after the RIT, and their difference (Posttreatment Attenuation  Pretreatment Attenuation) was de- fined as an attenuation change of the salivary gland. The mean of the above-mentioned percentage of volume reduction and attenua- tion change of the bilateral parotid and submandibular glands was used as a CT index for each salivary gland. Patients The protocol for this retrospective study was approved by our insti- tutional review board without requirement of informed consent. The study subjects were 40 patients: Thirteen were men (age range, 21–80 years; mean age, 61 years) and 27 were women (age range, 28–75 years; mean age, 55 years). All patients had undergone a total thyroid- ectomy for well-differentiated thyroid carcinoma and subsequent RIT from January 2006 to January 2011. We divided the patients into 4 groups according to the number of doses of RIT: 20, 10, 4, and 6 patients received 1, 2, 3, and 4 doses, respectively. The individual 1964 Nabaa  AJNR 33  Nov 2012  www.ajnr.org 1964 Nabaa  AJNR 33  Nov 2012  www.ajnr.org 1964 Table 2: The correlation of the grade of submandibular gland dysfunction on scintigraphy with the number of radioiodine treatments No. of Radioiodine Treatments Grade of Salivary Dysfunction on Scintigraphy 1 2 3 4 Total Pretreatment 31 9 0 0 40 1 11 5 4 0 20 2 0 4 4 2 10 3 0 1 2 1 4 4 0 2 2 2 6 Table 1: The correlation of the grade of parotid gland dysfunction on scintigraphy and the number of radioiodine treatments No. of Radioiodine Treatments Grade of Salivary Dysfunction on Scintigraphy 1 2 3 4 Total Pretreatment 32 8 0 0 40 1 8 3 5 4 20 2 0 1 1 8 10 3 0 0 0 4 4 4 1 0 0 5 6 doses ranged from 5.1 to 5.6 GBq (139–150 mCi), with a mean of 5.3 GBq (144.5 mCi). None of these patients had known salivary diseases, including Sjo¨gren disease, chronic sialolithiasis, sialadenitis, or sialosis. Table 1: The correlation of the grade of parotid gland dysfunction on scintigraphy and the number of radioiodine treatments No. of Radioiodine Treatments Grade of Salivary Dysfunction on Scintigraphy 1 2 3 4 Total Pretreatment 32 8 0 0 40 1 8 3 5 4 20 2 0 1 1 8 10 3 0 0 0 4 4 4 1 0 0 5 6 Imaging Examinations Salivary gland scintigraphy and non-contrast-enhanced neck CT were performed before the initial RIT in all patients. The average time interval from pretreatment salivary gland scintigraphy and CT to the initial RIT was 8 days (range, 1–15 days). Changes in the salivary glands after RIT were evaluated on scintigraphy and CT scans ob- tained on the same day, with an average interval of 9 months (range, 6–12 months) since the last therapy and of 17 months (range, 8–38 months) since the first dose of RIT. Table 2: The correlation of the grade of submandibular gland dysfunction on scintigraphy with the number of radioiodine treatments Scintigraphy Studies. Salivary gland function was estimated by salivary gland scintigraphy by using 185-MBq (5 mCi) technetium Tc99m pertechnetate. All salivary gland scintigraphic data were as- sessed by a radiologist (A.O., with 15 years’ experience in nuclear medicine). Depending on the salivary gland uptake and excretion, its function was classified into the following 4 grades: grade 1, normal uptake and excretion; grade 2, mild dysfunction, decreased salivary uptake, and delayed excretion, with oral activity equal to salivary up- take at 30–40 minutes; grade 3, moderate dysfunction, markedly de- creased salivary gland uptake, and delayed excretion, with higher sal- ivary gland activity than oral activity at 30–40 minutes; and grade 4, severe dysfunction, severely decreased salivary uptake and higher background than salivary activity during the entire study.7,8 To judge the grade of function in the parotid and submandibular glands, we initially determined the grade of each side gland separately. For the final grade, we used the worst grade in cases with different grades of the bilateral glands. No. of Radioiodine Treatments Grade of Salivary Dysfunction on Scintigraphy 1 2 3 4 Total Pretreatment 31 9 0 0 40 1 11 5 4 0 20 2 0 4 4 2 10 3 0 1 2 1 4 4 0 2 2 2 6 AJNR Am J Neuroradiol 33:1964–70  Nov 2012  www.ajnr.org 1965 Results Grade of Dysfunction on Scintigraphy and Its Correlation with the Treatment Dose Statistical Analyses All statistical analyses were performed by using a commercially avail- able software program (Statistical Package for the Social Sciences, Version 15.0 for Windows; SPSS, Chicago, Illinois). The correlation between the grade of dysfunction of the gland on scintigraphy and the number of RIT sessions was assessed by using the Spearman rank correlation test. The correlations of the volume reduction and atten- uation change of the salivary glands with the dysfunction grade of the salivary gland on scintigraphy were assessed by using a 1-way analysis of variance. Multiple comparisons of volume reduction and attenua- tion changes of the group with each dysfunction grade were assessed by using the Tukey Honestly Significant Difference test. The compar- isons of the incidences of each grade of dysfunction after RIT and those of the mean values of pretreatment attenuation and attenuation change after RIT between the parotid and submandibular glands were assessed by using the 2 test and a paired t test, respectively. We cal- culated a correlation coefficient to assess the correlation between 2 datasets. A P value  .05 was considered statistically significant. The cutoff value of the volume-reduction percentage and attenuation change of the salivary gland to diagnose grade 4 (severe) dysfunction of the gland was determined by an ROC curve. NECK ORIGINAL RESEARCH CT Studies. CT examinations were performed by using a 16- detector row scanner (Aquilion 16; Toshiba Medical Systems, Tokyo, Japan). CT images were obtained with the following parameters: the tube voltage, 120 kV; tube current, 280–400 mA (automatically adjusted for the patient body build); gantry rotation time, 0.5 sec- onds; detector collimation, 1 mm; and a table feed, 12 mm per gantry rotation. Contiguous 3-mm-thick-section axial images were reconstructed. All CT studies were performed without contrast enhancement. Grade of Dysfunction on Scintigraphy and Its Correlation with the Treatment Dose A 71-year-old woman who underwent 3 radioiodine treatments showed severe dysfunction of the parotid gland on scintigraphy. Non-contrast-enhanced CT images (A) before and (B) after the treatment. The bilateral parotid glands (arrows) showed prominent volume reduction of 53% and an increase in attenuation of 32 HU on CT after the treatment. Fig 2. A 71-year-old woman who underwent 3 radioiodine treatments showed severe dysfunction of the parotid gland on scintigraphy. Non-contrast-enhanced CT images (A) before and (B) after the treatment. The bilateral parotid glands (arrows) showed prominent volume reduction of 53% and an increase in attenuation of 32 HU on CT after the treatment. 13.9% (28.7%–41.4%) for grade 4 (n  21) (Figs 1 and 2). The parotid gland volume after the treatment significantly de- creased with an increase in the grade of dysfunction on scin- tigraphy (P  .001, 1-way analysis of variance). A significant difference in the reduction volume was present between grade 1 and grade 4 (P  .001) and grades 2 and 3 and grade 4 (P  .001) groups, but not between grade 1 and grades 2 and 3 groups, as determined by the Tukey Honestly Significant Dif- ference test (Fig 3). of dysfunction in the submandibular glands also increased with an increase in the number of treatments (r  0.636, P  .001, Spearman rank correlation test) (Table 2). When we compared the incidence of each grade of dysfunc- tion after RIT in all patients, the parotid gland showed a sig- nificantly higher grade of dysfunction than the submandibular gland (P  .002, 2 test). Results on CT and Their Correlation with the Grade of Dysfunction on Scintigraphy The reduction volume (mean  SD, 95% confidence inter- val) in the submandibular gland after RIT, depending on the grade of scintigraphy, was 4.0  3.1% (2.0%–6.1%) for grade 1 (n  11); 14.7  9.2% (10.8%–18.5%) for grades 2 and 3 (n  24); and 42.3  11.3% (28.3%–56.3%) for grade 4 (n  5) (Figs 4 and 5). The submandibular gland volume after the treatment significantly decreased with an increase in the grade of dysfunction on scintigraphy (P  .001, 1-way analysis of variance). There was a significant difference in the reduc- tion volume between grade 1 and grades 2 and 3 (P  .001), grades 2 and 3 and grade 4 (P  .001), and grade 1 and grade 4 (P  .001) groups, as determined by the Tukey Honestly Sig- nificant Difference test (Fig 3). Due to the small number of total patients and to simplify the analysis, we combined grades 2 and 3 salivary dysfunction into 1 category and divided all of the patients into 3 groups, de- pending on the results of scintigraphy; grade 1, grades 2 and 3, and grade 4 in the subsequent analyses. Grade of Dysfunction on Scintigraphy and Its Correlation with the Treatment Dose The grade of function in the parotid gland in all patients before RIT was grade 1 in 32, grade 2 in 8, grade 3 in 0, and grade 4 in 0 patients, and that after RIT was grade 1 in 9, grade 2 in 4, grade 3 in 6, and grade 4 in 21 patients. The deterioration in the parotid gland function on scintigraphy increased with an increase in the number of radiation treatments (r  0.593, P  .001, Spearman rank correlation test) (Table 1). The grade of function in the submandibular gland in all patients before RIT was grade 1 in 31, grade 2 in 9, grade 3 in 0, and grade 4 in 0 patients, and that after RIT was grade 1 in 11, grade 2 in 12, grade 3 in 12, and grade 4 in 5 patients. The grade AJNR Am J Neuroradiol 33:1964–70  Nov 2012  www.ajnr.org 1965 AJNR Am J Neuroradiol 33:1964–70  Nov 2012  www.ajnr.org 1965 Fig 1. A 73-year-old woman who underwent 2 radioiodine treatments showed mild dysfunction of the parotid gland on scintigraphy. Non-contrast-enhanced CT images (A) before and (B) after the treatment. Bilateral parotid glands (arrows) showed only mild shrinkage (volume reduction of 12%) and an increase in attenuation (6 HU) on the CT scan obtained after the treatment. Fig 1. A 73-year-old woman who underwent 2 radioiodine treatments showed mild dysfunction of the parotid gland on scintigraphy. Non-contrast-enhanced CT images (A) before and (B) after the treatment. Bilateral parotid glands (arrows) showed only mild shrinkage (volume reduction of 12%) and an increase in attenuation (6 HU) on the CT scan obtained after the treatment. Fig 2. A 71-year-old woman who underwent 3 radioiodine treatments showed severe dysfunction of the parotid gland on scintigraphy. Non-contrast-enhanced CT images (A) before and (B) after the treatment. The bilateral parotid glands (arrows) showed prominent volume reduction of 53% and an increase in attenuation of 32 HU on CT after the treatment. Fig 1. A 73-year-old woman who underwent 2 radioiodine treatments showed mild dysfunction of the parotid gland on scintigraphy. Non-contrast-enhanced CT images (A) before and (B) after the treatment. Bilateral parotid glands (arrows) showed only mild shrinkage (volume reduction of 12%) and an increase in attenuation (6 HU) on the CT scan obtained after the treatment. Fig 2. Attenuation Change in the Gland on CT versus the Grade of Dysfunction on Scintigraphy The attenuation change (mean  SD, 95% confidence inter- val) in the parotid gland after RIT, depending on the grade of scintigraphy, was 4.3  3.7 HU (1.5–7.2 HU) for grade 1 (n  9); 4.3  3.4 HU (1.8–6.8 HU) for grades 2 and 3 (n  10); and 23.9  17.7 HU (15.8–31.9 HU) for grade 4 (n  21) (Figs 1 and 2). The attenuation change in the parotid gland after the treatment significantly increased with an in- crease in the grade of dysfunction on scintigraphy (P  .001, 1-way analysis of variance). A significant difference in the attenuation change was present between the grade 1 and grade 4 (P  .002) and between the grades 2 and 3 and grade 4 (P  .001) groups, but not between the grade 1 and grades 2 and 3 groups, as determined by the Tukey Honestly Significant Difference test (Fig 6). Generally, salivary dysfunction after RIT occurs in a dose- dependent manner.2,7 If the patient receives a single dose of 6 GBq, it typically results in a 30% loss of parenchymal func- tion, while a cumulative administered dose of 35 GBq results in complete loss of glandular function, as determined by sali- vary gland scintigraphy.15 Radiation-induced salivary gland dysfunction is also a common complication in patients under- going external radiation therapy for head and neck can- cers.10,11 A complete loss of salivary gland function from ex- ternal radiation therapy occurs at a dose level of 60 Gy. Given the absorbed functional effect of complete loss in the salivary gland by RIT and external radiation, the absorbed dose per administered 131I activity was 1.7 Gy/GBq. However, the pub- lished absorbed dose per administered activity exhibits a large variation, with a range of 0.03–14 Gy/GBq.15 These data sug- gest that the biologic effectiveness of external radiation and RIT is not equivalent. The attenuation change (mean  SD, 95% confidence in- terval) in the submandibular gland after RIT, depending on the grade of scintigraphy, was 4.4  3.5 HU (2.0–6.7 HU) for grade 1 (n  11); 5.3  5.9 HU (2.8–7.8 HU) for grades 2 and 3 (n  24); and 10.2  2.8 HU (6.8–13.7 HU) for grade 4 (n  5) (Figs 4 and 5). In the submandibular glands, there was no significant correlation between the attenuation change and the grade of dysfunction on scintigraphy. Discussion Radioactive iodine (131I) is a well-known adjunctive treatment for thyroid carcinoma, especially in patients with a high risk of recurrence.1 In addition to accumulation in the thyroid tissue, radioactive iodine also concentrates in the salivary glands and typically leads to transient or permanent damage to the glands. In the salivary glands, serous cells are more susceptible to the deleterious effects of ionizing radiation than mucous acini. Therefore, the serous-dominant parotid gland demonstrates a more intense radiation sialoadenitis than the mixed mucous and serous cell–containing submandibular and sublingual glands.14 In line with this, our results showed that the grades of salivary dysfunction after RIT were significantly higher in the parotid glands than in the submandibular glands. ROC Curves and Cutoff Values We determined the cutoff values of volume reduction and attenuation changes to diagnose grade 4 dysfunction of the salivary glands by using ROC curves in both the parotid and submandibular glands (On-line Figs 1 and 2). The best cutoff value for the volume reduction to diagnose severe dysfunction was 19.5% in the parotid gland and 31.0% in the submandib- ular gland, and that in the attenuation change was 9.8 HU in the parotid gland. The sensitivity, specificity, positive predic- tive value, negative predictive value, positive likelihood ratio, and negative likelihood ratio for each cutoff value are shown in Table 3. Fig 3. The volume reduction of the parotid and submandibular glands on CT after radioiodine therapy in relation to the dysfunction grade on scintigraphy (Tukey Honestly Significant Difference test). In the parotid gland, a significant difference in the volume reduction is present between the following groups: grade 1 and grade 4 (P  .001) and grades 2 and 3 and grade 4 (P  .001), but not between grade 1 and grades 2 and 3. In the submandibular gland, a significant difference in the volume reduction is present between the following groups: grade 1 and grades 2 and 3 (P  .001), grades 2 and 3 and grade 4 (P  .001), and grade 1 and grade 4 (P  .001). The horizontal line in the box indicates median, E, outliers; asterisk, extreme outliers (3 times the box height). The means of attenuation change after RIT of the parotid gland and submandibular gland were 14.6  16.3 HU and 5.6  5.2 HU, respectively and the attenuation change of the parotid gland was significantly larger than that in the sub- mandibular gland (P  .001). The attenuation change after RIT in the parotid gland showed a significant inverse correlation with its pretreatment attenuation (r  0.47, P  .002), while no significant corre- lation was noted between the attenuation change after RIT in the submandibular gland and its pretreatment attenuation (r  0.12, P  .449). AJNR Am J Neuroradiol 33:1964–70  Nov 2012  www.ajnr.org 1967 Volume Reduction of the Gland on CT versus the Grade of Dysfunction on Scintigraphy The reduction volume (mean  SD; 95% confidence interval) in the parotid gland after RIT, depending on the grade of scin- tigraphy, was 2.5  4.2% (0%–5.8%) for grade 1 (n  9); 7.9  5.2% (4.2%–11.7%) for grades 2 and 3 (n  10); and 35.0  1966 Nabaa  AJNR 33  Nov 2012  www.ajnr.org Attenuation Change in the Gland on CT versus the Grade of Dysfunction on Scintigraphy No significant dif- ferences were noted among the attenuation changes for each group (Fig 6). The means of pretreatment attenuation of the parotid and submandibular gland were 5.5  25.6 HU and 40.5  10.8 HU, respectively, and the attenuation of the submandib- ular gland was significantly higher than that of the parotid gland (P  .001). The dose-effect relationship in the salivary glands is well- known with regard to external radiation therapy,10-13 whereas for RIT, predicting the severity of radiation-induced salivary dysfunction can be difficult for the following reasons: First, AJNR Am J Neuroradiol 33:1964–70  Nov 2012  www.ajnr.org 1967 Fig 4. A 46-year-old woman who underwent 2 radioiodine treatments shows mild dysfunction of the submandibular gland on scintigraphy. Non-contrast-enhanced CT images (A) before and (B) after the treatment. The bilateral submandibular glands (arrows) show a mild volume reduction of 11% and an increase in attenuation of 12 HU on CT after the treatment. Fig 4. A 46-year-old woman who underwent 2 radioiodine treatments shows mild dysfunction of the submandibular gland on scintigraphy. Non-contrast-enhanced CT images (A) before and (B) after the treatment. The bilateral submandibular glands (arrows) show a mild volume reduction of 11% and an increase in attenuation of 12 HU on CT after the treatment. Fig 5. A 68-year-old woman who underwent 4 radioiodine treatments shows severe dysfunction of the submandibular gland on scintigraphy. Non-contrast-enhanced CT images (A) before and (B) after the treatment. The bilateral submandibular glands (arrows) show a prominent volume reduction of 56% but a minimal increase in attenuation of 14 HU on CT after the treatment. Fig 4. A 46-year-old woman who underwent 2 radioiodine treatments shows mild dysfunction of the submandibular gland on scintigraphy. Non-contrast-enhanced CT images (A) before and (B) after the treatment. The bilateral submandibular glands (arrows) show a mild volume reduction of 11% and an increase in attenuation of 12 HU on CT after the treatment. ( ) g ( ) Fig 5. A 68-year-old woman who underwent 4 radioiodine treatments shows severe dysfunction of the submandibular gland on scintigraphy. Non-contrast-enhanced CT images (A) before and (B) after the treatment. The bilateral submandibular glands (arrows) show a prominent volume reduction of 56% but a minimal increase in attenuation of 14 HU on CT after the treatment. Fig 5. A 68-year-old woman who underwent 4 radioiodine treatments shows severe dysfunction of the submandibular gland on scintigraphy. 1968 Nabaa  AJNR 33  Nov 2012  www.ajnr.org Our results suggest that we can assess the extent of radia- tion-induced salivary gland dysfunction in patients after RIT on a noncontrast-enhanced CT study, which is performed as a routine examination to screen for recurrence and metastasis. We might thereby avoid the routine survey with salivary scin- tigraphy after RIT because the CT prediction of salivary dys- function allows clinicians to inform patients of the extent of salivary gland dysfunction without scintigraphy. Scintigraphy could be reserved for cases in which acute deterioration of the salivary gland function is suggested from clinical signs and symptoms. The advantages of CT over scintigraphy for the assessment of salivary function are its widespread availability in most hospitals; the simplicity and objectiveness of the widely used method of measurement of volume and atten- uation of the structures of interest, including salivary glands; and less expensive cost. There are some limitations to this study. First, although the aging process could significantly affect salivary gland function and attenuation, we did not consider the patient age owing to the small number of subjects examined in this study. Second, we could not determine the appropriate interval for perform- ing CT after RIT for the assessment of associated salivary dys- function because we did not sequentially perform CT studies after the treatment. Third, because scintigraphy and CT were performed on the same day for correlation in this study, we could not assess the potential sensitivity of scintigraphy versus CT as a function of time. Scintigraphy as a measure of salivary function might be able to detect salivary dysfunction sooner than CT, which is presumably measuring chronic changes such as fibrosis. Fig 6. The attenuation change of the parotid and submandibular glands on CT after radioiodine therapy, depending on the dysfunction grade on scintigraphy (Tukey Honestly Significant Difference test). In the parotid gland, a significant difference in the attenuation increase is present between the following groups: grade 1 and grade 4 (P  .002), grades 2 and 3 and grade 4 (P  .001), but not between grade 1 and grades 2 and 3. In the submandibular gland, no significant difference is noted among the attenuation changes for each group. The horizontal line in the box indicates the median; E, outliers; asterisk, extreme outliers (3 times the box height). Conclusions A reduction in volume of the parotid and submandibular glands and an increase in attenuation in the parotid gland on noncontrast-enhanced CT can be indicators of the grade of salivary dysfunction in patients who have undergone postop- erative RIT for thyroid carcinomas. Acknowledgments h k h l We thank the nuclear medicine technologist Junichi Sato and the CT technologist Masahiko Suzuki for their contribution and expertise in scanning technique. increase of nearly 10 HU indicated severe dysfunction of the gland, with a sensitivity of 81% and a specificity of 95%. Such a correlation was not present in the submandibular glands, possibly because of the high attenuation already present in the glands before treatment.17 In the current study, the pretreat- ment attenuation of the submandibular gland was signifi- cantly higher than that in the parotid gland. Moreover, its attenuation before treatment showed an inverse correlation with the attenuation change in the parotid gland, while that in the submandibular gland did not show such a correlation. Salivary dysfunction occasionally becomes more evident dur- ing a period of several months to 1 year after the last RIT, indicating that slowly progressing fibrosis is part of the patho- logic process involved in radiation-induced sialoadenitis.2,8 We think that replacement of the fat tissue by fibrosis within increase of nearly 10 HU indicated severe dysfunction of the gland, with a sensitivity of 81% and a specificity of 95%. Such a correlation was not present in the submandibular glands, possibly because of the high attenuation already present in the glands before treatment.17 In the current study, the pretreat- ment attenuation of the submandibular gland was signifi- cantly higher than that in the parotid gland. Moreover, its attenuation before treatment showed an inverse correlation with the attenuation change in the parotid gland, while that in the submandibular gland did not show such a correlation. Salivary dysfunction occasionally becomes more evident dur- ing a period of several months to 1 year after the last RIT, indicating that slowly progressing fibrosis is part of the patho- logic process involved in radiation-induced sialoadenitis.2,8 We think that replacement of the fat tissue by fibrosis within Table 3: The performance of the determined cutoff values of the percentage of volume reduction and attenuation increase in the salivary gland for the diagnosis of severe salivary dysfunction Parotid Gland Submandibular Gland Cutoff Value of the Attenuation Increase (HU) Cutoff value of volume reduction 19.5% 31.0% 9.8 Sensitivity 0.86 1.00 0.81 Specificity 1.00 0.97 0.95 PPV 1.00 0.83 0.94 NPV 0.86 1.00 0.82 Positive LR – 34.5 15.3 Negative LR 0.14 0 0.20 Note:—NPV indicates negative predictive value; PPV, positive predictive value; LR  likelihood ratio; –, not appreciable. Table 3: The performance of the determined cutoff values of the percentage of volume reduction and attenuation increase in the salivary gland for the diagnosis of severe salivary dysfunction Table 3: The performance of the determined cutoff values of the percentage of volume reduction and attenuation increase in the salivary gland for the diagnosis of severe salivary dysfunction Attenuation Change in the Gland on CT versus the Grade of Dysfunction on Scintigraphy Non-contrast-enhanced CT images (A) before and (B) after the treatment. The bilateral submandibular glands (arrows) show a prominent volume reduction of 56% but a minimal increase in attenuation of 14 HU on CT after the treatment. the activity distribution of 131I in the salivary gland is in- homogeneous. Second, the uptake of 131I in the salivary gland is individually variable and is also affected by the cumulative activity of previous treatments.4,7 Third, the biologic half-life of 131I differs in each patient. Therefore, a clinically available indicator for the salivary dysfunction is required in patients undergoing RIT. contraindicated for a few weeks before the RIT, we obtained noncontrast CT images. Our current results suggested that the volume change of the salivary gland measured on CT could be an indicator for the assessment of salivary dysfunction after RIT. A volume reduction of nearly 20% in the parotid and 30% in the submandibular gland suggested severe dys- function of the gland, with sensitivities of 86% and 100% and specificities of 100% and 97%, respectively. A few authors have reported radiation-induced volume loss in the salivary glands in patients receiving external radiation,10-13 and Teshima et al10 suggested a correlation between the loss of gland volume and decreased saliva production. However, no previous reports have investigated the relationship between the changes in the volume of the salivary gland on CT and the grade of salivary gland dysfunction in patients who undergo RIT. Salivary gland scintigraphy has been widely accepted as a sensitive and valid method for evaluation of the function of the salivary glands.5-9,16 Several authors have used salivary gland scintigraphy for the assessment of salivary gland dys- function after RIT and reported a good correlation with the clinical and histopathologic features of the salivary glands.5-9,16 In our study, salivary gland scintigraphy showed a dose-dependent salivary dysfunction after RIT, similar to the findings described in previous reports.5-9 The increase in attenuation after RIT on non-contrast- enhanced CT was also significantly correlated with gland dys- function on scintigraphy in the parotid glands. An attenuation CT examinations of the head and neck are commonly performed during the follow-up period after RIT to screen for recurrence and metastasis. Because iodine contrast material is 1968 Nabaa  AJNR 33  Nov 2012  www.ajnr.org the salivary gland contributes to its attenuation increase after RIT. References 1. Samaan NA, Schults PN, Hickey RC, et al. The results of various modalities of treatment of well differentiated thyroid carcinomas; a retrospective review of 1599 patients. J Clin Endocrinol Metab 1992;75:714–20 2. AlexanderC,BaderJB,SchaeferA,etal.Intermediateandlongtermsideeffects of high-dose radioiodine therapy for thyroid carcinoma. J Nucl Med 1998;39: 1551–54 3. Newkirk KA, Ringel MD, Wartofsky L, et al. The role of radioactive iodine in salivary gland dysfunction. Ear Nose Throat J 2000;79:460–68 y g y 4. Almeida JP, Sanabria AE, Lima EN, et al. Late side effects of radioactive iodine on salivary gland in patients with thyroid cancer. Head Neck 2011;33:686–90 5. Bohuslavizki KH, Brenner W, Lassmann S, et al. Quantitative salivary gland scintigraphy in the diagnosis of parenchymal damage after treatment with radioiodine. Nucl Med Commun 1996;17:681–86 6. Malpani BL, Samuel AM, Ray S. Quantification of salivary gland function in thyroid cancer patients treated with radioiodine. Int J Radiat Oncol Biol Phys 1996;35:535–40 7. Solans R, Bosch JA, Gaofre P, et al. Salivary and lacrimal gland dysfunction (Sicca syndrome) after radioiodine therapy. J Nucl Med 2001;42:738–43 AJNR Am J Neuroradiol 33:1964–70  Nov 2012  www.ajnr.org 1969 AJNR Am J Neuroradiol 33:1964–70  Nov 2012  www.ajnr.org 1969 8. Caglar M, Tuncel M, Alpar R. Scintigraphic evaluation of salivary gland dys- function in patients with thyroid cancer after radioiodine treatment. Clin Nucl Med 2002;27:767–71 cancer using integrated CT/linear acceleratory system. Int J Radiat Oncol Biol Phys 2004;59:960–70 cancer using integrated CT/linear acceleratory system. Int J Radiat Oncol Biol Phys 2004;59:960–70 13. Robar JL, Day A, Clancey J, et al. Spatial and dosimetric variability of organs at risk in head-and-neck intensity-modulated radiotherapy. Int J Radiat Oncol Biol Phys 2007;68:1121–30 9. Raza H, Khan AH, Hameed A, et al. Quantitative evaluation of salivary gland dysfunction after radioiodine therapy using salivary gland scintigraphy. Nucl Med Commun 2006;27:495–99 14. Mandel SJ, Mandel L. Radioactive iodine and salivary glands. Thyroid 2003;13:265–71 10. Teshima K, Murakami R, Tomitaka E, et al. Radiation-induced parotid gland changes in oral cancer patients: correlation between parotid volume and sa- liva production. Jpn J Clin Oncol 2010;40:42–46 15. Jentzen W, Schneider E, Freudenberg L. Relationship between cumulative radiation dose and salivary gland associated with radioiodine therapy of thyroid cancer. Nucl Med Commun 2006;27:669–76 11. Wang ZH, Yan C, Zhang ZY, et al. Radiation induced changes in parotid and submandibular gland in patients with head and neck cancer receiving postoperative radiotherapy: a longitudinal study. 8. Caglar M, Tuncel M, Alpar R. Scintigraphic evaluation of salivary gland dys- function in patients with thyroid cancer after radioiodine treatment. Clin Nucl Med 2002;27:767–71 References Laryngoscope 2009;119: 1966–74 16. Loutfi I, Nair MK, Ebrahim AK. Salivary gland scintigraphy: the use of semi- quantitative analysis for uptake and clearance. J Nucl Med Technol 2003;31: 81–85 12. Baker JL Jr, Garden AS, Ang KK, et al. Quantification of volumetric and geo- metricchangesoccurringduringfractionatedradiotherapyforheadandneck 17. Bryan RN, Miller RH, Ferreyro RI, et al. Computed tomography of the major salivary glands. AJR Am J Roentgenol 1982;139:547–54 1970 Nabaa  AJNR 33  Nov 2012  www.ajnr.org
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https://journal.binayfoundation.org/article/73764.pdf
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Disease characteristics and clinical outcomes of endometrial cancer in Asian Indian and Pakistani American (AIPA) women
International journal of cancer care and delivery
2,023
cc-by-sa
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International Journal of Cancer Care and Delivery International Journal of Cancer Care and Delivery Vol. 3, Issue Supplement 1, 2023 Vol. 3, Issue Supplement 1, 2023 619 (59%) were Asian Indians, 362 (35%) were Indian/Pak­ istanis, not specified, and 62 (6%) were Pakistanis. AIPAs were significantly younger at diagnosis (Median age: 60 and 63 years, p = 0.00). Endometroid carcinoma was the most common cancer. When compared, 87% of AIPA and 91% of NHW had endometroid cancer. Likewise, serous cell carci­ noma was seen in 11% AIPAs and 8% of the NHW popula­ tion. Endometroid cancer was more common compared to serous cell cancer among NHW when compared to AIPAs (p < 0.001). AIPAs were less likely to receive chemother­ apy than NHW (81% and 86%, p = 0.000). AIPAs were found to have better survival than NHW in univariate analysis (p=0.0001) and multivariate analysis after adjusting other co-variates (HR = 0.68, CI: 0.72 - 0.98, p = 0.04). METHODS We utilized the SEER database to derive disease charac­ teristics, therapy, and survival data for AIPA and Non-His­ panic White women aged 18 years and above from 2000 to 2018. Baseline characteristics were compared among these groups. Univariate survival analysis was done using a Ka­ plan Meier plot with a log-rank test, and the Cox pro­ portional-hazards model was used for multivariate survival analysis. BACKGROUND Endometrial cancer is the fourth most common cancer in the United States females. While the disparity in the out­ comes of this cancer has been studied extensively among other minority populations, it has not yet been studied for Asian Indian and Pakistani American populations (AIPAs)- one of the fastest-growing major ethnic groups. CONCLUSION AIPA women had better survival than the NHW population with endometrial cancer when adjusted for covariates. But there remains a need to define further the unique charac­ teristics of endometrial cancer in AIPA women. Disease characteristics and clinical outcomes of endometrial cancer in Asian Indian and Pakistani American (AIPA) women Anish Shah, MD 1, Pravash Budhathoki, MD 1, Suman Gaire, MD 2, Ujjwal Karki, MD 3, Siddhartha Yadav, MD 4 1 Bronxcare Health System, Bronx, NY, USA, 2 Mount Sinai Hospital Chicago, Chicago, IL, USA, 3 Beaumont Hospital, Troy, MI, USA, 4 Mayo Clinic, Rochester, MN, USA Keywords: endometrial cancer, disparities, minorities Keywords: endometrial cancer, disparities, minorities https://doi.org/10.53876/001c.73764 https://doi.org/10.53876/001c.73764 This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CCBY-SA-4.0). View this license’s legal deed at https://creativecommons.org/licenses/by-sa/4.0 and legal code at https://cre­ ativecommons.org/licenses/by-sa/4.0/legalcode for more information. Shah A, Budhathoki P, Gaire S, Karki U, Yadav S. Disease characteristics and clinical outcomes of endometrial cancer in Asian Indian and Pakistani American (AIPA) women. IJCCD. 2023;3(Supplement 1). doi:10.53876/001c.73764 Shah A, Budhathoki P, Gaire S, Karki U, Yadav S. Disease characteristics and clinical outcomes of endometrial cancer in Asian Indian and Pakistani American (AIPA) women. IJCCD. 2023;3(Supplement 1). doi:10.53876/001c.73764 Shah A, Budhathoki P, Gaire S, Karki U, Yadav S. Disease characteristics and clinical outcomes of endometrial cancer in Asian Indian and Pakistani American (AIPA) women. IJCCD. 2023;3(Supplement 1). doi:10.53876/001c.73764 Conference Abstracts RESULTS A total of 116,883 cases were included - among which 115,840 were NHW and 1043 were AIPAs. Among AIPAs, This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CCBY-SA-4.0). View this license’s legal deed at https://creativecommons.org/licenses/by-sa/4.0 and legal code at https://cre­ ativecommons.org/licenses/by-sa/4.0/legalcode for more information.
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https://www.nature.com/articles/s41598-020-71548-z.pdf
English
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Development of humanized mouse and rat models with full-thickness human skin and autologous immune cells
bioRxiv (Cold Spring Harbor Laboratory)
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9,844
Development of humanized mouse and rat models with full‑thickness human skin and autologous immune cells Yash Agarwal1,4, Cole Beatty1,4, Sara Ho1,4, Lance Thurlow2, Antu Das1, Samantha Kelly1, Isabella Castronova1, Rajeev Salunke1, Shivkumar Biradar1, Tseten Yeshi3, Anthony Richardson2 & Moses Bility  1* The human skin is a significant barrier for protection against pathogen transmission. Rodent models used to investigate human-specific pathogens that target the skin are generated by introducing human skin grafts to immunocompromised rodent strains. Infection-induced immunopathogenesis has been separately studied in humanized rodent models developed with human lymphoid tissue and hematopoietic stem cell transplants. Successful co-engraftment of human skin, autologous lymphoid tissues, and autologous immune cells in a rodent model has not yet been achieved, though it could provide a means of studying the human immune response to infection in the human skin. Here, we introduce the human Skin and Immune System (hSIS)-humanized NOD-scid IL2Rγnull (NSG) mouse and Sprague–Dawley-Rag2tm2hera Il2rγtm1hera (SRG) rat models, co-engrafted with human full-thickness fetal skin, autologous fetal lymphoid tissues, and autologous fetal liver-derived hematopoietic stem cells. hSIS-humanized rodents demonstrate the development of human full-thickness skin, along with autologous lymphoid tissues, and autologous immune cells. These models also support human skin infection following intradermal inoculation with community-associated methicillin-resistant Staphylococcus aureus. The co-engraftment of these human skin and immune system components into a single humanized rodent model could provide a platform for studying human skin infections. The human skin provides the first line of host protection against environmental injury and host defense against ­pathogens1, 2. Several emerging pathogens, including community-associated methicillin-resistant Staphylococ- cus aureus (CA-MRSA), target the skin for infection and ­disease1, 3–5. Also, vector-borne infectious diseases such as Lyme disease and dengue fever are transmitted via inoculation into the skin by ticks and mosquitos, ­respectively6. Interactions between keratinocytes, skin fibroblasts, and cutaneous immune cells are involved in initiating the systemic immune response and abrogate pathogen replication and dissemination to other sites of ­replication7–10. Thus, the skin provides an ideal vaccination target for inducing immunity against various pathogens, as reflected by the development of several novel vaccine technologies directed at the skin, including skin-patch intradermal ­vaccines11, 12. p In vivo models for studying environmental insults and pathogens that target the skin and associated cutaneous immune cells primarily involve mice and ­rats3. These rodent models have improved mechanistic understanding of human diseases; however, significant differences exist between the skin and immune system of humans and ­rodents3, 13. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports | (2020) 10:14598 Results The hSIS‑humanized NSG mouse model supports the development of full‑thickness human skin, autologous lymphoid tissues (thymus and spleen), and human immune cells. We previ- ously demonstrated that NSG mice support the development of human lymphoid tissues (thymus and spleen) along with autologous immune cells following engraftment of fetal tissues and autologous hematopoietic stem ­cells17. Several reports demonstrated that immunodeficient mice support the development of human skin fol- lowing engraftment of human fetal ­skin43, 44. Here, we hypothesize that NSG mice will support co-engraftment of human full-thickness fetal skin, autologous fetal lymphoid tissues, and autologous hematopoietic stem cells. Furthermore, we hypothesize that NSG mice will facilitate human skin and lymphoid tissue development, as well as enable systemic human immune cell reconstitution in transplanted human tissues and the blood. We pro- cessed human fetal spleen, thymus, and liver organs into ~ 1 ­mm3 pieces and isolated autologous human ­CD34+ hematopoietic stem cells from the fetal liver, then transplanted the tissues and hematopoietic stem cells into irradiated NSG mice. Human skin tissues were obtained from the scalp and dorsum of donors and were used in developing human skin engraftments with and without hair in the mouse model, respectively. Full-thickness human fetal skin was processed via removal of excess fat tissues attached to the subcutaneous layer of the skin, then engrafted over the rib cage, where the mouse skin was previously excised. In three cohorts, the overall suc- cess of the human immune system and autologous full-thickness skin development and maintenance for ten weeks or greater was over 75% (Supplementary Fig. 1). Gross analysis of the human skin xenograft in the hSIS- NSG mouse model beginning at two weeks post-transplantation demonstrates wound healing and maturation into adult-like human skin, which is evident at ten weeks post-transplantation (Fig. 1A, Supplementary Fig. 2). A limitation of the human skin in the hSIS-NSG mouse model is the development of dry skin (resulting in harden- ing) and early signs of murine hair loss (suggestive of graft-versus-host disease) at 20 weeks post-transplantation (Supplementary Fig. 2). The human skin in hSIS-NSG mice also develops human skin appendages, with human hair evident at 12 weeks post-transplantation (Fig. 1B). Gross analysis of human spleen and thymus xenografts in hSIS-humanized NSG mice at ten weeks post-transplantation demonstrated the growth of those lymphoid tissues under the kidney capsule (Fig. 1C)17. www.nature.com/scientificreports/ thus highlighting the need for humanized rodent models that can support the engraftment of both human skin and immune system ­components1.ti To address the species gap between rodents and humans, researchers have engrafted the immunodeficient NOD-scid IL2Rγnull (NSG) mouse model, which lacks mature lymphocytes and natural killer (NK) cells and possesses defects in innate immunity, with various human cells and ­tissues19, 20. Termed humanized-NSG mice, these models exhibit both human immune cell reconstitution and human lymphoid tissue growth and have been used to recapitulate clinical features of human diseases (including skin-associated diseases)21–25. Several separate reports demonstrate that immunodeficient mice support the engraftment of human ­skin26, 27. Adult human skin-derived from either medical wastes (elective plastic surgery)28, 29 or tissue culture-derived engi- neered skin (keratinocytes and fibroblasts)24 engrafts successfully in immunodeficient mice. Allogeneic adult human-peripheral blood mononuclear cells (PBMCs) have been introduced into these models to mimic human immune cell-skin interactions with infectious ­agents24, 29. Although these mouse models demonstrate successful engraftment and development of transplanted human skin and are amenable to the transplantation of allogeneic PBMCs, said platforms are not currently coupled with the engraftment of autologous lymphoid tissues that are critical for a de novo immune response. Humanized mouse models that combine human skin, human immune cells, and human lymphoid structures have yet to be established, despite their potential for developing a func- tional in vivo system, which could enable studies on human skin-immune cell ­interactions30. Humanized mouse models with human fetal-derived hematopoietic system and autologous lymphoid tissues are well-established19, 20. Additionally, full-thickness human fetal skin readily engrafts onto immunodeficient mice and develops into adult- like skin due to its high regenerative ­capability31, 32. Furthermore, human fetal skin exhibits low levels of major histocompatibility complex (MHC) I and II expression, which results in reduced immunogenicity compared to adult ­skin31, 32. Thus, human fetal-derived tissues and cells provide a feasible means to develop a humanized mouse model with autologous human skin and immune system. g y Rats are a major model specie for long-term (greater than one year) ­studies33; thus, humanized rat models that combine human skin, human immune cells, and human lymphoid structures are being explored for use in long-term in vivo studies of human skin-immune interactions. Reports have demonstrated that nude rats (with moderate immunodeficiency) support adult human skin (split-thickness skin) engraftment and development, albeit host-mediated immune rejection occurs within a few ­months34–36. www.nature.com/scientificreports/ Evidence also suggests that nude rats support engraftment and development of full-thickness neonatal ­foreskin37. Recently, an immunodeficient rat, termed Sprague–Dawley-Rag2tm2hera Il2rγtm1hera (SRG) rat, was developed to support engraftment of human cells and tissues into a larger and longer-life span rodent model; this model lacks mature T, B and NK ­cells38, 39. To date, humanized rat models co-engrafted with human full-thickness skin and autologous lymphoid tissues and immune cells (including cutaneous immune cells) remain to be developed and ­established28, 40–42.i Here, we utilize the immunodeficient NSG mouse and SRG rat models to generate rodent models with human skin, along with autologous lymphoid tissue(s) and autologous immune cells, termed human Skin and Immune System (hSIS)-NSG mice and SRG rats. Adolescent rodents received co-transplants of human full-thickness fetal skin, autologous lymphoid tissues, and autologous hematopoietic stem cells. Additionally, the human skin xenografts were inoculated with CA-MRSA to establish the susceptibility of these hSIS-humanized rodent models to human skin pathogens. Development of humanized mouse and rat models with full‑thickness human skin and autologous immune cells Rodent skin microanatomy differs from human skin microanatomy due to the rodent skin lacking a multi-layered epidermis, eccrine and apocrine glands, and the papillary, reticular, and hypo-dermal regions of the dermal ­layer14. Human primary and secondary lymphoid tissues microanatomy differs significantly from that of rodent lymphoid tissues as well, with significant differences in red pulp to white pulp ratio in the spleen and lobulation of the ­thymus15–17. It is well-established that lymphoid tissue microenvironment, including stro- mal cells, plays a significant role in immune cell ­development18. Interactions between immune cells and stromal cells in non-lymphoid tissues, such as the skin, play a significant role in modulating tissue-associated immune ­responses14. Translational gaps may form between clinical studies performed with traditional rodent models, 1Department of Infectious Diseases and Microbiology, University of Pittsburgh, Pittsburgh, USA. 2Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, USA. 3Hera Biolabs, Inc, Lexington, USA. 4These authors contributed equally: Yash Agarwal, Cole Beatty and Sara Ho. *email: mtbility@pitt.edu | https://doi.org/10.1038/s41598-020-71548-z www.nature.com/scientificreports/ Results The human skin xenograft exhibited multiple layers of human keratinocytes (AE1/AE3, pan-cytokeratin antibody+ cells) in the epidermis and dermal fibroblasts (Anti-Fibroblasts Anti- body+ cells) in the dermis (Fig. 2B). Additionally, the human skin exhibited human immune cell repopula- tion (human ­CD45+ cells), including Langerhans cells (hCD207+ cells), macrophages (hCD68+), and T cells (hCD3+) (Fig. 2B, Supplementary Figs. 4 and 5). The human skin in hSIS-humanized NSG mice also exhibited upregulation of alpha-smooth muscle actin-positive (α-SMA+) cells (i.e., blood vessel ­cells45) during revascu- larization and wound healing (~ 2 weeks post-transplantation), followed by a reduction in α-SMA+ cells in the healed skin (~ 10 weeks post-transplantation) (Fig. 2B). Histochemical analysis demonstrates the development of the co-transplanted human lymphoid tissues (spleen and thymus) in the renal capsule (~ 10 weeks post-trans- plantation) (Fig. 2C)17. Human thymus tissue in hSIS-humanized NSG mice exhibits T-cell (human ­CD3+ cells) reconstitution, with few B cells in the tissue (Fig. 2C)17, 46, 47. Macrophage reconstitution (human ­CD68+ cells) in the human thymus tissue is restricted to the medulla (Fig. 2C)17, 46, 47. Human spleen tissue in hSIS-humanized NSG mice exhibits macrophage reconstitution, with macrophages predominately in the red-pulp (Fig. 2C)15, 16. The human spleen tissue in hSIS-humanized NSG mice exhibits T and B cell repopulation (human ­CD3+ and ­CD20+ cells), with lymphocytes predominately in the white-pulp (Fig. 2C)15. Analysis of the PBMCs in hSIS- humanized NSG mice showed human immune cell reconstitution (hCD45+ cells) (Fig. 3A,B). Various human immune cell (hCD45+) subtypes, namely, αβ T cells (hαβ T cells with a CD4:CD8 ratio average of 1.34:1), γδ T cells (γδ T), NK cells (hNK), B cells (hB), monocytes (hMo) and granulocytes/polymorphonuclear neutrophils (hPMN) were reconstituted in the peripheral blood of the hSIS-humanized NSG mouse model (Fig. 3C,D), with analysis performed using appropriate flow cytometry assay controls (Supplementary Fig. 6). The hSIS‑humanized SRG rat model supports the development of full‑thickness human skin, thymus tissues, and human immune cells. Although humanized mice have provided in vivo platforms for modeling human diseases, the short-life span and small tissue size/volume of mice are a major limitation for long-termed and preclinical studies, respectively. We hypothesized that a larger immunodeficient rodent model, with a longer life span, namely rat, would support the development of an in vivo model for long-term studies (> 6 months) and provide large tissue volume/size. Results In addition to supporting the growth of human spleen and thymus tissues, the hSIS-humanized NSG mouse model supports the reconstitution of the immunodeficient-murine lymph nodes and spleen (Fig. 1C)17. Histochemical analysis of the human skin in hSIS-humanized NSG mice https://doi.org/10.1038/s41598-020-71548-z Scientific Reports | (2020) 10:14598 | www.nature.com/scientificreports/ Figure 1. Development of human skin and lymphoid tissues in the human Skin and Immune System- humanized NSG mouse model. Transplantation of full-thickness human skin (derived from the scalp for skin with hair, or derived from dorsum for skin without hair) on the dorsum (A, B) and autologous lymphoid tissues in the kidney capsule (C) of NSG mice results in the engraftment and development of full-thickness human skin and lymphoid tissues. (A) Representative gross-photos at 0 (the day of transplantation), 2-, 4- and 10-weeks post-transplantation, with intact mouse skin as control (n = 4 per group). (B) Transplantation of full-thickness human skin from regions with significant hair follicles (scalp) in the human Skin and Immune System-humanized NSG mouse model results in the development of human hair, as exhibited in representative gross-photos at 12 weeks post-transplantation (n = 6). (C) Representative gross-photos of human lymphoid tissues (spleen and thymus tissues) and humanized lymphoid tissues (reconstituted-immunodeficient murine lymph node and spleen) at ten weeks post-transplantation (n = 4). Figure 1. Development of human skin and lymphoid tissues in the human Skin and Immune System- humanized NSG mouse model. Transplantation of full-thickness human skin (derived from the scalp for skin with hair, or derived from dorsum for skin without hair) on the dorsum (A, B) and autologous lymphoid tissues in the kidney capsule (C) of NSG mice results in the engraftment and development of full-thickness human skin and lymphoid tissues. (A) Representative gross-photos at 0 (the day of transplantation), 2-, 4- and 10-weeks post-transplantation, with intact mouse skin as control (n = 4 per group). (B) Transplantation of full-thickness human skin from regions with significant hair follicles (scalp) in the human Skin and Immune System-humanized NSG mouse model results in the development of human hair, as exhibited in representative gross-photos at 12 weeks post-transplantation (n = 6). (C) Representative gross-photos of human lymphoid tissues (spleen and thymus tissues) and humanized lymphoid tissues (reconstituted-immunodeficient murine lymph node and spleen) at ten weeks post-transplantation (n = 4). Results demonstrates the development of the human skin xenograft; the microanatomy of the human skin at ten weeks post-transplantation is comparable to adult human skin, with multiple layers of cells present in the epidermis (Fig. 2A, Supplementary Fig. 3). The human skin xenograft exhibited multiple layers of human keratinocytes (AE1/AE3, pan-cytokeratin antibody+ cells) in the epidermis and dermal fibroblasts (Anti-Fibroblasts Anti- body+ cells) in the dermis (Fig. 2B). Additionally, the human skin exhibited human immune cell repopula- tion (human ­CD45+ cells), including Langerhans cells (hCD207+ cells), macrophages (hCD68+), and T cells (hCD3+) (Fig. 2B, Supplementary Figs. 4 and 5). The human skin in hSIS-humanized NSG mice also exhibited upregulation of alpha-smooth muscle actin-positive (α-SMA+) cells (i.e., blood vessel ­cells45) during revascu- larization and wound healing (~ 2 weeks post-transplantation), followed by a reduction in α-SMA+ cells in the healed skin (~ 10 weeks post-transplantation) (Fig. 2B). Histochemical analysis demonstrates the development of the co-transplanted human lymphoid tissues (spleen and thymus) in the renal capsule (~ 10 weeks post-trans- plantation) (Fig. 2C)17. Human thymus tissue in hSIS-humanized NSG mice exhibits T-cell (human ­CD3+ cells) reconstitution, with few B cells in the tissue (Fig. 2C)17, 46, 47. Macrophage reconstitution (human ­CD68+ cells) in the human thymus tissue is restricted to the medulla (Fig. 2C)17, 46, 47. Human spleen tissue in hSIS-humanized NSG mice exhibits macrophage reconstitution, with macrophages predominately in the red-pulp (Fig. 2C)15, 16. The human spleen tissue in hSIS-humanized NSG mice exhibits T and B cell repopulation (human ­CD3+ and ­CD20+ cells), with lymphocytes predominately in the white-pulp (Fig. 2C)15. Analysis of the PBMCs in hSIS- humanized NSG mice showed human immune cell reconstitution (hCD45+ cells) (Fig. 3A,B). Various human immune cell (hCD45+) subtypes, namely, αβ T cells (hαβ T cells with a CD4:CD8 ratio average of 1.34:1), γδ T cells (γδ T), NK cells (hNK), B cells (hB), monocytes (hMo) and granulocytes/polymorphonuclear neutrophils (hPMN) were reconstituted in the peripheral blood of the hSIS-humanized NSG mouse model (Fig. 3C,D), with analysis performed using appropriate flow cytometry assay controls (Supplementary Fig. 6). demonstrates the development of the human skin xenograft; the microanatomy of the human skin at ten weeks post-transplantation is comparable to adult human skin, with multiple layers of cells present in the epidermis (Fig. 2A, Supplementary Fig. 3). Results The black arrows denote representative IHC+ cells. (C) Representative histological and immunohistochemical analysis of the human spleen and thymus (both under the kidney capsule) in human Skin and Immune System-humanized NSG mice demonstrate the development of those lymphoid tissues at ten weeks post-transplantation, with human macrophages (hCD68+), T cells (hCD3+), B cells (hCD20+) present in the tissues (n = 4) Scale bars: 200 μm. fetal thymus and liver tissues (~ 1 ­mm3 pieces) were implanted into the kidney capsules of irradiated SRG rats, and those rats were immediately transplanted using autologous hematopoietic stem cells. Excess fat tissue was removed from the human fetal skin, and the tissue was subsequently engrafted over the rib cage, where the rat skin was previously excised. SRG rats were transplanted using human skin plus CD34+ human hematopoietic stem cells and thymus tissue, or with human skin only; 100% of the rats successfully engrafted and developed the transplanted human tissues (Supplementary Fig. 7). However, a human immune cell reconstitution of the blood in the transplanted rats was not observed in any group using this transplantation methodology (data not shown). Gross analysis of the human skin in rats transplanted with human CD34+ human hematopoietic stem cells plus thymus and skin demonstrated wound healing, beginning at three weeks post-transplantation, and matura- tion into adult-like human skin over time (Fig. 4A). The human skin transplanted onto SRG (with or without lymphoid tissue plus CD34+ human hematopoietic stem cells) also support the development of human skin appendages (hair) (Fig. 4B). Gross analysis of the lymphoid tissues in hSIS-humanized SRG rats at nine months post-transplantation showed the growth of the human thymic tissue in the kidney capsule and marginal increase in the size of the rat spleen (Fig. 4C). The human skin in hSIS-humanized SRG rats exhibited development of multi-layered human keratinocytes (AE1/AE3, pan-cytokeratin antibody+ cells) in the epidermis, and dermal fibroblasts (Anti-Fibroblasts Antibody+ cells) in the dermis, both of which are comparable to adult human skin (Adult-hSkin) and differ from rat skin (Fig. 5A, Supplementary Fig. 8). The human skin in hSIS-humanized SRG rats exhibited a reconstitution of human immune cells ­(hCD45+ cells), including Langerhans cells ­(hCD207+ cells), comparable to adult human skin (Adult-hSkin) (Fig. 5A). Histochemical analysis of human thymus tissue in hSIS-humanized SRG rats at nine months post-transplantation demonstrates thymus development, including the presence of thymic lobules (Fig. 5B). Results We further hypothesized that the co-transplantation of human full-thickness fetal skin, autologous fetal thymus tissues, autologous fetal liver tissues, and autologous fetal-liver derived hematopoietic stem cells into SRG rat would enable the development of a model containing human skin, autologous thymus, and autologous human immune cells, termed hSIS-humanized SRG rat. Human https://doi.org/10.1038/s41598-020-71548-z Scientific Reports | (2020) 10:14598 | www.nature.com/scientificreports/ Figure 2. Development of human skin and immune cells in the human Skin and Immune System-humanized NSG mouse model. (A) Representative histological (H&E) analysis of the human skin in human Skin and Immune System-humanized NSG mice (n = 4) demonstrate the development of human adult-like skin, including the dermis, multicellular layer (> 5 layers) epidermis, and cornified envelope. Representative histological (H&E) analysis of the mouse skin demonstrate a thin-layer of epidermal cells, with a thin dermal layer. (B) Various human skin cells are present in the human skin, including keratinocytes (AE1/AE3+ cells, hCytokeratins+ cells), dermal fibroblasts (TE7+ cells, hFibroblast+ cells), cutaneous immune cells (hCD45+ cells), and Langerhans cells (hCD207+); alpha-smooth muscle actin-expressing blood vessel cells (hα-SMA+ cells) are present in the human skin xenograft and expand during wound healing and contract after healing (n = 4). The black arrows denote representative IHC+ cells. (C) Representative histological and immunohistochemical analysis of the human spleen and thymus (both under the kidney capsule) in human Skin and Immune System-humanized NSG mice demonstrate the development of those lymphoid tissues at ten weeks post-transplantation, with human macrophages (hCD68+), T cells (hCD3+), B cells (hCD20+) present in the tissues (n = 4) Scale bars: 200 μm. Figure 2. Development of human skin and immune cells in the human Skin and Immune System-humanized NSG mouse model. (A) Representative histological (H&E) analysis of the human skin in human Skin and Immune System-humanized NSG mice (n = 4) demonstrate the development of human adult-like skin, including the dermis, multicellular layer (> 5 layers) epidermis, and cornified envelope. Representative histological (H&E) analysis of the mouse skin demonstrate a thin-layer of epidermal cells, with a thin dermal layer. (B) Various human skin cells are present in the human skin, including keratinocytes (AE1/AE3+ cells, hCytokeratins+ cells), dermal fibroblasts (TE7+ cells, hFibroblast+ cells), cutaneous immune cells (hCD45+ cells), and Langerhans cells (hCD207+); alpha-smooth muscle actin-expressing blood vessel cells (hα-SMA+ cells) are present in the human skin xenograft and expand during wound healing and contract after healing (n = 4). Results Human thymus tissues in the hSIS-humanized SRG rat exhibits human immune cell (human ­CD45+ cells) reconstitution, including T cell (human ­CD3+ cells) and macrophage (human ­CD68+ cells) reconstitution (Fig. 5B,C). Additionally, human thymic T cells exhibit cytokine response to stimu- lation using CD3/CD28 beads (Supplementary Fig. 9). Human immune cells in the hSIS-humanized SRG rat also reconstitute the immunodeficient-rat spleen (Fig. 5D). The human skin xenograft in immunodeficient rodent models supports community‑associated methicillin‑resistant Staphylococcus aureus infection. Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infection represents a significant public health threat 5; thus, in vivo models that enable investigation of human skin infections are of significance. In order to demonstrate that human fetal skin-derived skin xenografts in immunodeficient rodents provide a means for investigating human skin infec- tions, we inoculated (intradermal) the adult-like human skin xenografts with CA-MRSA USA300. We observed lesions in the human skin in SRG rats inoculated with CA-MRSA, which were comparable to lesions in CA- MRSA patients; those human skin-lesions in the rats exhibited CA-MRSA bacterial growth (Fig. 6). Inoculation of CA-MRSA in the human skin xenograft in the NSG mouse model at 10–12 weeks post-transplantation, also supports bacterial growth (Supplementary Fig. 10). https://doi.org/10.1038/s41598-020-71548-z Scientific Reports | (2020) 10:14598 www.nature.com/scientificreports/ Figure 3. Development of human peripheral blood mononuclear cells in the human Skin and Immune System- humanized NSG mouse model. (A) Representative flow cytometry analysis of human immune cell (hCD45+) reconstitution in peripheral blood mononuclear cells (PBMCs) of human Skin and Immune System-humanized NSG mice at 10–12 weeks post-transplantation demonstrates high levels (> 10%) of human immune cells in the blood. (B) Quantification of human immune cell reconstitution (n = 22; 3 independent experiments) in PBMCs of hSIS-humanized mice at 10 weeks post-transplantation. (C) Representative flow cytometry analysis of human PBMCs (hCD45+ PBMCs) in human Skin and Immune System-humanized mice at 10–12 weeks post-transplantation demonstrates readily detectable levels of various human immune cell types (B cells- hCD19+hCD3- human PBMCs, αβ T cells-hCD3+ human PBMCs, hCD4+ T cells, hCD8+ T cells, hγδTCR+ T cells- hγδ TCR+ CD3+ human PBMCs, natural killer cells (NK)-hCD57+ hCD3- human PBMCs, monocytes (hMo)-hCD14+ CD3- human PBMCs, and granulocytes (hPMN)-hCD66b+ hCD3- human PBMCs). (D) Quantification of human immune cell subtypes (n = 4) in human PBMCs (hCD45+ PBMCs) of human Skin and Immune System-humanized mice at 10–12 weeks post-transplantation. Figure 3. Results Development of human peripheral blood mononuclear cells in the human Skin and Immune System- humanized NSG mouse model. (A) Representative flow cytometry analysis of human immune cell (hCD45+) reconstitution in peripheral blood mononuclear cells (PBMCs) of human Skin and Immune System-humanized NSG mice at 10–12 weeks post-transplantation demonstrates high levels (> 10%) of human immune cells in the blood. (B) Quantification of human immune cell reconstitution (n = 22; 3 independent experiments) in PBMCs of hSIS-humanized mice at 10 weeks post-transplantation. (C) Representative flow cytometry analysis of human PBMCs (hCD45+ PBMCs) in human Skin and Immune System-humanized mice at 10–12 weeks post-transplantation demonstrates readily detectable levels of various human immune cell types (B cells- hCD19+hCD3- human PBMCs, αβ T cells-hCD3+ human PBMCs, hCD4+ T cells, hCD8+ T cells, hγδTCR+ T cells- hγδ TCR+ CD3+ human PBMCs, natural killer cells (NK)-hCD57+ hCD3- human PBMCs, monocytes (hMo)-hCD14+ CD3- human PBMCs, and granulocytes (hPMN)-hCD66b+ hCD3- human PBMCs). (D) Quantification of human immune cell subtypes (n = 4) in human PBMCs (hCD45+ PBMCs) of human Skin and Immune System-humanized mice at 10–12 weeks post-transplantation. Scientific Reports | (2020) 10:14598 Discussion d d l Rodent models are the primary platforms for investigating human skin-associated infections, injuries, and ­diseases10, 43. Although rodent models provide insights into the mechanisms of human diseases and host response/defense against skin-associated infectious agents, several limitations exist in rodent ­models10, 43. It is well established that the human skin exhibits significant structural differences compared to rodent ­skin10, 43. These structural differences could play a critical role in skin-microbes interactions and cutaneous immune signaling, resulting in significant differences in anti-microbial host defense and inflammatory response between human and rodent ­skin1, 2. These differences could also significantly impact the translation of mechanistic findings from traditional rodent models to humans.f In an attempt to address the species differences between traditional rodent models of human skin diseases and skin diseases in humans, we engrafted human skin, autologous lymphoid tissues, and autologous hematopoietic stem cells into immunodeficient rodents (SRG rats and NSG mice). The human skin engrafted into immunode- ficient rodents developed a microanatomical structure that resembled adult human skin. Human keratinocytes and dermal fibroblasts, which facilitate the development of multi-layered epidermis and dermis, respectively, were present in the human skin of both the hSIS-humanized rats and mice. To establish that the human skin xenograft on immunodeficient rats is susceptible to human pathogens, we infected the engrafted human skin with CA-MRSA via intradermal inoculation. The human skin xenograft in the hSIS rat model supports CA-MRSA- infection. The human skin in the hSIS-humanized mouse model also supports CA-MRSA-infection, albeit the infection appears to induce rapid death within three days. Prince et al. reported that human neutrophils and monocyte-derived macrophages are a target for CA-MRSA-associated Panton-Valentine leucocidin (PVL) in humanized mice with human immune cells and thymus xenograft 48; thus, enhanced inflammation could be mediating the rapid deaths. In the hSIS-humanized NSG mouse model, we observed readily detectable levels of human monocytes/macrophages in the blood and human skin. Future studies will investigate the interactions between human immune cell perturbations and CA-MRSA in the infected skin. A limitation of the hSIS-human- ized mouse model is the short experimental window (< 2.5 months) for infection, as drying of the human skin https://doi.org/10.1038/s41598-020-71548-z www.nature.com/scientificreports/ Figure 4. Development of human skin and lymphoid tissues in the human Skin and Immune System- humanized SRG rat model. Discussion d d l Transplantation of full-thickness human fetal skin on the dorsum (A, B) and autologous fetal lymphoid tissues (thymus and liver) in the kidney capsule (C) of SRG rats results in engraftment and development of full-thickness human skin and primary lymphoid tissue (thymus) (n = 4). (A) Representative gross-photos at 0- (the day of transplantation), 3-, 20-, and 36-weeks post-transplantation demonstrate human fetal skin engraftment and development (using donor skin obtained from the dorsum). (B) Transplantation of full-thickness human fetal skin, derived from regions with significant hair follicles (scalp), on SRG rats with (left panel) (n = 2) or without (right panel) (n = 2) co-transplantation of thymus and hematopoietic stem cells results in the development of human hair as exhibited in representative gross-photos at six months post-transplantation. In the right panel, autologous human skin from dorsum and scalp were co-transplanted to demonstrate human hair only grows in human skin tissue with preexisting hair follicles (scalp; identified with black box). (C) Representative gross-photos of lymphoid tissues (human thymus in the kidney capsule and rat spleen) at nine months post-transplantation demonstrates the development of lymphoid tissues compared to non-transplanted SRG rat (n = 4). The black circles denote human thymus tissues. Figure 4. Development of human skin and lymphoid tissues in the human Skin and Immune System- humanized SRG rat model. Transplantation of full-thickness human fetal skin on the dorsum (A, B) and autologous fetal lymphoid tissues (thymus and liver) in the kidney capsule (C) of SRG rats results in engraftment and development of full-thickness human skin and primary lymphoid tissue (thymus) (n = 4). (A) Representative gross-photos at 0- (the day of transplantation), 3-, 20-, and 36-weeks post-transplantation demonstrate human fetal skin engraftment and development (using donor skin obtained from the dorsum). (B) Transplantation of full-thickness human fetal skin, derived from regions with significant hair follicles (scalp), on SRG rats with (left panel) (n = 2) or without (right panel) (n = 2) co-transplantation of thymus and hematopoietic stem cells results in the development of human hair as exhibited in representative gross-photos at six months post-transplantation. In the right panel, autologous human skin from dorsum and scalp were co-transplanted to demonstrate human hair only grows in human skin tissue with preexisting hair follicles (scalp; identified with black box). Discussion d d l (C) Representative gross-photos of lymphoid tissues (human thymus in the kidney capsule and rat spleen) at nine months post-transplantation demonstrates the development of lymphoid tissues compared to non-transplanted SRG rat (n = 4). The black circles denote human thymus tissues. xenograft (resulting in hardening) and early signs of murine hair loss (suggestive of graft-versus-host disease) are evident at approximately five months post-transplantation. hSIS-humanized SRG rats do not exhibit signs of graft versus host disease, even at nine months post-transplantation; thus, hSIS-humanized SRG rats could provide an in vivo model for studies requiring a wider experimental window (> 3 months). Diverse leukocyte populations mediate human immune response; the hSIS-humanized mouse model supports the development of both innate and adaptive immune cells. The peripheral blood of hSIS-humanized mice is reconstituted with lymphocytes, granulocytes, monocytes, dendritic cells, and natural killer cells. Additionally, the hSIS-humanized mouse model supports the development of human thymus and spleen tissues. The presence of these human primary and secondary lymphoid tissues, coupled with the development of innate and adaptive immune cells, may allow this model to recapitulate de novo antigen-specific human immune responses to a skin infection. Interestingly, the hSIS-humanized rat model exhibited marginal human immune cell reconstitution in the peripheral blood, despite evidence of human immune cell reconstitution in the human skin and thymus, as well as the rat spleen. A transgenic variant of the SRG rat model, carrying human signal regulatory protein alpha, a negative regulator of macrophage phagocytosis, could facilitate human immune cell reconstitution in the ­blood49, 50. In summary, we report the development of humanized NSG mouse and SRG rat models that incorporate human lymphoid tissue(s) and autologous full-thickness human skin with cutaneous immune cells. hSIS-human- ized NSG mice and SRG rats could provide a means for studying skin ­infections25, 48, 51–60. Methods Construction of human Skin and Immune System‑humanized rodents. Adult (8–10 weeks) male and female severely immunodeficient rodents, namely Non-obese Diabetic (NOD) strain mice (Jackson Labo- ratory, Stock No: 005557) and Sprague Dawley (SD) strain rats (Hera Biolabs), carrying mutations in inter- leukin-2 receptor subunit gamma (IL2Rγ), as well as in the Protein Kinase, DNA-Activated, Catalytic Subunit (PRKDC) (mice only) or recombination activating gene 2 (RAG2) (rat only) were obtained from vendor and bred in the Division of Laboratory Animal Resources (DLAR) facility at the University of Pittsburgh. Human fetal tissues were obtained from the Health Sciences Tissue Bank at the University of Pittsburgh. Human fetal tissues for constructing humanized rodents were handled and processed under biosafety level 2 conditions. Male and female rodents were myoablated via gamma radiation using cesium-137 irradiator, with mice receiving a dose of 150 rads and rats receiving a dose of 500 rads. Myoablated male and female mice were transplanted with human fetal-thymus, liver, and spleen in the kidney capsule, autologous ­CD34+ hematopoietic stem cells (via retroorbital injection of 0.2 × ­106 cells)17, and full-thickness human fetal skin on the panniculus carnosus of the mouse skin-excised ­dorsum61–63. Myoablated male and female rats were transplanted with human fetal-thymus and liver in the renal capsule, autologous ­CD34+ hematopoietic stem cells (via retroorbital injection of 0.5 × ­106 cells), and autologous full-thickness human fetal skin (less than four days old) on the panniculus carnosus of the rat-skin excised ­dorsum61–63. In some instances, rodents were only transplanted with full-thickness human https://doi.org/10.1038/s41598-020-71548-z Scientific Reports | (2020) 10:14598 | www.nature.com/scientificreports/ Figure 5. Development of human skin and immune cells in the human Skin and Immune System-humanized SRG rat model. Representative immunohistochemical analysis of the human skin in the human Skin and Immune System-humanized rat (n = 4) demonstrates the development of human skin, including dermis, multi- layered (> 5 layers) epidermis, and cornified envelope, which are hallmarks of adult-human skin (Adult-hSkin) (n = 2) (A). Various human skin cells are present in the human skin, including keratinocytes (AE1/AE3+ cells, hCytokeratins+ cells), dermal fibroblasts (TE7+ cells, hFibroblast+ cells), cutaneous immune cells (hCD45+ cells), and Langerhans cells (hCD207+; black arrows denote IHC+ cells) (n = 4). Methods (B) Representative histological and immunohistochemical analysis of the human thymus (under the kidney capsule) in the human Skin and Immune System-humanized SRG rat demonstrates the development of human thymus tissue at nine months post-transplantation, with human immune cells (human CD45+), including (C) high levels of T cells (hCD3+) and macrophages (hCD68+) (n = 4). (D) The rat spleen in the human Skin and Immune System-humanized SRG rat model (n = 4) is also reconstituted with human immune cells (Humans CD45+); non-transplanted (NTP) SRG rat (n = 2) was used as a staining control. Scale bars: 200 μm. igure 5. Development of human skin and immune cells in the human Skin and Immune System-humanized Figure 5. Development of human skin and immune cells in the human Skin and Immune System-humanized SRG rat model. Representative immunohistochemical analysis of the human skin in the human Skin and Immune System-humanized rat (n = 4) demonstrates the development of human skin, including dermis, multi- layered (> 5 layers) epidermis, and cornified envelope, which are hallmarks of adult-human skin (Adult-hSkin) (n = 2) (A). Various human skin cells are present in the human skin, including keratinocytes (AE1/AE3+ cells, hCytokeratins+ cells), dermal fibroblasts (TE7+ cells, hFibroblast+ cells), cutaneous immune cells (hCD45+ cells), and Langerhans cells (hCD207+; black arrows denote IHC+ cells) (n = 4). (B) Representative histological and immunohistochemical analysis of the human thymus (under the kidney capsule) in the human Skin and Immune System-humanized SRG rat demonstrates the development of human thymus tissue at nine months post-transplantation, with human immune cells (human CD45+), including (C) high levels of T cells (hCD3+) and macrophages (hCD68+) (n = 4). (D) The rat spleen in the human Skin and Immune System-humanized SRG rat model (n = 4) is also reconstituted with human immune cells (Humans CD45+); non-transplanted (NTP) SRG rat (n = 2) was used as a staining control. Scale bars: 200 μm. Figure 6. Human skin xenograft on SRG rats supports CA-MRSA infection. Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) was inoculated into human skin xenograft (intradermal injection) on humanized (human skin) SRG rats (n = 9) to demonstrate that the human skin xenograft supports CA-MRSA infection. (A) Gross analysis of the skin tissues were examined in CA-MRSA inoculated humanized (human skin) SRG rats (n = 9) and compared to CA-MRSA infected human skin (Patient CA-MRSA skin photo credit: S. Methods Camazine); healthy control-human skin in humanized (human skin) SRG rats (n = 5) were also used as controls to determine skin lesions. (C) The skin lesions in the CA-MRSA inoculated humanized (human skin) SRG rats (n = 9) supports high CA-MRSA bacteria load as measured at six weeks post-infection. igure 6. Human skin xenograft on SRG rats supports CA-MRSA infection. Community-associated t Figure 6. Human skin xenograft on SRG rats supports CA-MRSA infection. Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) was inoculated into human skin xenograft (intradermal injection) on humanized (human skin) SRG rats (n = 9) to demonstrate that the human skin xenograft supports CA-MRSA infection. (A) Gross analysis of the skin tissues were examined in CA-MRSA inoculated humanized (human skin) SRG rats (n = 9) and compared to CA-MRSA infected human skin (Patient CA-MRSA skin photo credit: S. Camazine); healthy control-human skin in humanized (human skin) SRG rats (n = 5) were also used as controls to determine skin lesions. (C) The skin lesions in the CA-MRSA inoculated humanized (human skin) SRG rats (n = 9) supports high CA-MRSA bacteria load as measured at six weeks post-infection. Figure 6. Human skin xenograft on SRG rats supports CA-MRSA infection. Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) was inoculated into human skin xenograft (intradermal injection) on humanized (human skin) SRG rats (n = 9) to demonstrate that the human skin xenograft supports CA-MRSA infection. (A) Gross analysis of the skin tissues were examined in CA-MRSA inoculated humanized (human skin) SRG rats (n = 9) and compared to CA-MRSA infected human skin (Patient CA-MRSA skin photo credit: S. Camazine); healthy control-human skin in humanized (human skin) SRG rats (n = 5) were also used as controls to determine skin lesions. (C) The skin lesions in the CA-MRSA inoculated humanized (human skin) SRG rats (n = 9) supports high CA-MRSA bacteria load as measured at six weeks post-infection. https://doi.org/10.1038/s41598-020-71548-z Scientific Reports | (2020) 10:14598 | www.nature.com/scientificreports/ fetal skin. Rodents were housed under specific-pathogen-free conditions and fed irradiated chow and autoclaved water. Immune cell reconstitution and functional assays. For evaluating human immune cell reconstitu- tion at indicated time points, peripheral blood was collected from animals, and leukocytes were analyzed using flow ­cytometry17. Briefly, peripheral blood was collected from rodents and mixed with 20 mM Ethylenediami- netetraacetic acid (EDTA) at a 1:1 ratio, and single-cell leukocytes were prepared via red blood cells lysis using Ammonium-Chloride-Potassium (ACK) buffer. Methods CME 324 A, B, C; anti-human CD68-Biocare Medical catalog num- ber CM 033 A, B, C; anti-human CD20-Biocare Medical catalog number ACR 3004 A, B; anti-human alpha- smooth muscle actin; anti-pan cytokeratin, Clone AE1/AE3, Biocare Medical catalog number SKU: 011; anti- human fibroblast, Clone TE7, Millipore Sigma catalog number CBL271; anti-human CD207, Dendritics catalog number: DDX0362). The immunoreactivity of the antibodies was determined via incubation with DAB substrate (MACH 2 Detection Kit, Biocare Medical) and counterstaining with hematoxylin. CA‑MRSA infection in the human skin in immunodeficient rodents. The human skin xenografts on the immunodeficient rodents (SRG rats and NSG mice) were inoculated with CA-MRSA ­USA30064 via intra- dermal injection with 1 × ­108 CFU for rats and 1 × ­106 CFU for mice; non-transplanted rodents were inoculated via subqutaneous injection at the same dosage. After three days, portions of equal weight human or rodent skin was excised, and bacterial load was determined based on the number of colony-forming units (CFU); CA-MRSA bacterial strain was confirmed via polymerase chain reaction (PCR). Ethical approval. De-identified human fetal tissues at the gestational age of 18 to 20 weeks were obtained from medically or elective indicated termination of pregnancy through Magee-Womens Hospital of the Uni- versity of Pittsburgh Medical Center (UPMC), with the University of Pittsburgh, Health Sciences Tissue Bank. Written informed consent of the maternal donors was obtained in all cases, under a protocol reviewed and approved by the Institutional Review Board (IRB) of the University of Pittsburgh; approved guidelines and fed- eral/state regulations were adhered to for all procedures. The use of de-identified human fetal tissues to con- struct humanized rodents was reviewed and approved by the University of Pittsburgh IRB Office. The use of de-identified human fetal tissues did not constitute human subjects research as defined under federal regulations [45 CFR 46.102(d or f) and 21 CFR 56.102(c), (e), and (l)]. The use of human fetal liver-derived hematopoietic stem cells was reviewed and approved by the Human Stem Cell Research Oversight (hSCRO) at the University of Pittsburgh. The use of a biological agent (CA-MRSA), recombinant DNA, and transgenic animals were reviewed and approved by the Institutional Biosafety Committee (IBC) at the University of Pittsburgh. Methods For evaluating human immune reconstitution in the human skin xenograft in the hSIS-humanized mouse model, human skin tissue was excised from an hSIS-humanized mouse. Skin samples were cut into small and digested with collagenase. The dermis was separated from the epidermis, and a single-cell suspension of dermal tissue was created using a gentleMACS dissociator (Miltenyi Biotech). Epidermal tissues underwent trypsinization in order to obtain a single-cell suspension. Single-cell sus- pensions were stained with a LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (ThermoFisher Scientific) and fluo- rochrome-conjugated antibodies (anti-mouse CD45-BioLegend Cat. No. 103126, anti-human CD45-BioLegend Cat. No. 304014), fixed with formalin, and analyzed on a BD LSRFortessa™ cell analyzer—flow cytometer (BD Biosciences). Data were analyzed using FlowJo software (Dako). Leukocytes were selected based on forward, and side scatter measurements. Single-cell and live leukocytes were selected for further analysis of the percent- age of human leukocytes (anti-human ­CD45+, ­hCD3+, ­hCD4+, ­CD8+, hγδTCR​+, ­hCD19+, ­hCD14+, ­hCD16+, ­hCD66b+) and mouse leukocytes (anti-mouse ­CD45+). The analysis of the various human immune cell popula- tions and subsets were gated on human leukocytes. Human T cells were also isolated from the thymus tissue in humanized SRG rats via immunomagnetic selection using an anti-human CD3 antibody (EasySep™ Human CD3 Positive Selection, Catalog # 17951, Stemcell Technologies) and treated without (vehicle) or with Gibco™ Dynabeads™ Human T-Activator CD3/CD28 (Cat. No. 111.61D, ThermoFisher Scientific) in the presence of recombinant IL2 and BD GolgiPlug (BD Biosciences) for 12 h. Human cytokine expression (hTNFα and hIFNγ) in human T cells were analyzed using BD LSRFortessa™ cell analyzer—flow cytometer (BD Biosciences), and the data were analyzed using FlowJo software (Dako). Gross/In situ immune cell analysis. Gross analysis of tissues was performed using a camera (8 meg- apixels), with animals either euthanized or anesthetized prior to photographing. Indicated tissue samples from humanized rodents or humans (adult human skin from a 77-year-old male, BioChain, catalog number: T2234218 or adult female breast skin, University of Pittsburgh Biospecimen Repository), were fixed with for- malin and, subsequently, embedded in paraffin. Paraffin-embedded, fixed sections were stained via hematoxylin and eosin, or via indicated human ­antibodies17 (anti-human CD45-Biocare Medical Cat. No. CME PM016AA; anti-human CD3-Biocare Medical Cat. No. Methods All animal stud- ies/experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh and were conducted following approved guidelines, which adheres to the NIH guidelines for housing and care of laboratory animals. 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To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. © The Author(s) 2020, corrected publication 2021 © The Author(s) 2020, corrected publication 2021 Competing interests p g Yash Agarwal, Cole Beatty, Sara Ho, Lance Thurlow, Antu Das, Samantha Kelly, Rajeev Salunke, Isabella Cas- tronova, Shivkumar Biradar, Anthony Richardson, and Moses Bility have declared that no conflict of interest exists. Tseten Yeshi has a financial conflict of interest, as he works for HaraBiolabs, which provides the Sprague– Dawley-Rag2tm2hera Il2rγtm1hera (SRG) rat commercially. https://doi.org/10.1038/s41598-020-71548-z Scientific Reports | (2020) 10:14598 | www.nature.com/scientificreports/ Additional information Supplementary information is available for this paper at https://​doi.​org/​10.​1038/​s41598-​020-​71548-z. Correspondence and requests for materials should be addressed to M.B. 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. 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Development and validation of a microbiological assay for determination of chlorhexidine digluconate in aqueous solution
Brazilian Journal of Pharmaceutical Sciences
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Uniterms: Agar diffusion. Antiseptic. Chlorhexidine. Microbiological assay. Quality control. Validation. Uniterms: Agar diffusion. Antiseptic. Chlorhexidine. Microbiological assay. Quality control. Validation Clorexidina (CHX) é um antisséptico com amplo espectro de ação utilizada em muitos tipos de preparações farmacêuticas para uso tópico. Uma vez que não há na literatura ensaio microbiológico oficial para quantificar a clorexidina, este trabalho objetivou o desenvolvimento e validação de um ensaio microbiológico simples, sensível, exato e reprodutível, por difusão em ágar, para doseamento de digliconato de clorexidina (CHX-D) em solução aquosa. O ensaio é baseado no efeito da inibição de Staphylococcus aureus ATCC 25923, utilizado como microorganismo teste, pela CHX-D. Utilizou- se o delineamento 3x3. Os resultados foram verificados estatisticamente pela análise de variância (ANOVA) e apresentaram excelente linearidade (r = 0,9999), demonstrando que o método segue o modelo linear com regressão significativa entre o diâmetro da zona de inibição e o lagaritmo da concentração no intervalo de 0,5 a 4,5%. Os resultados obtidos foram precisos apresentando desvio padrão relativo (DPR) para precisão intra-dia de 2,03% e DPR para precisão inter-dias de 2,94%. A exatidão foi 99,03%. O método provou ser muito útil e apropriado para doseamento microbiológico da CHX-D em formas farmacêuticas e pode ser empregado para análise desta substância no controle de qualidade em indústrias farmacêuticas. *Correspondence: H. R. N. Salgado. Universidade Estadual Paulista. Ro­ dovia Araraquara-Jaú, km 1, 14801-902 – Araraquara - SP, Brasil. Phone: 55‑16‑33016967, Fax 55-16-33016900. E-mail: salgadoh@fcfar.unesp.br Article Brazilian Journal of Pharmaceutical Sciences vol. 49, n. 2, apr./jun., 2013 Unitermos: Difusão em ágar. Antisséptico. Clorexidina. Ensaio microbiológico. Controle de qualidade. Validação. Flávia Angélica Másquio Fiorentino, Marcos Antonio Corrêa, Hérida Regina Nunes Salgado* partment of Drugs And Medicines, School of Pharmaceutical Sciences, University Estadual Paulista Chlorhexidine (CHX) is a broad-spectrum antiseptic that is used in many topical pharmaceutical formulations. Because there is no official microbiological assay reported in the literature that is used to quantify CHX, this paper reports the development and validation of a simple, sensitive, accurate and reproducible agar diffusion method for the dosage of chlorhexidine digluconate (CHX-D) in an aqueous solution. The assay is based on the inhibitory effect of CHX-D upon the strain of Staphylococcus aureus ATCC 25923, which is used as the test microorganism. The design 3x3 parallel-line model was used. The results were treated statistically by analysis of variance (ANOVA), and they were excellent in terms of linearity (r = 0.9999), presenting a significant regression between the zone diameter of growth inhibition and the logarithm of the concentration within the range of 0.5 to 4.5%. The results obtained were precise, having relative standard deviations (RSD) for intra-day and inter-day precision of 2.03% and 2.94%, respectively. The accuracy was 99.03%. The method proved to be very useful and appropriate for the microbiological dosage of CHX-D in pharmaceutical formulations; it might also be used for routine drug analysis during quality control in pharmaceutical industries. Uniterms: Agar diffusion. Antiseptic. Chlorhexidine. Microbiological assay. Quality control. Validation. INTRODUCTION hands, and surgical sites and are sometimes used to impregnate catheters (Fiorentino, Corrêa, Salgado, 2010). hands, and surgical sites and are sometimes used to impregnate catheters (Fiorentino, Corrêa, Salgado, 2010). Chlorhexidine (CHX) is an excellent cationic antiseptic that belongs to a class of drugs called biguanides and has the chemical name (1,1’-hexamethylenebis{5- (p-chlorophenyl)biguanide)} (Buxbaum et al., 2006; USP, 2008; BP, 2010; EP, 2011). It exists as a variety of salts, including acetate, gluconate, hydrochloride Chlorhexidine (CHX) is an excellent cationic antiseptic that belongs to a class of drugs called biguanides and has the chemical name (1,1’-hexamethylenebis{5- (p-chlorophenyl)biguanide)} (Buxbaum et al., 2006; USP, 2008; BP, 2010; EP, 2011). It exists as a variety of salts, including acetate, gluconate, hydrochloride Antiseptics are products that are used to clean and disinfect traumatic wounds, burns, mucous membranes, 352 F. A. M. Fiorentino, M. A. Corrêa, H. R. N. Salgado and digluconate, but the digluconate form (Figure 1) is the most soluble in water and at physiological pH (EP, 2011; Thomas et al., 2000; Martindale, 2009). CHX is active against Gram-positive bacteria; less active against Gram-negative bacteria, fungi, and species of Proteus; and has activity against certain types of enveloped viruses, including hepatitis virus, herpes simplex, HIV, cytomegalovirus, influenza and respiratory syncytial virus. For mycobacteria, the CHX exhibits minimal activity; against endospores and cysts of protozoa the activity is nil (Fiorentino, Corrêa, Salgado, 2010; Cone et al., 2006; Martindale, 2009). contain CHX, several physicochemical methods are presented, such as HPLC (Gavlick, 1992; Há, Cheung, 1996; Izumoto et al., 1997; Havlíková et al., 2007), solid- phase extraction with UV spectrophotometry (Bonazzi et al., 1995), gas-liquid chromatography (Miribel et al., 1983), and capillary electrophoresis (Abad-Villar et al., 2006). A standard microbiological assay has not been reported to determine the potency of CHX digluconate in an aqueous solution using an agar diffusion test. However, in the literature, there are a large number of reports describing the use of microbiological assays to assess the potency of many antibiotics (Marona, Schapoval, 1998; Mendez et al., 2005; Gomes, Salgado, 2006; Salgado, Lopes, Lucchesi, 2006; Vaugher, Breier, Schapoval, 2006; Souza et al., 2007; Moreno, Salgado, 2007; Salgado, Tozo, 2007; Schmidt et al., 2008; Lopes, Salgado, 2010; Cazedey, Salgado, 2011; Paim et al., 2011; Solano et al., 2011). FIGURE 1 - Chemical structure of chlorhexidine digluconate (CAS: 18472-51-0). INTRODUCTION Although biological assays have a high variability, a microbiological assay can reveal subtle changes that are not demonstrable by chemical methods. A bioassay is an environmentally friendly technique because it does not require solvent or produce toxic by-products. Moreover, a microbiological assay requires no specialised equipment, is low in cost, is a simple procedure and requires only a simple facility to obtain results. These factors make the use of microbiological assays a very good alternative methodology for a potency assessment of antimicrobial drugs in a quality control laboratory (Salgado, Lopes, Lucchesi, 2006; Vaugher, Breier, Schapoval, 2006; Souza et al., 2007; Schmidt et al., 2008; Lopes, Salgado, 2010; Paim et al., 2011). Additionally, the excipients used for physicochemical techniques often cause interference and require expensive and sophisticated equipment that is not available in some quality control laboratories (Schmidt et al., 2008). FIGURE 1 - Chemical structure of chlorhexidine digluconate (CAS: 18472-51-0). There are many uses for CHX. It is most likely the most widely used biocide in antiseptic products, specifically in hand-washing and oral products; it is also used to impregnate catheters or other medical products, as a preservative in vaginal gel, as a disinfectant (Cone et al., 2006; Galice et al., 2006) and as a surgical scrub to clean the site of surgery (Buxbaum et al., 2006). Standard physicochemical methods for the determination of CHX are described in the European, British and United States Pharmacopoeias (USP, 2008; BP, 2010; EP, 2011). These techniques employ high performance liquid chromatography (HPLC) (USP, 2008; BP, 2010; EP, 2011) or titration with 0.1 M perchloric acid (BP, 2010; EP, 2011). The methods described in the literature to analyse degradation products of CHX and related substances are HPLC (USP, 2008) and colorimetric tests (BP, 2010; EP, 2011). The aim of the present study was to develop and validate a simple, sensitive, precise and accurate microbiological assay by agar diffusion using a template method to quantify CHX-D in aqueous solution as an ecological alternative to the physicochemical methods described in the literature. The literature describes several methods for the determination of CHX in biological fluids, which include direct UV spectroscopy (Jensen, Christensen, 1971), fluorometry (Vries, Ruben, Arends, 1991), HPLC (Gaffney, Cooke, 1984; Brougham, Cheng, Pittman, 1986; Lam et al., 1993; Pesonem, Holmalahti, Pohjola, 1995; Below, Lehan, Kramer, 2004) and liquid chromatography- electrospray ionisation mass spectrometry (LC-ESI-MS) (Usui et al., 2006). For pharmaceutical products that Method validation Aliquots of 250 µL, 750 µL and 2250 µL of a CHX digluconate aqueous solution were transferred to 10-mL volumetric flasks, which were filled with water to give concentrations of 0.5, 1.5 and 4.5% (T1, T2 and T3), respectively. These standard solutions were tested against S1, S2 and S3. The method was validated by the determination of linearity, precision and accuracy (USP, 2008; Marona, Schapoval, 1998; Gomes, Salgado, 2007; Salgado, Lopes, Lucchesi, 2006; Moreno, Salgado, 2007; Salgado, Tozo, 2007; Lopes, Salgado, 2010; Cazedey, Salgado, 2011; Paim et al., 2011; ICH, 2005; AOAC, 2005). Chemicals The CHX digluconate aqueous standard solution (purity 20.0%, batch number: 07/1306) was kindly donated by Rioquímica LTDA (Brazil). The CHX digluconate aqueous solution (Henrifarma®, Brazil) (rotulated purity 353 Development and validation of a microbiological assay for determination of chlorhexidine digluconate in aqueous solution 20.0%, batch number: CS 0120705) was kindly donated by the Cosmetology Laboratory of Universidade Estadual Paulista (UNESP) (Brazil). Purified water was used in all experiments. 20.0%, batch number: CS 0120705) was kindly donated by the Cosmetology Laboratory of Universidade Estadual Paulista (UNESP) (Brazil). Purified water was used in all experiments. x 20 mm Petri dish as a base layer. After solidification of this layer, a 5 mL portion of the inoculated BHI agar was poured onto the base layer. In each plate, a template was placed on the surface of the inoculated medium. Three alternating holes were filled with 200 µL of the reference solution (S1, S2 and S3), and the other three holes were filled with the sample solutions (T1, T2 and T3). Six plates were used in each assay. The plates were incubated at 35 ± 1 ºC under aerobic conditions for 18 hours. The diameters (mm) of the zones of inhibition were carefully measured using a digital calliper (Mitutoyo® IP 65) (Figure 2). All experiments were performed in a biological safety cabinet, and the infected material was decontaminated before being discarded. Precision The precision of the method was determined by repeatability (intra-assay) and the intermediate precision (inter-assay) and was expressed as the relative standard deviation (RSD). The repeatability was verified by assaying samples of CHX digluconate in aqueous solution, at the same concentration, within one day and under the same experimental conditions. The intermediate precision was evaluated by comparing the assay results from three different days. Chlorhexidine digluclonate reference solutions Chlorhexidine digluclonate reference solutions Aliquots of 250 µL, 750 µL and 2250 µL of the aqueous CHX digluconate reference solution were transferred to 10-mL volumetric flasks, which were filled with water to give concentrations of 0.5, 1.5 and 4.5% (S1, S2 and S3), respectively. These standard solutions were used in the bioassay. Linearity The cultures of S. aureus ATCC 25923 were cultivated on casein-soy agar (Acumedia®) at a temperature near freezing (2.0 to 8.0 ºC). The microorganism standardisation was carried out according to the procedures described in the Brazilian and United States Pharmacopoeias (USP, 2008; F. BRAS., 2010). Prior to use, the microorganism was grown in Brain Heart Infusion (BHI) broth (BD®) in a test tube that was incubated for 24 h at 35 ºC ± 2 ºC. Using a spectrophotometer (Beckman, DU® 530) with the wavelength set at 580 nm and a 10 mm absorption cell, the broth containing the microorganism was diluted to achieve a suspension turbidity of 25 ± 2% (transmittance) using the same sterile broth solution as for the blank. From this standardised suspension, 1.0 mL aliquots were added to each 100 mL of BHI agar (BD®) at 47 ºC ± 1 ºC and used as the inoculum layer in the plate. To assess the validity of the assay, three doses of the standard and three doses of the sample were used. The linearity was evaluated by linear regression analysis, which was calculated using the least squares method. Agar diffusion bioassay Accuracy was determined by adding 3 concentrations, which covered the specified range. Aliquots of precisely 500, 1000 and 1500 μL of the standard solution (20%) were transferred into 10 mL volumetric flasks, and diluted with water to give final concentrations of 1.5, 2.5 and 3.5%, respectively. The percentage recovery of the CHX digluconate standard was calculated using the equation proposed by the AOAC (2005). The bioassay described previously followed the 3 x 3 parallel line assay design (three doses of the standard and three doses of the sample in each plate), with six plates for each assay, according to the Brazilian Pharmacopoeia (F. BRAS., 2010). The agar was composed of two separate layers. The BHI agar (20 mL) was poured into a 100 mm 354 F. A. M. Fiorentino, M. A. Corrêa, H. R. N. Salgado RESULTS AND DISCUSSION The development and validation of analytical methods for the determination of active ingredients in medicines is very important for quality control and patient safety. When choosing an analytical method for use in routine quality control, the analyst must consider cost, complexity, required time, availability of equipment and reagents, purity, quantity of the sample and the generation of residues (ICH, 2005; Salgado, Lopes, Lucchesi, 2006; Moreno, Salgado, 2007). Calculation of potency TABLE I - Tested conditions to establish the parameters for microbiological assay of CHX digluconate microbiological assay of CHX digluconate Parameter Condition Standard curve (μg/mL) 1.5, 3.0, 6.0%; 0.25, 1.0, 4.0% and 0.5, 1.5, 4.5% Diluents Buffer solution pH 6.0 and pH 8.0 Purified water Inoculum (%) 0.5 and 1.0 Microorganism Bacillus subtilis ATCC 9372 Staphylococcus aureus ATCC 25923 Culture media Mueller Hinton agar Brain Heart Infusion (BHI) agar To calculate the activity of CHX digluconate in aqueous solution, the Hewitt [38] equation was used. The assays were statistically analysed using the linear parallel model and regression analysis and verified using analysis of variance (ANOVA) (USP, 2008; Marona, Schapoval, 1998; Salgado, Gomes, Salgado, 2006; Lopes, Lucchesi, 2006; Moreno, Salgado, 2007; Salgado, Tozo, 2007; F. BRAS., 2010; Lopes, Salgado, 2010; Cazedey, Salgado, 2011; Paim et al., 2011). Method validation In this experimental work, a 3 x 3 design was used, using three concentrations of both a standard and sample solution, according to the procedure described in the Brazilian Pharmacopoeia (F. BRAS., 2010). The calculation assumes a direct relationship between the observed diameter of the zone of inhibition and the logarithm of the applied dose. The growth inhibition zones of aqueous solutions of CHX digluconate and the reference substance are shown in Figure 2. g Moreover, the development and validation of analytical methods to determine potency, such as microbiological assays, have recently received considerable attention, mainly from regulatory agencies, because of their importance in pharmaceutical analysis (Mendez et al., 2005; Moreno, Salgado, 2007; Salgado, Tozo, 2007; Lopes, Salgado, 2010; Cazedey, Salgado, 2011). Microbiological assays have the potential to prevent the possible loss of activity, while physicochemical methods do not prevent the loss of the antimicrobial potency (USP, 2008). FIGURE 2 - An agar diffusion assay using a strain of S. aureus ATCC 25923 as the test microorganism. The chlorhexidine digluconate reference solutions are at concentrations of 0.5 (S1), 1.5 (S2) and 4.5% (S3) and the chlorhexidine digluconate samples are at concentrations of 0.5 (T1), 1.5 (T2) and 4.5% (T3). For this reason, a microbiological assay was proposed as a suitable method for the determination of CHX digluconate in aqueous solution. The experimental conditions were tested and adjusted to accurately determine the performance of the assay. Some parameters were tested previously to establish the described conditions and shown in Table I. The strain of Staphylococcus aureus that was used, ATCC 25923, appears to be an appropriate test microorganism because of its susceptibility to CHX-D and its capacity to form sharply defined zones of growth inhibition, allowing for precise measurements. FIGURE 2 - An agar diffusion assay using a strain of S. aureus ATCC 25923 as the test microorganism. The chlorhexidine digluconate reference solutions are at concentrations of 0.5 (S1), 1.5 (S2) and 4.5% (S3) and the chlorhexidine digluconate samples are at concentrations of 0.5 (T1), 1.5 (T2) and 4.5% (T3). The potency of an antibiotic may be demonstrated under suitable conditions by comparing the inhibition of growth of a microorganism caused by known concentrations of the antibiotic and a reference standard (USP, 2008; F. BRAS., 2010; EP, 2011). Table II gives the results, represented as the diameters of the zones of inhibition, for CHX digluconate in aqueous solution. Method validation 355 Development and validation of a microbiological assay for determination of chlorhexidine digluconate in aqueous solution TABLE II - Diameters of the zones of inhibition for CHX digluconate in aqueous solution and the reference solution, obtained using a standard curve TABLE II - Diameters of the zones of inhibition for CHX digluconate in aqueous solution and the reference solution, obtained using a standard curve Concentration (%) Range of zone size, mm Means diameters of growth inhibition zonesa, mm RSD% 0.5 19.27 – 19.32 19.30 0.9 1.5 20.79 – 20.84 20.81 1.0 4.5 22.33 – 22.39 22.37 1.1 aMean of 3 assays with 6 plates in each. aMean of 3 assays with 6 plates in each. The method precision in terms of repeatability (intra-assay) shows a mean R.S.D. of 2.03%, indicating good intra-day precision for the method. The intermediate precision shows a mean R.S.D. of 2.94%. The calibration curve for CHX digluconate in aqueous solution was constructed by plotting the zone diameter (mm) versus the log of the concentration (%) and showed good linearity in the 0.5 to 4.5% range. The mean accuracy was 99.03%, and the R.S.D. was 1.32% (Table IV), which confirms the ability of the method to accurately determine the concentration of CHX digluconate in aqueous solution and shows that the results obtained from the bioassay were close to the true concentrations of the samples. The representative linear equation for CHX digluconate was y = 1.3972 Ln(x) + 20.26, where x is the log dose and y is the zone diameter. The coefficient of regression was 0.9999, which is highly significant for the method. According to the Brazilian and the United States Pharmacopoeias (USP, 2008; F. BRAS., 2010), if a parallel-line model is chosen, the two log dose-response lines of the preparations to be examined, as well as that of the reference preparation, must be parallel, and they must be linear over the range of doses used in the calculation. These conditions must be verified by validity tests for a given probability, usually p = 0.05. The assays were validated by analysis of variance (ANOVA), as described in those official codes. There were no deviations from parallelism or linearity in the obtained results (p < 0.05) (Table III). Calculation of potency The CHX digluconate activity ranged from 92.52 to 92.61%, with an R.S.D. value of 0.1%. This value is acceptable because the American Pharmacopoeia (USP, 2008) allows for variations of up to 10% for mouth rinse. Moreover, CHX digluconate is a non-sterile product that is indicated for external use only, which allows a 10% variation (USP, 2008; EP, 2011). The low R.S.D. values that were obtained confirm that the proposed method can generate reproducible results with little variation between independent assays. TABLE III - Analysis of variance of determination of CHX digluconate by bioassay Parameters F calc F tab Linear regression 154264.9* 4.24 Linearity deviation 3.159 3.38 Parallelism 0.182 4.24 * p < 0.05). TABLE III - Analysis of variance of determination of CHX digluconate by bioassay The quantification of ingredients in antiseptics using chemical methods, such as HPLC and UV spectrophotometry, although precise, cannot provide a true indication of biological activity. Therefore, bioassays play an essential role in the manufacturing and quality control of antibiotics. This study confirms the validity of using bioassays for the quality control of antiseptic medicines Parameters F calc F tab Linear regression 154264.9* 4.24 Linearity deviation 3.159 3.38 Parallelism 0.182 4.24 * p < 0.05). TABLE IV - Accuracy of the microbiological assay of CHX digluconate TABLE IV - Accuracy of the microbiological assay of CHX digluconate Run Amount of standard (%) Added (%) Recovered (%) Recovery (%)a RSD (%) R1 5.00 4.93 98.6 R2 10.00 9.80 98.0 1.32 R3 15.00 15.07 100.5 aMean of 3 assays 356 F. A. M. Fiorentino, M. A. Corrêa, H. R. N. Salgado BONAZZI, D.; ANDRISANO, V.; GATTI, R.; CAVRINI, V. Analysis of pharmaceutical creams: a useful approach base on solid-phase extraction (SPE) and UV spectrophotometry. J. Pharm. Biomed. Anal., v.13, p.1321-1329, 1995. this for antiseptic medicines, and this technique still demands considerable skill and expertise to ensure success (Vaugher, Breier, Schapoval, 2006; Lopes, Salgado, 2010; Cazedey, Salgado, 2011; Paim et al., 2011). Although biological assays might have a high variability, the analysis of the results demonstrates that the proposed method might be very useful for determination of the potency of CHX digluconate in aqueous solution. BRITISH PHARMACOPOEIA. London: Her Majesty’s Stationery Office, 2010. p.467-470, p.2485-2488. BROUGHAM, L.R.; CHENG, H.; PITTMAN, K.A. Sensitive high-performance liquid chromatographic method for the determination of chlorhexidine in human serum and urine. J. Chromatogr., v.383, p.365-373, 1986. ACKNOWLEDGEMENTS EUROPEAN PHARMACOPOEIA. 7.ed. Strasbourg: Council of Europe, 2011. p.1659-1663. The authors are grateful to Maria de Fátima Rodrigues for technical support and to Dr. Greici C. G. Tozo, Rioquimica Ltda (São José Rio Preto, Brazil) for providing the standard of CHX. CNPq – Brazil, CAPES – Brazil and PADC – FCF – Araraquara Program supported this work. FARMACOPEIA BRASILEIRA. 5.ed. Brasília: ANVISA, 2010. p.258-270. FIORENTINO, F.A.M.; CORRÊA, M.A.; SALGADO, H.RN. Analytical methods for the determination of chlorhexidine: a review. Critical Rev. Anal. Chem., v.40, p.89-101, 2010. CONCLUSION The results indicated that the microbiological assay demonstrated excellent linearity, precision and accuracy at concentrations ranging from 0.5 to 4.5%. Therefore, it is an acceptable alternative method for the routine quality control of CHX digluconate in aqueous solution. The method uses simple reagents, requires minimal sample preparation, produces no toxic residues and there is no sophisticated equipment required, supporting its use in routine analysis. CAZEDEY, E.C.L.; SALGADO, H.R.N. Development and validation of a microbiological agar assay for determination of orbifloxacin in pharmaceutical preparations. Pharmaceutics, v.3, p.572-580, 2011. CONE, R.A.; HOEN, T.; WONG, X.; ABUSUWWA, R.; ANDERSON, D.J; MOENCH, T.R. Vaginal microbicides: detecting toxicities in vivo that paradoxically increase pathogen transmission. BMC Infect. Dis., v.6, p.1-16, 2006. 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Chromatogr. B. Biomed. Appl., v.665, p.222-225, 1995. IZUMOTO, S.; MACHIDA, Y.; NISHI, H.; NAKAMURA, K.; NAKAI, H.; SATO, T. Chromatography of crotamiton and its applications to the determination of active ingredients in ointment. J. Pharm. Biomed. Anal., v.15, p.1457-1466, 1997. SALGADO, H.R.N.; LOPES, C.C.G.O.; LUCCHESI, M.B.B. Microbiological assay of gatifloxacin in pharmaceutical formulations. J. Pharm. Biomed. Anal., v.40, p.443-446, 2006. JENSEN, J.E.; CHRISTENSEN, F. AOAC INTERNATIONAL. Official methods of analysis. 18ed. Gaithersburg: AOAC INTERNATIONAL, 2005. A study of the elimination of chlorhexidine from the oral cavity using a new spectrophotometric method. J. Periodont. Res., v.6, p.306- 311, 1971. SALGADO, H.R.N.; TOZO, G.C.G. Microbiological assay for cefoxitin sodium in dosage form. J. AOAC Int., v.90, n.2, p.452-455, 2007. LAM, Y.W.F.; CHAN, D.C.N.; RODRIGUEZ, S.; LINTAKOON, J.H.; LAM, T-H. Sensitive high-performance liquid chromatographic assay for the determination of chlorhexidine in saliva. J. Chromatogr., v.612, p.166-171, 1993. SCHMIDT, C.A.; CARAZZO, M.; LAPORTA, L.V.; BITTENCOURT, C.F.; SANTOS, M.R.; FRIEDRICH, M. Development and validation of an agar diffusion assay for determination of ceftazidime in pharmaceutical preparations. J. AOAC Int., v.91, p.59-66, 2008. LOPES, C.C.G.O.; SALGADO, H.R.N. Development and validation of a stability-indicative agar diffusion assay to determine the potency of linezolid in tablets in the presence of photodegradation products. Talanta, v.82, p.918-922, 2010. 358 F. A. M. Fiorentino, M. A. Corrêa, H. R. N. Salgado SOLANO, A.G.R.; PREIRA, L.M.C.S.; LEONEL, M.F.V.; NUNAN, E.A. Development of agar diffusion method for dosage of gramididin. Braz. J. Pharm. Sci., v. 47, p. 555- 572, 2011. USUI, K.; HISHINUMA, T.; YAMAMGUCHI, H.; TACHIIRI, N.; GOTO, J. Determination of chlorhexidine (CHD) and nonylphenolethoxylates (NPEOn) using LC-ESI-MS method and application to hemolyzed blood. J. Chromatogr. B, v.831, p.105-109, 2006. SOUZA, M.J.E.; ROLIM, C.M.B.; MELO, J.; SOUZA-FILHO, P.S.; BERGOLD, A.M. Development of a microbiological assay to determine the potency of ceftiofur sodium powder. J. AOAC Int., v.90, p.1724-1728, 2007. VAUGHER, L.C.; BREIER, A.R.; SCHAPOVAL, E.E.S. Microbiological assay for the determination of telithromycin in tablets. J. AOAC Int., v.89, p.1398-1402, 2006. VRIES, J.; RUBEN, J.; ARENDS, J. Determination of chlorhexidine in saliva and in aqueous solutions. Caries Res., v.25, p.410-414, 1991. Received for publication on: 08th May 2012 Accepted for publication on: 12th December 2012 VRIES, J.; RUBEN, J.; ARENDS, J. Determination of chlorhexidine in saliva and in aqueous solutions. Caries Res., v.25, p.410-414, 1991. UNITED STATES PHARMACOPOEIA. 31ed. Rockville: The United States Pharmacopeia Convention, 2008. p.1731- 1733. Received for publication on: 08th May 2012 Accepted for publication on: 12th December 2012 THOMAS, L.; MAILLARD, J.Y.; LAMBERT, R.J.W.; RUSSELL, A.D. Development of resistance to chlorhexidine diacetate in Pseudomonas aeruginosa and the effect of a residual concentration. J. Hosp. Infect., v.46, p.297-303, 2000.
https://openalex.org/W3190928269
https://journal.unnes.ac.id/nju/index.php/beaj/article/download/30142/11595
Indonesian
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Profitabilitas dalam Memediasi Pengaruh Kepemilikan Institusional, Komite Audit, dan Ukuran Perusahaan terhadap Pengungkapan Sustainability Report
Business and Economic Analysis Journal
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BEAJ Vol 1 (1) (April) 2021 : 14-26 BEAJ Vol 1 (1) (April) 2021 : 14-26 Permalink/DOI: http://dx.doi.org/10.15294/beaj.v1i1.30142 Permalink/DOI: http://dx.doi.org/10.15294/beaj.v1i1.30142 Permalink/DOI: http://dx.doi.org/10.15294/beaj.v1i1.30142  Corresponding author : E-mail: muh_khafid@mail.unnes.ac.id Kata Kunci : Kepemilikan Institusional; Komite Audit; Profitabilitas; Sustainability Report; Ukuran Perusahaan Devi Istiani Roviqoh, Muhammad Khafid Devi Istiani Roviqoh, Muhammad Khafid Jurusan Akuntansi, Fakultas Ekonomi, Universitas Negeri Semarang, Gedung L2, Kampus Sekaran, Gunungpati, Semarang, Jawa Tengah, 50229, Indonesia Abstrak Tujuan penelitian dilakukan adalah menguji pengaruh kepemilikan institusional, komite audit, dan ukuran perusahaan terhadap pengungkapan sustainability report dengan variabel intervening profitabilitas. Populasi dalam penelitian ini adalah perusahaan yang termasuk kategori indeks LQ45 periode 2013-2017 sebanyak 70 perusahaan. Teknik sampel yang digunakan pada penelitian ini yaitu purposive sampling. Sampel yang diperoleh sebanyak 14 perusahaan sehingga diperoleh total 70 unit analisis. Teknik analisis yang digunakan adalah analisis statistik deskriptif dan path analysis dengan software IBM SPSS Statistics 23. Penelitian ini menunjukkan adanya pengaruh positif profitabilitas terhadap pengungkapan sustainability report, komite audit berpengaruh positif terhadap profitabilitas, dan ukuran perusahaan berpengaruh negatif terhadap profitabilitas. Sedangkan kepemilikan institusional, ukuran perusahaan, dan komite audit tidak mempengaruhi pengungkapan sustainability report, serta kepemilikan institusional tidak berpengaruh terhadap profitabilitas. Hasil Sobel Test menunjukkan profitabilitas mampu memediasi pengaruh ukuran perusahaan dan komite audit terhadap pengungkapan sustainability report, namun tidak mampu memediasi pengaruh kepemilikan institusional terhadap pengungkapan sustainability report. Simpulan penelitian yaitu pengungkapan sustainability report akan lebih tinggi ketika ukuran perusahaan dan komite audit yang dimiliki, ditunjang dengan tingginya tingkat profitabilitas perusahaan. Hal tersebut menunjukkan bahwa manajemen hendaknya menjaga profitabilitas perusahaan karena terbukti berpengaruh langsung dan mampu memediasi terhadap pengungkapan sustainability report. Kata Kunci : Kepemilikan Institusional; Komite Audit; Profitabilitas; Sustainability Report; Ukuran Perusahaan  Corresponding author : E-mail: muh_khafid@mail.unnes.ac.id 15 BEAJ Vol 1 (1) (2021): 14-26 15 Kurangnya antusias dan pemahaman perusahaan pada pentingnya peran dari lingkungan dan sosial menjadi penyebab rendahnya angka pengungkapan tersebut. Alasan lainnya yaitu peraturan yang mengatur tentang sustainability report di Indonesia baru hadir di tahun 2017 melalui POJK 51/POJK.03/2017. PENDAHULUAN Haladu & Salim (2016), Afsari et al., (2017), dan Nurleni et al., (2017) menyatakan ada dampak positif dari kepemilikan institusional pada pengungkapan laporan keberlanjutan. H1: Kepemilikan institusional berpengaruh positif terhadap pengungkapan sustainability report proporsi inverstor institusional mampu mempengaruhi pengungkapan laporan keberlanjutan. Haladu & Salim (2016), Afsari et al., (2017), dan Nurleni et al., (2017) menyatakan ada dampak positif dari kepemilikan institusional pada pengungkapan laporan keberlanjutan. profitabilitas mencerminkan kehandalan suatu organisasi dalam menciptakan profit berdasarkan aktivitas normal bisnisnya. Seiring meningkatnya profit maka suatu organisasi memiliki sumber modal untuk menjalankan kegiatan sosial kepada masyarakat maupun lingkungan sebagai tanggung jawab perusahaan. Sehingga, kegiatan tersebut harus diinformasikan dan diungkapkan kepada para pemangku kepentingan perusahaan melalui sustainability report. Dalam teori agensi, struktur kepemilikan mayoritas dianggap mampu mempengaruhi mekanisme corporate governance. Pihak institusi sebagai investor berperan signifikan dalam memonitor manajemen seiring besarnya investasi yang diberikan kepada perusahaan. Monitoring ditujukan untuk memastikan kesejahteraan para stakeholder. Jika performa manajer dirasa kurang maksimal, akibatnya investor akan berhenti menanamkan modalnya pada perusahaan. Meminimalisir hal tersebut, manajer akan bersikap lebih akuntabel dalam menjalankan aktivitas perusahaan. Akibatnya, kinerja keuangan perusahaan akan meningkat melalui tingginya profitabilitas. Sehingga besarnya proporsi kepemilikan institusional, akan membuat tingkat profitabilitas juga mengalami peningkatan. Nurkhin et al., (2017) dan Kumai et al., (2014) menyatakan ada dampak positif dari kepemilikan institusional pada profitabilitas. Penelitian ini berlandaskan pada agency theory, legitimacy theory, dan stakeholders theory. Agency theory secara implisit menyatakan bahwa manajer perlu mengungkapkan sustainability report untuk menjamin akuntabilitas manajemen terhadap stakeholder, sehingga harapannya dapat memperkecil konflik kepentingan antara agen dan principal. Legitimacy theory menjelaskan bahwa suatu organisasi harus mendapatkan dukungan dari masyarakat dengan cara beraktivitas sesuai dengan aturan yang ditetapkan. Sedangkan teori stakeholder menunjukkan bahwa hubungan perusahaan dengan stakeholdernya juga harus dijaga, salah satu caranya dengan mengungkapkan informasi secara transparan. Kepemilikan institusional merupakan banyaknya pihak lembaga eksternal yang memiliki saham di perusahaan. Besarnya kepemilikan institusional mampu mempengaruhi pengendalian investor kepada manajemen agar mengungkapkan sustainability report. Hal ini penting sebagai bentuk transparansi perusahaan atas informasi yang di dalamnya berisi tindakan yang telah dilakukan untuk menjaga kondisi perusahaan dari segi ekonomi, lingkungan, dan sosial. Atas dasar teori agensi, pengungkapan sustainability report bisa memperkecil ketidakseimbangan informasi antara agen dan pemilik. Sehingga, besarnya H2: Kepemilikan institusional berpengaruh positif terhadap profitabilitas Tingkat kepercayaan investor salah satunya ditentukan oleh ukuran perusahaan. Perusahaan besar memiliki konsekuensi lebih diperhatikan publik seiring besarnya pengaruh perusahan terhadap masyarakat dan lingkungan. PENDAHULUAN Seiring dengan perkembangan zaman, perlahan konsep maksimalisasi laba bukan menjadi satu-satunya tujuan perusahaan. Selain berfokus pada laba yang tinggi, perusahaan juga harus memenuhi tanggung jawab sosial dan lingkungan akibat aktivitas operasi yang dilakukannya (Racelia et al., 2017). Sejalan dengan perkembangan tersebut, perusahaan diharapkan dapat memberikan kontribusinya pada sektor ekonomi, sosial, dan lingkungan. Berdasarkan penelusuran riset-riset sebelumnya mengenai pengungkapan sustainability report, masih ditemukan adanya research gap. Penelitian Haladu & Salim (2016), Nurleni et al., (2017), dan Afsari et al., (2017) memperoleh bukti kepemilikan institusional mempengaruhi pengungkapan laporan keberlanjutan. Sementara Pratama & Yulianto (2015) serta Setyawan et al., (2018) memperoleh bukti kepemilikan institusional tidak mempengaruhi pengungkapan laporan keberlanjutan. Bhatia & Tuli (2017) dan Karaman et al., (2018) membuktikan ukuran perusahaan mempengaruhi pengungkapan laporan keberlanjutan. Sedangkan Nasir et al., (2014) dan Aliniar & Wahyuni (2017) memperoleh bukti ukuran perusahaan tidak mempengaruhi pengungkapan laporan keberlanjutan. Aniktia & Khafid (2015) dan Racelia et al., (2017) dalam penelitiannya berhasil memperoleh bukti komite audit bisa mempengaruhi laporan keberlanjutan. Sedangkan Pratama & Yulianto (2015) dan Aliniar & Wahyuni (2017) membuktikan komite audit tidak mempengaruhi pengungkapan laporan keberlanjutan. Namun kenyataannya, kerusakan lingkungan akibat aktivitas bisnis perusahaan masih sering terjadi. Hal tersebut menandakan bahwa perusahaan-perusahaan di Indonesia kurang mempedulikan dampak negatif dari kegiatan operasional perusahaan tersebut yang berimbas pada ruginya masyarakat di sekitar.Hal ini tentunya akanmembuat timbulnya citra negatif perusahaan di mata masyarakat dan para pemangku kepentingan. Sehingga diperlukan suatu informasi yang transparan mengenai cara perusahaan mengelola aktivitas bisnisnya. Informasi mengenai dampak aktivitas bisnis perusahaan dapat diungkapkan melalui laporan keberlanjutan (sustainability report). Saat ini di Indonesia, jumlah pengungkapan sustainability reportnya masih terbilang rendah. Terbukti menurut OJK (2017) hingga Maret 2017, jumlah perusahaan terdaftar BEI yang telah menerbitkan sustainability report baru sekitar 9%. Setyawan et al., (2018) dalam penelitiannya juga menunjukkan rendahnya tingkat pengungkapan sustainability report, dapat dilihat dari rata-ratanya pada objek penelitian yaitu 37,31%. Selain itu, penelitian Kusuma & Priantinah (2018) juga menemukan rendahnya pengungkapan sustainability report yaitu sebesar 33,20%. Tujuan dilakukannya riset adalah menguji pengaruh kepemilikan institusional, komite audit, serta ukuran perusahaan terhadap pengungkapan laporan keberlanjutan dengan variabel intervening profitabilitas. Orisinalitas dalam penelitian yaitu menghadirkan profitabilitas sebagai variabel intervening untuk memediasi pengaruh kepemilikan institusional, komite audit, serta ukuran perusahaan terhadap pengungkapan laporan keberlanjutan. Rasio Devi Istiani Roviqoh et al., Profitabilitas dalam Memediasi Pengaruh Kepemilikan 16 proporsi inverstor institusional mampu mempengaruhi pengungkapan laporan keberlanjutan. PENDAHULUAN Berdasarkan teori legitimasi, perusahaan yang ingin berkembang secara berkelanjutan haruslah memperoleh izin dan keberpihakan dari masyarakat, sebagai pemilik sumber daya yang akan digunakan perusahaan. Sehingga, perusahaan dengan ukuran makin besar, berdampak pada makin banyaknya item 17 BEAJ Vol 1 (1) (2021): 14-26 17 akan melakukan tugasnya dalam mengawasi kegiatan pelaporan yang dilakukan oleh manajemen. Berdasarkan teori stakeholders, pihak manajemen perusahaan dapat dimaksimalkan fungsinya melalui adanya dorongan dari komite audit agar melakukan transparansi informasi sesuai kebutuhan stakeholder. Salah satu informasi yang dibutuhkan stakeholder adalah pengungkapan laporan keberlanjutan. Aniktia & Khafid (2015), Racelia et al., (2017), serta Buallay & Aldhaen (2018) membuktikan adanya pengaruh antara komite audit terhadap pengungkapan laporan keberlanjutan. yang harus diungkap pada sustainability report. Hal tersebut penting untuk meyakinkan masyarakat bahwa kegiatan dan kinerja perusahaan sudah sesuai dan tidak melanggar aturan yang berlaku. Bhatia & Tuli (2017), Khafid et al., (2018), Karaman et al., (2018) menyatakan adanya dampak positif dari kepemilikan institusional pada pengungkapan laporan keberlanjutan. H3: Ukuran perusahaan berpengaruh positif terhadap pengungkapan sustainability report Besar kecilnya aktivitas operasional di dalam perusahaan dapat tercermin melalui ukuran perusahaan. Perusahaan besar memiliki kemampuan berproduksi pada skala ekonomis, akibatnya produk yang dihasilkan memiliki harga satuan yang rendah. Harga satuan yang rendah tersebut membuat perusahaan memiliki daya saing yang tinggi di pasaran. Akibatnya terjadi peningkatan penjualan perusahaan yang pada akhirnya meningkatkan laba/keuntungan perusahaan. Keunggulan lain dari perusahaan-perusahaan besar adalah dimilikinya akses pasar yang lebih baik dibanding perusahaan dengan skala kecil (Purnomosidi et al., 2014). Dengan demikian, perusahaan dengan skala besar lebih berkesempatan untuk meningkatkan profitabilitasnya. Atas dasar teori stakeholder, peningkatan profitabilitas merupakan tanggung jawab perusahaan untuk memenuhi ekspektasi seluruh pemangku kepentingan. Penelitian John & Adebayo, (2013), Wikardi & Wiyani (2017), serta Giriyani & Diyani (2018) menemukan bukti adanya dampak positif dari ukuran perusahaan pada profitabilitas. H5: Komite audit berpengaruh positif terhadap pengungkapan sustainability report Komite audit dalam membahas permasalahan yang ada di internal perusahaan perlu mengadakan pertemuan secara berkala. Atas dasar teori agensi, pertemuan secara berkala tersebut mampu membantu memperkecil ketidak seimbangan informasi yang dapat menimbulkan masalah, seperti kemungkinan adanya manipulasi data-data keuangan dan prosedur akuntansi. Akibatnya, kinerja keuangan perusahaan akan meningkat. Kinerja keuangan yang meningkat dapat ditandai dengan tingginya rasio profitabilitas yang ada. Sehingga semakin sering rapat komite audit dilakukan, mampu menciptakan tren positif dengan tingginya profitabilitas perusahaan. Abernathy et al., (2015), Racelia et al., (2017), serta Buallay & Aldhaen (2018) menunjukkan hasil komite audit mempengaruhi profitabilitas perusahaan. PENDAHULUAN H6: Komite audit berpengaruh positif terhadap profitabilitas Profitabilitas menjadi hal penting dalam mengungkapkan sustainability report. Alasannya, perusahaan yang memiliki kemampuan lebih dalam memperoleh profit menandakan sehatnya kondisi keuangan yang dimiliki perusahaan. Sehingga perusahaan mampu menghasilkan sumber daya keuangan H4: Ukuran perusahaan berpengaruh positif terhadap profitabilitas Komunikasi dan koordinasi antar anggota komite audit merupakan suatu keharusan dalam menjalankan tanggungjawab pekerjaannya. Komite audit Devi Istiani Roviqoh et al., Profitabilitas dalam Memediasi Pengaruh Kepemilikan 18 H8: Kepemilikan institusional berpengaruh terhadap pengungkapan sustainability report melalui profitabilitas yang besar untuk membuat komitmen pengungkapan sustainability report dengan kualitas tinggi (Kuzey & Uyar, 2017). Menurut teori stakeholder, dalam rangka mewujudkan kondisi senantiasa harmonis antara perusahaan dan stakeholdernya, maka perusahaan akan berupaya memenuhi kebutuhan stakeholder termasuk kebutuhan informasi yang diwujudkan melalui pengungkapan laporan keberlanjutan. Nazari et al. (2015), dan Martínez-Ferrero et al., (2015) membuktikan adanya pengaruh antara profitabilitas terhadap pengungkapan laporan keberlanjutan. Perusahaan yang besar akan semakin disorot para pemangku kepentingan untuk melakukan transparansi insformasi dalam mewujudkan legitimasi perusahaan. Atas dasar teori legitimasi, perusahaan dengan skala besar mengindikasikan makin luasnya item yang mampu perusahaan tersebut ungkapkan pada laporan keberlanjutan yang diterbitkan sebagai laporan pertanggungjawaban. Khafid & Mulyaningsih (2015), Bhatia & Tuli (2017), serta Karaman et al., (2018) membuktikan ukuran perusahaan mampu mempengaruhi pengungkapan pada laporan keberlanjutan. Besarnya perusahaan biasanya diiringi dengan tingginya perolehan laba. Perusahaan yang mempunyai kemampuan tinggi dalam memperoleh laba akan memiliki kemampuan lebih untuk memenuhi semua kepentingan para pemangku kepentingan, termasuk pengungkapan informasi melalui sustainability report. Nazari et al. (2015) serta Martínez- Ferrero et al., (2015) dalam penelitiannya berhasil membuktikan profitabilitas mampu mempengaruhi pengungkapan pada laporan keberlanjutan. H7: Profitabilitas berpengaruh positif terhadap pengungkapan sustainability report terhadap pengungkapan sustainability report Dalam teori legitimasi, organisasi harus mendapatkan dukungan dari masyarakat dengan cara beraktivitas sesuai dengan aturan yang ditetapkan. Investor institusional sebagai pemilik saham dalam perusahaan tentunya memerlukan informasi untuk memperoleh keyakinan bahwa perusahaan telah memenuhi aspek legitimasi tersebut melalui pengungkapan sustainability report. Akibatnya, investor institusi cenderung lebih mendukung manajemen untuk menerbitkan sustainability report. Haladu & Salim (2016), Afsari et al., (2017), serta Nurleni et al., (2017) menunjukkan dampak positif kepemilikan institusional pada pengungkapan laporan keberlanjutan. Besarnya proporsi investor institusional dapat mendorong organisasi untuk mengoptimalkan kinerja keuangannya yang tercermin melalui tingginya profitabilitas. Dengan tingginya profitabilitas bisa disimpulkan bahwa besarnya biaya- biaya dalam rangka mengungkapkan sustainability report mampu perusahaan tanggung. Nazari et al. (2015) dan Martínez- Ferrero et al., (2015) menunjukkan peran positif antara profitabilitas dan pengungkapan laporan keberlanjutan. PENDAHULUAN H9: Ukuran perusahaan berpengaruh terhadap pengungkapan sustainability report melalui profitabilitas Komite audit memiliki tanggung jawab tugas pada dewan komisaris untuk mengawasi proses pelaporan yang dilakukan manajemen. Atas dasar teori stakeholders disebutkan bahwa manajer perusahaan dapat dimaksimalkan fungsinya melalui adanya dorongan dari komite audit supaya mengungkapkan semua informasi yang dibutuhkan secara transparan. Informasi yang dibutuhkan stakeholder salah satunya diwujudkan dalam pengungkapan sustainability report untuk mendapat legitimasi dari masyarakat. Racelia et al., (2017) dan Buallay & Aldhaen (2018) berhasil menemukan bukti komite audit dapat mempengaruhi 19 BEAJ Vol 1 (1) (2021): 14-26 19 pengungkapan laporan keberlanjutan. Seringnya pertemuan komite audit diselenggarakan, berdampak pada semakin baiknya pengawasan terhadap manajemen perusahaan. Pengawasan yang baik mampu membuat meningkatnya performa perusahaan yang tercermin melalui tingginya profitabilitas. Dengan tingginya profitabilitas, bisa disimpulkan bahwa besarnya biaya-biaya dalam rangka mengungkapkan sustainability report mampu perusahaan tanggung. Nazari et al. (2015) serta Martínez-Ferrero et al., (2015) menunjukkan peran positif antara profitabilitas dan pengungkapan laporan keberlanjutan. H10: Komite audit berpengaruh terhadap pengungkapan sustainability report melalui profitabilitas METODE PENELITIAN (Khafid, 2012) (Pratama & Yulianto, 2015) Ukuran Perusahaan (SIZE) Besar kecilnya perusahaan yang tercermin dari jumlah aset perusahaan pada neraca akhir periode (Mahardika et al., 2014) Ln(Total Asset) (Adhipradana & Daljono, 2014) Devi Istiani Roviqoh et al., Profitabilitas dalam Memediasi Pengaruh Kepemilikan 20 20 Devi Istiani Roviqoh et al., Profitabilitas dalam Memediasi Pengaruh Kepemilikan Variabel Definisi Pengukuran Komite Audit (KA) Komite bentukan dewan komisaris dalam rangka membantu melaksanakan tugas dan fungsinya (Khafid, 2012) ∑ rapat komite audit dalam satu periode (Pratama & Yulianto, 2015) Profitabilitas (ROA) kehandalan suatu organisasi dalam menciptakan profit berdasarkan aktivitas normal bisnisnya. (Agustina & Rusmana, 2016) (Aniktia & Khafid, 2015) Variabel Definisi Pengukuran Komite Audit (KA) Komite bentukan dewan komisaris dalam rangka membantu melaksanakan tugas dan fungsinya (Khafid, 2012) ∑ rapat komite audit dalam satu periode (Pratama & Yulianto, 2015) Profitabilitas (ROA) kehandalan suatu organisasi dalam menciptakan profit berdasarkan aktivitas normal bisnisnya. (Agustina & Rusmana, 2016) (Aniktia & Khafid, 2015) Definisi Sumber: Berbagai referensi diolah, 2019 Sumber: Berbagai referensi diolah, 2019 Teknik pengambilan data menggunakan teknik dokumenter. Sedangkan model penelitian dianalisis dengan analisis jalur menggunakan aplikasi SPSS versi 23. Analisis jalur dengan meregresikan KI, SIZE, dan KA terhadap ROA sehingga diperoleh persamaan 1: ROA = α + β1KI + β2SIZE + β3KA + e1 (1) Teknik pengambilan data menggunakan teknik dokumenter. Sedangkan model penelitian dianalisis dengan analisis jalur menggunakan aplikasi SPSS versi 23. Analisis jalur dengan meregresikan KI, SIZE, dan KA terhadap ROA sehingga diperoleh persamaan 1: ROA = α + β1KI + β2SIZE + β3KA + e1 (1) menunjukkan nilai yang cukup tinggi yaitu masing-masing 66.5% dan 18 kali. Selanjutnya, rata-rata ukuran perusahaan menunjukkan nilai sebesar 32.04. Sedangkan untuk profitabilitas menunjukkan rata-rata sebesar 6.4%. Hasil uji normalitas Kolmogorov-Smirnov menunjukkan nilai signifikansi 0.081 di atas 0.050, maka dapat diambil kesimpulan data residual penelitian terdistribusi secara normal. Uji multikolinearitas menunjukkan VIF<10 dan tolerance>0.01 maka artinya seluruh variabel yang digunakan terbebas dari gejala multikolinearitas. Selanjutnya dilakukan uji white dengan c2 hitung<c2 tabel (17.29<89.39). Maka disimpulkan tidak terjadi adanya gejala heteroskedastisitas. Durbin-Watson Test digunakan untuk menguji ada tidaknya autokorelasi, dimana diperoleh nilai DW sebesar 1.742 berada diantara 1.735 dan 2.265, sehingga disimpulkan model penelitian terbebas dari autokorelasi. Pengaruh KI, SIZE, KA dan ROA terhadap SR ditunjukkan pada R2 yaitu 0.104 atau 10,4%, sedangkan 89,6% karena adanya variabel independen lain. METODE PENELITIAN Secara ringkas, pengujian hipotesis hasilnya dapat dilihat pada Tabel 3 Langkah selanjutnya yaitu meregresikan KI, SIZE, KA serta ROA terhadap variabel dependen SR sehingga diperoleh persamaan 2: SR = α + β1KI + β2SIZE + β3KA + β4ROA + e2 (2) (2) Pengujian pengaruh variabel intervening menggunakan uji Sobel Daniel Soper dengan tingkat signifikansi 5%. METODE PENELITIAN Jenis penelitian ini adalah kuantitatif diambil dari laporan tahunan dan laporan keberlanjutan yang diakses melalui laman BEI dan website perusahaan terkait sebagai sumber data sekunder. 70 perusahaan LQ45 periode 2013 hingga 2017 menjadi populasi dalam riset ini. Teknik pemilihan sampel dipilih menggunakan purposive sampling dan didapat 14 perusahaan. Pengamatan dilakukan selama 5 tahun sehingga total unit analisis sebanyak 70. j gg y 7 Tabel 1. Kriteria Pemilihan Sampel No Kriteria Tidak Sesuai Kriteria Jumlah Populasi 70 1 Perusahaan BEI yang terdaftar secara konsisten dalam indeks LQ45 dalam kurun waktu 2013-2017 (39) 31 2 Perusahaan LQ45 yang menerbitkan sustainability report secara terpisah dari annual report dalam kurun waktu 2013- 2017 (17) 14 3 Perusahaan yang memberikan informasi lengkap terkait variabel penelitian. - 14 Sisa (Sampel Penelitian) 14 Periode pengamatan 5 Total unit analisis 70 Sumber: Data sekunder diolah, 2019 Adapun penjelasan variabel penelitian dapat dijelaskan pada Tabel 2. Tabel 2. Definisi Operasional Variabel Penelitian Variabel Definisi Pengukuran Pengungkapan Sustainability Report (SR) Media perwujudan transparansi perusahaan yang mencakup 3 aspek pelaporan, yakni sosial, ekonomi dan lingkungan (Khafid & Mulyaningsih, 2015). (Doktoralina et al., 2018) Kepemilikan Institusional (KI) Banyaknya pihak lembaga eksternal yang memiliki saham di perusahaan. (Khafid, 2012) (Pratama & Yulianto, 2015) Ukuran Perusahaan (SIZE) Besar kecilnya perusahaan yang tercermin dari jumlah aset perusahaan pada neraca akhir periode (Mahardika et al., 2014) Ln(Total Asset) (Adhipradana & Daljono, 2014) Tabel 1. Kriteria Pemilihan Sampel Tabel 1. Kriteria Pemilihan Sampel No Kriteria Tidak Sesuai Kriteria Jumlah Populasi 70 1 Perusahaan BEI yang terdaftar secara konsisten dalam indeks LQ45 dalam kurun waktu 2013-2017 (39) 31 2 Perusahaan LQ45 yang menerbitkan sustainability report secara terpisah dari annual report dalam kurun waktu 2013- 2017 (17) 14 3 Perusahaan yang memberikan informasi lengkap terkait variabel penelitian. - 14 Sisa (Sampel Penelitian) 14 Periode pengamatan 5 Total unit analisis 70 Sumber: Data sekunder diolah, 2019 j p Tabel 2. Definisi Operasional Variabel Penelitian Variabel Definisi Pengukuran Pengungkapan Sustainability Report (SR) Media perwujudan transparansi perusahaan yang mencakup 3 aspek pelaporan, yakni sosial, ekonomi dan lingkungan (Khafid & Mulyaningsih, 2015). (Doktoralina et al., 2018) Kepemilikan Institusional (KI) Banyaknya pihak lembaga eksternal yang memiliki saham di perusahaan. Tabel 3. Hasil Pengujian Hipotesis Hipotesis β Sig Hasil H1 Kepemilikan Institusional berpengaruh positif terhadap Pengungkapan Sustainability Report 0,156 0,535 Ditolak H2 Kepemilikan Institusional berpengaruh positif terhadap Profitabilitas -0,132 0,058 Ditolak H3 Ukuran Perusahaan berpengaruh positif terhadap Pengungkapan Sustainability Report 0,000 0,993 Ditolak H4 Ukuran perusahaan berpengaruh positif terhadap profitabilitas -0,021 0,000 Ditolak HASIL DAN PEMBAHASAN Pengujian statistik deskriptif menunjukkan bahwa pengungkapan sustainability report selama tahun pengamatan menunjukkan rata-rata yang rendah yaitu sebesar 32.5%. Rata-rata kepemilikan institusional dan komite audit Pengujian statistik deskriptif menunjukkan bahwa pengungkapan sustainability report selama tahun pengamatan menunjukkan rata-rata yang rendah yaitu sebesar 32.5%. Rata-rata kepemilikan institusional dan komite audit Tabel 3. Hasil Pengujian Hipotesis Hipotesis β Sig Hasil H1 Kepemilikan Institusional berpengaruh positif terhadap Pengungkapan Sustainability Report 0,156 0,535 Ditolak H2 Kepemilikan Institusional berpengaruh positif terhadap Profitabilitas -0,132 0,058 Ditolak H3 Ukuran Perusahaan berpengaruh positif terhadap Pengungkapan Sustainability Report 0,000 0,993 Ditolak H4 Ukuran perusahaan berpengaruh positif terhadap profitabilitas -0,021 0,000 Ditolak Tabel 3. Hasil Pengujian Hipotesis Hipotesis β Sig Hasil H1 Kepemilikan Institusional berpengaruh positif terhadap Pengungkapan Sustainability Report 0,156 0,535 Ditolak H2 Kepemilikan Institusional berpengaruh positif terhadap Profitabilitas -0,132 0,058 Ditolak H3 Ukuran Perusahaan berpengaruh positif terhadap Pengungkapan Sustainability Report 0,000 0,993 Ditolak H4 Ukuran perusahaan berpengaruh positif terhadap profitabilitas -0,021 0,000 Ditolak 21 BEAJ Vol 1 (1) (2021): 14-26 21 Hipotesis β Sig Hasil H5 Komite audit berpengaruh positif terhadap pengungkapan sustainability report 0,000 0,913 Ditolak H6 Komite audit berpengaruh positif terhadap profitabilitas 0,060 0,032 Diterima H7 Profitabilitas berpengaruh positif terhadap pengungkapan sustainability report 1,275 0,005 Diterima H8 Kepemilikan institusional berpengaruh positif terhadap pengungkapan sustainability report melalui profitabilitas -0,168 0,053 Ditolak H9 Ukuran perusahaan berpengaruh positif terhadap pengungkapan sustainability report melalui profitabilitas -0,027 0,005 Ditolak H10 Komite audit berpengaruh positif terhadap pengungkapan sustainability report melalui profitabilitas 0,001 0,029 Diterima S b D k d di l h Hipotesis Hipotesis saham di perusahaan. Akibatnya, tidak diperlukan monitoring pihak institusi sebagai pemilik saham mayoritas yang dapat mendorong peningkatan kinerja manajemen, dalam rangka meningkatkan kemampuan perusahaan memperoleh profit yang diukur dengan profitabilitas. Pemikiran tersebut mendukung konsep teori stewardship, dimana manajer berupaya memaksimalkan kinerja perusahaan demi mencapai tujuan organisasi sesuai dengan kepentingan investor. Riset ini mendukung temuan Wiranata & Nugrahanti (2013) dan Hu et al., (2018). Pengaruh Kepemilikan Institusional terhadap Pengungkapan Sustainability Report Kepemilikan institusional tidak mempengaruhi pengungkapan sustainability report. Kondisi ini dapat diamati pada salah satu perusahaan sampel selama lima tahun pengamatan menunjukkan tingkat kepemilikan institusional yang tinggi yaitu sekitar 84-90%. Namun kenyataannya, tingginya investor institusi tersebut belum mampu memberi dampak peningkatan pada pengungkapan sustainability report, dibuktikan dengan rendahnya rata-rata pengungkapan sustainability report sebesar 32.5% pada objek penelitian. HASIL DAN PEMBAHASAN Hal ini diduga karena pihak institusi di Indonesia lebih berfokus pada pencapaian jangka pendek dan kurang sadar akan pentingnya keberlangsungan masa depan perusahaan yang hanya bisa tercapai apabila dilakukan pengungkapan laporan keberlanjutan. Penelitian ini mendukung penelitian Setyawan et al., (2018) serta Pratama & Yulianto (2015). Pengaruh Ukuran Perusahaan terhadap Pengungkapan Sustainability Report Pengaruh Ukuran Perusahaan terhadap Pengungkapan Sustainability Report Riset ini tidak berhasil menemukan adanya pengaruh dari ukuran perusahaan terhadap pengungkapan laporan keberlanjutan. Kondisi tersebut dapat terjadi karena ukuran perusahaan sampel yang didasarkan pada pengklasifikasian distribusi frekuensi menunjukkan nilai yang besar yaitu 32.05. Namun kenyataannya, besarnya ukuran perusahaan tersebut tidak diimbangi dengan peningkatan item dalam sustainability report, dibuktikan dengan rendahnya nilai pengungkapan sustainability report yang didasarkan pada pengklasifikasian distribusi frekuensi sebesar 32.5%. Hasil riset tidak sejalan dengan teori legitimasi, yang menyebutkan perusahaan besar seharusnya mengungkapkan sustainability report secara lebih luas. Hal ini karena adanya pemikiran Pengaruh Kepemilikan Institusional terhadap Profitabilitas Pengaruh Kepemilikan Institusional terhadap Profitabilitas Kepemilikan institusional tidak mempengaruhi profitabilitas. Hal ini karena manajemen perusahaan diyakini akan terus berupaya memenuhi kehendak para pemegang saham, tanpa memperhatikan berapa jumlah institusi yang berinvestasi Devi Istiani Roviqoh et al., Profitabilitas dalam Memediasi Pengaruh Kepemilikan 22 bahwa perusahaan besar dapat mempertahankan eksistensi usahanya dan telah diakui oleh publik, selain itu perusahaan juga terlalu fokus dalam memperoleh profit sehingga mengesampingkan tanggung jawabnya mengungkapkan laporan keberlanjutan. Riset ini mendukung temuan Nasir et al., (2014) dan Aliniar & Wahyuni (2017). diperhatikan pula efektivitas rapat yang dilihat dari seberapa banyak masalah perusahaan yang dapat diselesaikan dan kebijakan-kebijakan yang diambil dalam rapat sehingga manajemen dapat mengambil keputusan untuk melakukan pengungkapan laporan keberlanjutan. Riset ini sejalan dengan Aliniar & Wahyuni (2017) serta Pratama & Yulianto (2015). bahwa perusahaan Pengaruh Komite Audit terhadap Profitabilitas g p f Hasil riset membuktikan komite audit mempengaruhi profitabilitas positif signifikan. Dalam melaksanakan perannya, komite audit telah berhasil memonitor dengan baik pengendalian internal perusahaan untuk membantu dewan komisaris. Menurut teori agensi, pada hakikatnya konflik kepentingan dapat menimbulkan masalah, seperti kemungkinan adanya manipulasi data-data keuangan dan prosedur akuntansi. Namun, pertemuan secara berkala komite audit mampu membantu memperkecil peluang terjadinya masalah tersebut, Akibatnya kinerja keuangan perusahaan akan meningkat (Mamun, 2014). Hasil riset ini sesuai dengan riset terdahulu Racelia et al., (2017) dan Buallay & Aldhaen (2018). Pengaruh Ukuran Perusahaan terhadap Profitabilitas Ukuran perusahaan mempengaruhi negatif signifikan profitabilitas. Hal ini sesuai dengan teori critical resources (Grossman & Hart, 1986) yang menyatakan bahwa ukuran perusahaan pada jumlah tertentu akhirnya akan menurunkan laba perusahaan. Alasan yang mendasarinya yakni semakin besar perusahaan dengan kepemilikan aset yang banyak pada akhirnya kurang bisa memanfaatkan aset tersebut secara optimal. Karena seiring meningkatnya ukuran perusahaan juga berpengaruh pada tingginya pembiayaan operasional yang harus dicurahkan untuk membiayai karyawan, perawatan aset, dan lain-lain. Dimana nilai tersebut dapat melebihi profit yang didapat perusahaan dari memaksimalkan ukuran perusahaan tersebut. Riset ini sejalan dengan Anindito (2015) dan Marwanti & Yulianti (2015). Pengaruh Profitabilitas terhadap Pengungkapan Sustainability Report Profitabilitas mempengaruhi secara positif signifikan pengungkapan laporan keberlanjutan. Beradasrkan teori stakeholders dinyatakan bahwa hubungan perusahaan dengan stakeholdernya juga harus dijaga, salah satu caranya dengan melakukan pertanggungjawaban sosial dan lingkungan beserta pengungkapannya. Tinggi profitabilitas membuat perusahaan dapat melakukan hal tersebut. Dengan diungkapkannya sustainability report, maka image dan reputasi perusahaan akan lebih baik di mata masyarakat, serta memiliki risiko yang lebih minim atas peristiwa sosial negatif yang bisa menciderai reputasi perusahaan. Hasil riset ini sesuai dengan riset terdahulu Pratama & Yulianto (2015) serta Agustina & Rusmana (2016). Pengaruh Komite audit terhadap Pengungkapan Sustainability Report melalui Profitabilitas Profitabilitas mampu memediasi positif signifikan komite audit pada pengungkapan laporan keberlanjutan melalui profitabilitas. Berdasarkan teori agensi, konflik keagenan akan lebih rentan terjadi pada perusahaan yang membedakan antara fungsi pemilik dan manajer, sehingga perusahaan perlu menerapkan tata kelola yang baik. Salah satunya melalui penerapan rapat rutin komite audit. Keefektifan rapat dapat menghasilkan keputusan-keputusan terbaik yang mampu membawa peningkatan kinerja perusahaan melalui tingginya profitabilitas. Profitabilitas yang tinggi menandakan semakin baiknya kinerja keuangan perusahaan, sehingga mampu mendorong pengungkapan sustainability report. Dimana laporan tersebut akan memberikan pemahaman untuk para pemangku kepentingan tentang dampak keberlanjutan bagi kegiatan bisnis perusahaan. Sehingga, profitabilitas berhasil menunjukkan perannya sebagai pemediasi. Pengaruh Ukuran Perusahaan terhadap Pengungkapan Sustainability Report melalui Profitabilitas Profitabilitas mampu memediasi pengaruh negatif ukuran perusahaan terhadap pengungkapan sustainability report. Besarnya perusahaan akan berdampak pada semakin besarnya biaya yang harus perusahaan keluarkan untuk menjalankan aktivitas bisnisnya. Sehingga, menjadikan perusahaan kurang menghasilkan profit akibat dari tingginya biaya yang ditanggung perusahaan. Perusahaan dengan profitabilitas rendah cenderung mengungkapkan informasi seperlunya saja. Hal ini karena pengungkapan informasi membutuhkan biaya yang cukup besar sementara beban yang ditanggung perusahaan sudah terlalu banyak. Data Pengaruh Komite Audit terhadap Pengungkapan Sustainability Report Pengaruh Komite Audit terhadap Pengungkapan Sustainability Report Hasil riset membuktikan komite audit tidak mempengaruhi pengungkapan laporan keberlanjutan. Berdasarkan statistik deskriptif, komite audit mealksanakan pertemuan kurun waktu satu periode rata- ratanya mencapai 18 kali, dimana sebesar 56% data penelitian berada di bawah nilai rata-rata. Artinya perusahaan sampel cenderung jarang melakukan rapat komite audit. Hal tersebut diduga menjadi penyebab ditolaknya hipotesis. Selain itu, perlu 23 BEAJ Vol 1 (1) (2021): 14-26 23 Pengaruh Kepemilikan Institusional terhadap Pengungkapan Sustainability Report melalui Profitabilitas penelitian menunjukkan profitabilitas padasalah satu sampel mempunyai penurunan nilai profitabilitas tiap tahunnya. Sedangkan untuk total aset perusahaan justru mengalami peningkatan tiap tahunnya. Sementara item yang diungkapkan dalam sustainability report terus mengalami penurunan sebanding dengan menurunnya profitabilitas. Dengan demikian, penelitian ini memberikan bukti bahwa secara positif ukuran perusahaan memberikan pengaruh terhadap pengungkapan laporan keberlanjutan melalui profitabilitas. Riset ini tidak berhasil membuktikan pengaruh kepemilikan institusional pada pengungkapan laporan keberlanjutan melalui profitabilitas. Berdasarkan data salah satu sampel penelitian selama periode pengamatan diketahui persentase proporsi kepemilikan institusional yang cenderung tetap, yaitu sebesar 50% namun tingkat profitabilitas yang diperoleh perusahaan berubah-ubah dan cenderung rendah. Hal ini dapat ditafsirkan bahwa keterlibatan investor institusional relatif kecil di dalam mendorong dan mengawasi kinerja manajemen perusahaan. Akibatnya, kendali investor institusi sebagai pemegang saham mayoritas tidak menjamin kinerja manajer agar dapat menghasilkan profitabilitas untuk mengungkapkan informasi secara transparan melalui sustainability report. Sehingga profitabilitas gagal dalam menjadi pemediasi antara kepemilikan institusional dengan pengungkapan laporan keberlanjutan. Pengaruh Komite audit terhadap Pengungkapan Sustainability Report melalui Profitabilitas KESIMPULAN Hasil riset ini dapat ditarik kesimpulan bahwa pengungkapan laporan keberlanjutan dapat lebih tinggi ketika ukuran perusahaan dan komite audit yang dimiliki, ditunjang dengan tingginya tingkat profitabilitas perusahaan. Hal Devi Istiani Roviqoh et al., Profitabilitas dalam Memediasi Pengaruh Kepemilikan 24 Influence Of Corporate Governance And Compay Characteristics To The Disclosure Of Corporate Sosial Responsibility (Empirical Study In Mining Companies That Publish Sustainability Report). Simposiun Nasional Akuntansi Xix Lampung, 1-22. Influence Of Corporate Governance And Compay Characteristics To The Disclosure Of Corporate Sosial Responsibility (Empirical Study In Mining Companies That Publish Sustainability Report). Simposiun Nasional Akuntansi Xix Lampung, 1-22. tersebut karena profitabilitas terbukti berpengaruh langsung dan mampu menjadi pemediasi antara ukuran perusahaan dan komite audit pada pengungkapan laporan keberlanjutan. Hasil statistik deskriptif memperlihatkan bahwa pengungkapan sustainability report di Indonesia terbilang rendah, dilihat dari rata-rata pengungkapan sebesar 32.5%. Manajemen perusahaan hendaknya menjaga profitabilitas perusahaan yang tercermin melalui ROA karena variabel tersebut terbukti mempengaruhi secara positif pengungkapan sustainability report dan mampu menunjukkan perannya sebagai pemediasi. Bagi penelitian selanjutnya diharapkan mempertimbangkan indeks referensi POJK sebagai pedoman pengungkapan sustainability report seiring dikeluarkannya peraturan OJK nomor 51/POJK.03/2017. Aliniar, D., & Wahyuni, S. (2017). 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The expression landscape of JAK1 and its potential as a biomarker for prognosis and immune infiltrates in NSCLC
BMC bioinformatics
2,021
cc-by
8,566
© The Author(s), 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the mate- rial. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​ creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​mmons.​org/​publi​ cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background:  Janus-activated kinase-1 (JAK1) plays a crucial role in many aspects of cell proliferation, differentiation, apoptosis and immune regulation. However, cor- relations of JAK1 with prognosis and immune infiltration in NSCLC have not been documented. Methods:  We analyzed the relationship between JAK1 expression and NSCLC progno- sis and immune infiltration using multiple public databases. Results:  JAK1 expression was significantly decreased in NSCLC compared with that in paired normal tissues. JAK1 overexpression indicated a favourable prognosis in NSCLC. In subgroup analysis, high JAK1 expression was associated with a preferable prognosis in lung adenocarcinoma (OS: HR, 0.74, 95% CI from 0.58 to 0.95, log-rank P = 0.017), not squamous cell carcinoma. In addition, data from Kaplan–Meier plotter revealed that JAK1 overexpression was associated with a preferable prognosis in male and stage N2 patients and patients without distant metastasis. Notably, increased levels of JAK1 expression were associated with an undesirable prognosis in patients with stage 1 (OS: HR, 1.46, 95% CI from 1.06 to 2.00, P = 0.02) and without lymph node metastasis (PFS: HR, 2.18, 95% CI from 1.06 to 4.46, P = 0.029), which suggests that early-stage NSCLC patients with JAK1 overexpression may have a bleak prognosis. Moreover, multiple immune infiltration cells, including NK cells, CD8 + T and CD4 + T cells, B cells, mac- rophages, neutrophils, and dendritic cells (DCs), in NSCLC were positively correlated with JAK1 expression. Furthermore, diverse immune markers are associated with JAK1 expression. Conclusions:  JAK1 overexpression exhibited superior prognosis and immune infiltra- tion in NSCLC. Keywords:  JAK1, Immune infiltrating, Prognosis, NSCLC The expression landscape of JAK1 and its potential as a biomarker for prognosis and immune infiltrates in NSCLC Kaikai Shen1†, Yuqing Wei2†, Tangfeng Lv3, Yong Song3, Xiaogan Jiang1, Zhiwei Lu2, Ping Zhan3, Xianghai Wang2, Meng Fan4 and Weihua Lu1* *Correspondence: lwh683@126.com †Kaikai Shen and Yuqing Wei contributed equally to this work 1 Department of Critical Care Medicine, The First Affiliated Hospital of Wannan Medical College (Yijishan Hospital of Wannan Medical College), Wuhu 241000, China Full list of author information is available at the end of the article Open Access Introduction Lung cancer, as a malignant tumour with high morbidity and mortality, poses a serious threat to people’s physical and mental health [1]. Non-small-cell lung cancer (NSCLC) accounts for approximately 80% of all lung cancer cases [2]. With the advent of precision Shen et al. BMC Bioinformatics (2021) 22:471 https://doi.org/10.1186/s12859-021-04379-y Shen et al. BMC Bioinformatics (2021) 22:471 https://doi.org/10.1186/s12859-021-04379-y Shen et al. BMC Bioinformatics (2021) 22:471 Page 2 of 14 therapy, lung cancer treatment has entered molecular therapy, including targeted ther- apy, anti-angiogenesis therapy and immunotherapy [3]. However, the prognosis has not improved significantly, and the 5-year survival rate remains poor [1]. In recent years, clinical studies have shown that immunotherapy (PD-1/L1 monoclonal antibody, CTLA- 4 inhibitor) has great potential in the treatment of lung cancer patients without epider- mal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) mutations [4]. Nevertheless, immunotherapy only activates immune cells in a subset of patients. With the continuous exploration of the tumour immune microenvironment (TME), which can directly or indirectly affect the development of tumours, including promoting tumour angiogenesis, changing the biological characteristics of the tumour, promoting immune escape, and even regulating the activity of cancer stem cells (CSCs) [5, 6]. Many studies have found that TAMs (tumour-associated macrophages), TILs (tumour infil- trating lymphocytes) and TINs (tumour-infiltrating neutrophils) in the TME can affect the efficacy of immunotherapy [7, 8]. Hence, it is imperative to find immune infiltration- related biomarkers that are related to the prognosis of NSCLC. p g Janus-activated kinase (JAK) is an inactive tyrosine protein kinase that consists of four family members, including JAK1, JAK2, TYK2, and JAK3 [9]. JAKs approach each other and are activated by interactive tyrosine phosphorylation, ultimately leading to signal transducer and activator of transcription (STAT​) proteins forming a homo/heterodimer that is incorporated into the nucleus and binding to the target gene promoter to activate transcription and expression [10]. Previous studies have shown that the JAK1/STAT3 pathway is widely involved in many significant biological processes, such as cell prolif- eration, differentiation, apoptosis and immune regulation [11–13]. JAK family kinases play an essential role in cytokine signalling. Functionally acquired JAK1 mutations can encourage the development of cancers, especially leukaemia. Abnormal JAK1 expression either promotes or suppresses tumour growth [10, 14, 15]. Chen et al. [10] showed that high expression of JAK1 mRNA was associated with TNM (Tumor, Node, Metastasis) stage and superior prognosis of breast cancer. In addition, infiltration and enrichment of immunoregulatory cells were significantly positively correlated with JAK1 expression. In contrast, Zhang et al. [16] showed that JAK1 signal activation could promote the prolif- eration of bladder cancer cells and lead to a poor prognosis. Hu et al. also indicated that JAK1/STAT3 plays a crucial role in ovarian cancer as a pro-oncogenic signalling path- way [17]. Whether JAK1 expression is involved in the prognosis and the level of immune infiltration in NSCLC still needs to be further explored. i In our descriptive study, we explored the expression landscape of JAK1 in NSCLC and its relationship with prognosis using shared databases, including TIMER, GEPIA, Kaplan–Meier Plotter and PrognoScan. We also visualized the relationship between JAK1 and immune infiltration using TIMER and TISIDB. Moreover, correlations between JAK1 expression and multiple gene marker sets related to immune infiltrates were also analysed via TIMER and GEPIA. TISIDB analysis TISIDB is also a web portal for tumour and immune system interaction, which inte- grates multiple heterogeneous data types. (http://​cis.​hku.​hk/​TISIDB/​index.​php) [19]. We explored the correlation between JAK1 expression in NSCLC and the abundance of multiple immune cells, including activated CD4 T cells (Act_CD4), activated dendritic cells (Act_DCs), immature dendritic cells (iDCs), neutrophils, natural killer cells (NKs), plasmacytoid dendritic cells (pDCs), central memory CD4 cells (Tcm_CD4), and effec- tor memory CD8 cells (Tem_CD8). The relative abundance of each immune cell was inferred by using gene set variation analysis (GSVA) based on the gene expression pro- file. JAK1 expression was drawn on the x-axis, while the abundance of immune cells was drawn on the y-axis. A scatterplot was used to display the correlation between the abun- dance of each immune cell and JAK1 expression. GEPIA database analysis To further verify the gene marker associated with immune infiltration in NSCLC. We used the public database Gene Expression Profiling Interactive Analysis (GEPIA) (http://​ gepia.​cancer-​pku.​cn/​index.​html) [20], which analyses the RNA sequencing expression from the TCGA and GTEx projects of 9736 tumours and 8587 normal samples. The correlation coefficient was determined by the Spearman method. The tumour and nor- mal tissue datasets were used for analysis. JAK1 expression profiles across LUAD (lung adenocarcinoma) and LUSC (lung squamous cell carcinoma) samples and paired normal tissues from GEPIA were also analysed. Materials and methods TIMER database analysis The TIMER (Tumour Immune Estimation Resource) web server is a comprehensive resource for the systematic analysis of immune infiltrates across diverse cancer types. Shen et al. BMC Bioinformatics (2021) 22:471 Page 3 of 14 (https://​cistr​ome.​shiny​apps.​io/​timer/) [18]. The abundances of six immune infiltrates (B cells, ­CD4+ T cells, ­CD8+ T cells, neutrophils, macrophages, and dendritic cells) were estimated by the TIMER algorithm. We evaluated the correlation between JAK1 expres- sion levels and various immune infiltrating cells via the TIMER algorithm. In addition, JAK1 expression profiles across various tumour samples and paired normal tissues from the TCGA data in TIMER were also determined. Finally, to further identify other poten- tial subtypes of immune cell infiltration, we also analysed the correlation between JAK1 expression and diverse immune cell markers, including monocytes, tumour-associated macrophages (TAMs), M1 macrophages, M2 macrophages, ­CD8+ T cells, B cells, neu- trophils, dendritic cells, natural killer (NK) cells, T-helper 1 (Th1) cells, T-helper 2 (Th2) cells, Tregs and exhausted T cells (https://​www.​rndsy​stems.​com/​cn/​resou​rces/​cell-​ marke​rs/​immune-​cells). Tumour purity was also determined. The gene expression level was described in terms of ­log2TAM. JAK1 expression was drawn in the x-axis, while marker genes were drawn in the y-axis. A scatterplot was used to describe the specific connection between every immune gene marker and JAK1 expression. (https://​cistr​ome.​shiny​apps.​io/​timer/) [18]. The abundances of six immune infiltrates (B cells, ­CD4+ T cells, ­CD8+ T cells, neutrophils, macrophages, and dendritic cells) were estimated by the TIMER algorithm. We evaluated the correlation between JAK1 expres- sion levels and various immune infiltrating cells via the TIMER algorithm. In addition, JAK1 expression profiles across various tumour samples and paired normal tissues from the TCGA data in TIMER were also determined. Finally, to further identify other poten- tial subtypes of immune cell infiltration, we also analysed the correlation between JAK1 expression and diverse immune cell markers, including monocytes, tumour-associated macrophages (TAMs), M1 macrophages, M2 macrophages, ­CD8+ T cells, B cells, neu- trophils, dendritic cells, natural killer (NK) cells, T-helper 1 (Th1) cells, T-helper 2 (Th2) cells, Tregs and exhausted T cells (https://​www.​rndsy​stems.​com/​cn/​resou​rces/​cell-​ marke​rs/​immune-​cells). Tumour purity was also determined. The gene expression level was described in terms of ­log2TAM. JAK1 expression was drawn in the x-axis, while marker genes were drawn in the y-axis. A scatterplot was used to describe the specific connection between every immune gene marker and JAK1 expression. Prognostic analysis We used public databases including Kaplan–Meier Plotter (https://​kmplot.​com/​analy​ sis/) [21] and PrognoScan (http://​dna00.​bio.​kyute​ch.​ac.​jp/​Progn​oScan/​index.​html) [22] to examine the relationship between JAK1 expression level and NSCLC prognosis. The Kaplan–Meier plotter is competent for assessing the effect of 54,000 genes on prognosis in 21 cancer types. Sources included the GEO, EGA, and TCGA databases. The hazard Shen et al. BMC Bioinformatics (2021) 22:471 Page 4 of 14 ratio (HR) and its 95% confidence interval (95% CI) for OS (overall survival) and PFS (progression-free survival) in NSCLC were calculated. The log-rank P value was likewise computed. Similarly, the prognostic database PrognoScan was designed to analyse the correlation between JAK1 expression and overall survival (OS). The threshold was set as a Cox P value < 0.05. Statistical analysish The results examined in TIMER and GEPIA are displayed with P values determined by t tests, fold changes, and gene ranks. Survival outcomes were presented with Kaplan– Meier plots and PrognoScan, and the results are displayed with HR and Cox P values from a log-rank test. The correlation between JAK1 expression and each gene marker was assessed by Spearman’s correlation test and statistical significance. The strength of the correlation was defined as follows: 0.00–0.19 “very weak”, 0.20–0.39 “weak”, 0.40– 0.59 “moderate”, 0.60–0.79 “strong”, and 0.80–1.0 “very strong”. For all analyses, a P value less than 0.05 indicates statistical significance. JAK1 expression in multiple human tumours We evaluated the differences in JAK1 expression in various human tumour tissues and paired normal tissues using RNA sequencing data from the TCGA. The detailed expres- sion of JAK1 in the tumour and adjacent tissues is shown in Fig. 1A. JAK1 expression was significantly decreased in BLCA (bladder urothelial carcinoma), BRCA (breast inva- sive carcinoma), COAD (colon adenocarcinoma), KICH (kidney chromophobe), LUAD, LUSC, PRAD (prostate adenocarcinoma), READ (rectum adenocarcinoma), and UCEC (uterine corpus endometrial carcinoma) compared to that in adjacent normal tissues, while the expression of JAK1 was significantly higher in CHOL (cholangiocarcinoma), ESCA (oesophageal carcinoma), HNSC (head and neck squamous cell carcinoma), KIRC (kidney renal clear cell carcinoma), KIRP (kidney renal papillary cell carcinoma), LIHC (liver hepatocellular carcinoma), STAD (stomach adenocarcinoma), and THCA (thyroid carcinoma) than that in adjacent normal tissues. Fig. 1  JAK1 expression profile in various types of human tumours. A Human JAK1 expression profile across various tumour samples and paired normal tissues from the TCGA data in TIMER. B Human JAK1 expression profile in NSCLC from the GEPIA database. The ordinate of B indicates the expression level of JAK1 ­(log2 TPM). LUAD lung adenocarcinoma, LUSC lung squamous cell carcinoma, NSCLC non-small-cell lung cancer, TPM transcripts per million, N normal, T tumour. *P < 0.05, **P < 0.01, ***P < 0.001 Fig. 1  JAK1 expression profile in various types of human tumours. A Human JAK1 expression profile across various tumour samples and paired normal tissues from the TCGA data in TIMER. B Human JAK1 expression profile in NSCLC from the GEPIA database. The ordinate of B indicates the expression level of JAK1 ­(log2 TPM). LUAD lung adenocarcinoma, LUSC lung squamous cell carcinoma, NSCLC non-small-cell lung cancer, TPM transcripts per million, N normal, T tumour. *P < 0.05, **P < 0.01, ***P < 0.001 Shen et al. BMC Bioinformatics (2021) 22:471 Page 5 of 14 To further evaluate the expression patterns of JAK1 in NSCLC, the GEPIA database was further selected. Similar results were likewise obtained, namely, JAK1 expression in LUAD and LUSC was significantly lower than that in the paired normal tissues (Fig. 1B). JAK1 expression predicts the prognosis of NSCLC Next, we explored the prognostic value of JAK1 for NSCLC by adopting two public databases. First, we investigated JAK1 expression and the prognosis of NSCLC, LUAD and LUSC using Kaplan–Meier Plotter, which principally focused on the strength of the information from the GEO, EGA and TCGA miRNA gene chips. The results showed that high JAK1 expression indicated a favourable prognosis in NSCLC (OS: HR, 0.62, 95% CI from 0.53 to 0.74, log-rank P < 0.001; PFS: HR, 0.65, 95% CI from 0.50 to 0.86, log-rank P = 0.002). In the subgroup analysis, the high expression of JAK1 in LUAD lasted longer in OS (HR: 0.74, 95% CI from 0.58 to 0.95, log-rank P = 0.017), but there was no benefit in PFS (HR: 0.83, 95% CI from 0.60 to 1.14, log-rank P = 0.24). In LUSC, high expres- sion of JAK1 was associated with longer duration of PFS (HR: 0.65, 95% CI from 0.39 to 1.09, log-rank P = 0.097), while the difference was not statistically significant. In addi- tion, there was no benefit in OS (HR: 0.95, 95% CI from 0.69 to 1.29, log-rank P = 0.73). (Fig. 2). Next, we investigated the association of JAK1 expression and prognosis with distinct clinicopathological features in NSCLC (Table 1). JAK1 overexpression related to supe- rior OS and PFS in males (HR: 0.64, 0.62, 95% CI from 0.52 to 0.79, P < 0.001) rather than females. In addition, the higher expression of JAK1 is associated with preferable OS in patients with N2 lymph node metastasis (HR: 0.39, 95% CI from 0.17 to 0.86, P = 0.016) Fig. 2  Kaplan–Meier survival curves comparing high and low expression of JAK1 in NSCLC. A, B OS and PFS survival curves of NSCLC (n = 1144, n = 596). C, D OS and PFS survival curves of LUAD (n = 672, n = 443). E, F OS and PFS survival curves of LUSC (n = 271, n = 141). OS overall survival, PFS progression-free survival, LUAD lung adenocarcinoma, LUSC lung squamous cell carcinoma, NSCLC non-small-cell lung cancer, HR hazard ratio Fig. 2  Kaplan–Meier survival curves comparing high and low expression of JAK1 in NSCLC. A, B OS and PFS survival curves of NSCLC (n = 1144, n = 596). C, D OS and PFS survival curves of LUAD (n = 672, n = 443). JAK1 expression predicts the prognosis of NSCLC E, F OS and PFS survival curves of LUSC (n = 271, n = 141). OS overall survival, PFS progression-free survival, LUAD lung adenocarcinoma, LUSC lung squamous cell carcinoma, NSCLC non-small-cell lung cancer, HR hazard ratio Page 6 of 14 Shen et al. BMC Bioinformatics (2021) 22:471 Table 1  Association between JAK1 expression and prognosis with different clinicopathological features of NSCLC by Kaplan–Meier plotter N Number, HR Hazard Ratio Clinicopathological characteristics Overall survival (n = 1144) Progression-free survival (n = 596) N HR P value N HR P value Gender  Male 659 0.64 (0.52–0.79) 2.20E−05 343 0.61 (0.43–0.86) 0.0043  Female 374 0.86 (0.61–1.21) 0.38 253 0.77 (0.49–1.20) 0.25 Stage  1 449 1.46 (1.06–2.00) 0.02 596 1.26 (0.81–1.95) 0.31  2 161 0.99 (0.63–1.55) 0.95 125 0.64 (0.38–1.11) 0.108  3 44 1.11 (0.55–2.25) 0.77 17 – – AJCC stage T  1 224 0.86 (0.57–1.28) 0.44 54 0.95 (0.23–3.53) 0.94  2 190 0.86 (0.58–1.27) 0.44 121 1.75 (0.94–3.27) 0.073  3 29 0.83 (0.38–1.85) 0.65 2 – –  4 23 0.74 (0.30–1.84) 0.52 0 – – AJCC stage N  0 324 0.87 (0.63–1.19) 0.38 126 2.18 (1.06–4.46) 0.029  1 102 0.64 (0.38–1.08) 0.09 51 0.98 (0.40–2.41) 0.96  2 32 0.39 (0.17–0.86) 0.016 0 – – AJCC stage M  0 462 0.73 (0.56–0.93) 0.013 177 1.66 (0.95–2.9) 0.07 Smoking history  Yes 300 1.59 (1.04–2.24) 0.029 297 0.73 (0.48–1.08) 0.11  No 141 2.24 (0.96–5.25) 0.056 141 1.39 (0.75–2.58) 0.29 Chemotherapy  Yes 34 0.82 (0.26–2.64) 0.74 34 1.18 (0.45–3.11) 0.74  No 21 0.18 (0.02–1.52) 0.075 21 0.63 (0.19–2.07) 0.44 Table 1  Association between JAK1 expression and prognosis with different clinicopathological features of NSCLC by Kaplan–Meier plotter without distant metastasis (HR: 0.73, 95% CI from 0.56 to 0.93, P = 0.013) of NSCLC. Notably, overexpression of JAK1 is associated with undesirable prognosis in patients with stage 1 NSCLC (OS: HR, 1.46, 95% CI from 1.06 to 2.00, P = 0.02) and without lymph node metastasis (PFS: HR, 2.18, 95% CI from 1.06 to 4.46, P = 0.029), which implicit early NSCLC patients with JAK1 overexpression may have a poor prognosis. Regrettably, there were no statistically significant differences between JAK1 expression and prognosis in females, stage 2 to 3, stage T1 to T4, N1 lymph node metastasis or prior chemotherapy. The exact survival time is shown in Additional file 1: Table S1. JAK1 expression predicts the prognosis of NSCLC hi Finally, we selected the PrognoScan database to further verify the relationship between JAK1 expression and prognosis in NSCLC. Five cohorts containing a total of 530 patients with NSCLC and LUAD showed that high expression of JAK1 was associ- ated with favourable OS (Table 2). Correlation of JAK1 expression and immune infiltration Tumour infiltrating lymphocytes (TILs) are closely related to prognosis and sub- sequent immunotherapy in lung cancer [23, 24]. We investigated the correlation between JAK1 expression level and immune cell infiltration in LUAD and LUSC Shen et al. BMC Bioinformatics (2021) 22:471 Page 7 of 14 Table 2  Survival analysis of JAK1 mRNA in NSCLC from the PrognoScan database Dataset Subtype Endpoint Number Ln (HR-high/ HR-low) COX P value ln HR HR [95% CIlow– CIup] jacob-00182- CANDF LUAD OS 82 − 1.11 0.002460 − 1.37 0.25 [0.10–0.62] GSE31210 LUAD OS 204 − 1.18 0.026306 − 1.13 0.32 [0.12–0.88] GSE11117 NSCLC OS 41 − 1.55 0.034669 − 0.78 0.46 [0.22–0.95] MICHIGAN-LC LUAD OS 86 − 1.25 0.094138 − 0.85 0.43 [0.16–1.16] GSE13213 LUAD OS 117 − 0.73 0.057336 − 0.43 0.65 [0.42–1.01] Table 2  Survival analysis of JAK1 mRNA in NSCLC from the PrognoScan database f JAK1 mRNA in NSCLC from the PrognoScan database Fig. 3  TIMER database showing the relationship between JAK1 expression level and immune infiltration in LUAD and LUSC. A JAK1 expression was negatively correlated with tumour purity and significantly positively correlated with infiltrating levels of B cells, ­CD8+ T cells, ­CD4+ T cells, macrophages, neutrophils, and dendritic cells in LUAD. B JAK1 expression was negatively correlated with tumour purity and significantly positively correlated with infiltrating levels of B cells, ­CD8+ T cells, ­CD4+ T cells, macrophages, neutrophils, and dendritic cells in LUSC. LUAD lung adenocarcinoma, LUSC lung squamous cell carcinoma. A P value less than 0.05 indicated statistical significance Fig. 3  TIMER database showing the relationship between JAK1 expression level and immune infiltration in LUAD and LUSC. A JAK1 expression was negatively correlated with tumour purity and significantly positively correlated with infiltrating levels of B cells, ­CD8+ T cells, ­CD4+ T cells, macrophages, neutrophils, and dendritic cells in LUAD. B JAK1 expression was negatively correlated with tumour purity and significantly positively correlated with infiltrating levels of B cells, ­CD8+ T cells, ­CD4+ T cells, macrophages, neutrophils, and dendritic cells in LUSC. LUAD lung adenocarcinoma, LUSC lung squamous cell carcinoma. A P value less than 0.05 indicated statistical significance Fig. 3  TIMER database showing the relationship between JAK1 expression level and immune infiltration in LUAD and LUSC. A JAK1 expression was negatively correlated with tumour purity and significantly positively correlated with infiltrating levels of B cells, ­CD8+ T cells, ­CD4+ T cells, macrophages, neutrophils, and dendritic cells in LUAD. Correlation of JAK1 expression and immune infiltration B JAK1 expression was negatively correlated with tumour purity and significantly positively correlated with infiltrating levels of B cells, ­CD8+ T cells, ­CD4+ T cells, macrophages, neutrophils, and dendritic cells in LUSC. LUAD lung adenocarcinoma, LUSC lung squamous cell carcinoma. A P value less than 0.05 indicated statistical significance Fig. 3  TIMER database showing the relationship between JAK1 expression level and immune infiltration in LUAD and LUSC. A JAK1 expression was negatively correlated with tumour purity and significantly positively correlated with infiltrating levels of B cells, ­CD8+ T cells, ­CD4+ T cells, macrophages, neutrophils, and dendritic cells in LUAD. B JAK1 expression was negatively correlated with tumour purity and significantly positively correlated with infiltrating levels of B cells, ­CD8+ T cells, ­CD4+ T cells, macrophages, neutrophils, and dendritic cells in LUSC. LUAD lung adenocarcinoma, LUSC lung squamous cell carcinoma. A P value less than 0.05 indicated statistical significance from TIMER. The results showed that JAK1 expression was negatively correlated with tumour purity (r = − 0.229, P = 2.73e-07) and significantly positively correlated with infiltrating levels of B cells (r = 0.155, P = 6.20e−04), ­CD8+ T cells (r = 0.307, P = 4.41e−12), ­CD4+ T cells (r = 0.422, P = 2.42e−22), macrophages (r = 0.342, P = 9.91e−15), neutrophils (r = 0.459, P = 1.41e−26), and dendritic cells (r = 0.479, P = 2.25e−29). Similar results were also observed in LUSC. JAK1 expression was negatively correlated with tumour purity (r = − 0.309, P = 5.34e−12) and signifi- cantly positively correlated with infiltrating levels of B cells (r = 0.224, P = 8.82e−07), ­CD8+ T cells (r = 0.26, P = 9.26e−09), ­CD4+ T cells (r = 0.517, P = 7.50e−34), mac- rophages (r = 0.405, P = 2.92e−20), neutrophils (r = 0.522, P = 1.36e−34), and den- dritic cells (r = 0.5, P = 2.20e−31) (Fig. 3). In addition, the public database TISIDB also explored the correlation between the abundance of multiple immune cells and JAK1 expression in NSCLC. The enrich- ment of diversified immune cells, such as Act_CD4, Act_DCs, iDCs, neutrophils, NK cells, pDCs, Tcm_CD4 and Tem_CD8, was positively correlated with JAK1 expression in LUAD and LUSC. What needs illustration is that JAK1 expression has Shen et al. BMC Bioinformatics (2021) 22:471 Page 8 of 14 Page 8 of 14 Fig. Correlation of JAK1 expression and immune infiltration 4  Association between JAK1 expression level and immune cell infiltration in LUAD from the TISIDB web portal (517 samples). A–H JAK1 expression was significantly positively correlated with infiltrating levels of Act_CD4, Act_DCs, iDCs, neutrophils, NK cells, pDCs, Tcm_CD4 and Tem_CD8. LUAD lung adenocarcinoma, Act_CD4 activated CD4 T cells, Act_DCs activated dendritic cells, iDCs immature dendritic cells, NK cells natural killer cells, pDCs plasmacytoid dendritic cells, Tcm_CD4 central memory CD4 cells, Tem_CD8 effector memory CD8 cells. A P value less than 0.05 indicated statistical significance Fig. 4  Association between JAK1 expression level and immune cell infiltration in LUAD from the TISIDB web ii portal (517 samples). A–H JAK1 expression was significantly positively correlated with infiltrating levels of Act_CD4, Act_DCs, iDCs, neutrophils, NK cells, pDCs, Tcm_CD4 and Tem_CD8. LUAD lung adenocarcinoma, Act_CD4 activated CD4 T cells, Act_DCs activated dendritic cells, iDCs immature dendritic cells, NK cells natural killer cells, pDCs plasmacytoid dendritic cells, Tcm_CD4 central memory CD4 cells, Tem_CD8 effector memory CD8 cells. A P value less than 0.05 indicated statistical significance no significant corrections with infiltrating levels of Act_CD4 in LUSC. For details, please refer to Fig. 4 and Additional file 1: Fig S1. Correlations between JAK1 expression and immune gene markers To further understand the interaction between JAK1 expression and TME in NSCLC. We further explored the potential correlation between JAK1 and immune gene markers in the public databases TIMER and GEPIA (Tables 3, 4). These gene markers depicted diverse immune infiltration cells, including monocytes, TAMs, M1 macrophages, M2 macrophages, ­CD8+ T cells, B cells, neutrophils, dendritic cells and NK cells. In addi- tion, various T cells, including Th1, Th2, Tregs, and T cell exhaustion, which play dif- ferent functions in the TME, were included. Although they were adjusted for tumour purity, most immune markers remained significantly related to JAK1 expression levels in LUAD and LUSC. Interestingly, the results from TIMER and GEPIA showed that most gene sets of monocytes, M1 macrophages, and TAMs were significantly associated with JAK1 expression levels in LUAD. However, we discovered that JAK1 expression was also associated with most gene sets of monocytes and TAMs rather than M1 macrophages. Notably, the majority chemokine ligand, which induced cells of the immune system to enter the site of infection, CCL-2, CD80 and CD68 of TAMs, IRF5 and NOS2 of M1, CD163 and MS4A4A of M2 were strongly related to JAK1 expression in LUAD (all P value < 0.0001). These consequences suggest that JAK1 may play a vital role in the TME by regulating the function of macrophages. In addition, some of the gene markers, such as MPO, CCR7 and CD11b (ITGAM), of neutrophils and CD8A of ­CD8+ T cells were associated with JAK1 expression in LUAD and LUSC. Page 9 of 14 Shen et al. Correlations between JAK1 expression and immune gene markers BMC Bioinformatics (2021) 22:471 Shen et al. BMC Bioinformatics LUAD lung adenocarcinoma, LUSC lung squamous cell carcinoma, TAM tumour-associated macrophage, M1 M1 macrophage, M2 M2 macrophage, Th T helper cell, Treg regulatory T cell, Cor R value of Spearman’s correlation, None correlation without adjustment, Purity correlation adjusted by purity *P < 0.01; **P < 0.001; ***P < 0.0001 Table 3  (continued) Description Gene markers LUAD LUSC None Purity None Purity Cor P Cor P Cor P Cor P Treg FOXP3 0.354 *** 0.297 *** 0.481 *** 0.402 *** STAT5B 0.46 *** 0.45 *** 0.274 *** 0.288 *** TGFβ (TGFB1) 0.487 *** 0.451 *** 0.312 *** 0.266 *** CCR8 0.422 *** 0.378 *** 0.442 *** 0.358 *** CD25(IL2RA) 0.357 *** 0.305 *** 0.368 *** 0.273 *** T cell exhaustion PD-1(PDCD1) 0.225 *** 0.144 * 0.357 *** 0.267 *** CTLA4 0.274 *** 0.197 *** 0.331 *** 0.221 *** LAG3 0.173 *** 0.104 0.021 0.257 *** 0.181 *** TIM-3 (HAVCR2) 0.371 *** 0.314 *** 0.391 *** 0.292 *** Table 4  Correlation analysis between JAK1 and relevant genes and markers of monocytes and macrophages in GEPIA Table 4  Correlation analysis between JAK1 and relevant genes and markers of monocytes and macrophages in GEPIA LUAD lung adenocarcinoma, LUSC lung squamous cell carcinoma, TAM tumour-associated macrophage, M1 M1 macrophage, M2 M2 macrophage *P < 0.01; **P < 0.001; ***P < 0.0001 Description Gene markers LUAD LUSC Tumor Normal Tumor Normal R P R P R P R P Monocyte CD14 0.3 *** − 0.051 0.7 0.27 *** 0.027 0.85 CD115 0.51 *** 0.2 0.13 0.43 *** 0.32 0.024 CD86 0.4 *** − 0.22 0.093 0.28 *** − 0.035 0.81 TAM CCL2 0.23 *** 0.16 0.24 0.23 *** − 0.02 0.89 CD80 0.35 *** 0.21 0.11 0.21 *** 0.22 0.13 CD68 0.4 *** 0.01 0.94 0.28 *** 0.14 0.32 M1 IRF5 0.25 *** − 0.1 0.43 0.0053 0.91 0.11 0.45 ROS 0.2 *** 0.51 *** 0.31 *** 0.34 0.015 NOS2 0.0033 0.94 0.56 *** − 0.042 0.36 0.49 ** M2 CD163 0.33 *** − 0.18 0.18 0.29 *** 0.074 0.61 ARG1 0.05 0.27 0.27 0.036 − 0.027 0.55 0.14 0.34 MS4A4A 0.33 *** − 0.29 0.024 0.22 *** − 0.038 0.79 Moreover, the vast majority of gene sets of dendritic cells, including HLA-DPB1, HLA-DQB1, HLA-DRA, HLA-DPA1, BDCA-1, BDCA-4 and CD11C, were positively correlated with JAK1 expression levels in LUAD and LUSC. Correlations between JAK1 expression and immune gene markers BMC Bioinformatics (2021) 22:471 from the TIMER database Description Gene markers LUAD LUSC None Purity None Purity Cor P Cor P Cor P Cor P Monocyte CD14 0.262 *** 0.2 *** 0.425 *** 0.323 *** CSF1R 0.503 *** 0.463 *** 0.553 *** 0.482 *** CD86 0.395 *** 0.344 *** 0.444 *** 0.349 *** TAM CCL2 0.254 *** 0.19 *** 0.368 *** 0.296 *** CD80 0.385 *** 0.331 *** 0.383 *** 0.294 *** CD68 0.383 *** 0.339 *** 0.382 *** 0.29 *** M1 IRF5 0.323 *** 0.277 *** 0.068 0.126 0.049 0.281 NOS2 0.22 *** 0.194 *** 0.062 0.165 0.071 0.122 M2 CD163 0.45 *** 0.414 *** 0.458 *** 0.378 *** ARG1 0.113 * 0.11 0.015 − 0.069 0.125 − 0.074 0.107 MS4A4A 0.352 *** 0.3 *** 0.345 *** 0.24 *** CD8+ T cell CD8A 0.248 *** 0.177 *** 0.262 *** 0.175 ** CD8B 0.118 * 0.051 0.257 0.188 *** 0.135 * B cell CD19 0.121 * 0.02 0.653 0.277 *** 0.146 * CD79A 0.137 * 0.047 0.295 0.323 *** 0.198 *** Neutrophils CEACAM8 0.259 *** 0.252 *** 0.072 0.106 0.048 0.293 MPO 0.22 *** 0.183 *** 0.342 *** 0.29 *** CCR7 0.361 *** 0.295 *** 0.39 *** 0.291 *** CD11b(ITGAM) 0.472 *** 0.435 *** 0.578 *** 0.517 *** Dendritic cell HLA-DPB1 0.313 *** 0.249 *** 0.457 *** 0.371 *** HLA-DQB1 0.253 *** 0.192 *** 0.328 *** 0.245 *** HLA-DRA 0.281 *** 0.215 *** 0.387 *** 0.292 *** HLA-DPA1 0.353 *** 0.3 *** 0.454 *** 0.371 *** BDCA-1(CD1C) 0.294 *** 0.243 *** 0.341 *** 0.227 *** BDCA-4(NRP1) 0.386 *** 0.377 *** 0.523 *** 0.473 *** CD11C(ITGAX) 0.383 *** 0.327 *** 0.513 *** 0.427 *** CD141(THBD) 0.366 *** 0.344 *** 0.032 0.469 − 0.006 0.89 NK cell KIR2DL1 0.046 0.296 0.023 0.612 0.143 * 0.091 0.047 KIR2DL3 0.104 0.019 0.06 0.187 0.158 ** 0.117 0.010 KIR2DL4 0.062 0.163 0.015 0.737 0.113 0.011 0.051 0.265 KIR3DL1 0.064 0.15 0.024 0.601 0.241 *** 0.187 *** KIR3DL2 0.139 * 0.087 0.053 0.141 * 0.069 0.134 KIR3DL3 0.002 0.964 − 0.019 0.67 0.029 0.52 0.002 0.962 KIR2DS4 0.119 * 0.082 0.068 0.122 * 0.093 0.043 CD7 0.168 ** 0.091 0.043 0.339 *** 0.237 *** XCL1 0.036 0.416 0.004 0.931 − 0.015 0.742 0.017 0.705 Th1 T-bet (TBX21) 0.322 *** 0.263 *** 0.363 *** 0.273 *** STAT4 0.336 *** 0.274 *** 0.49 *** 0.415 *** STAT1 0.398 *** 0.364 *** 0.298 *** 0.251 *** IFN-γ (IFNG) 0.129 * 0.06 0.181 0.121 * 0.057 0.216 TNF-α (TNF) 0.271 *** 0.229 *** 0.402 *** 0.345 *** Th2 GATA3 0.435 *** 0.387 *** 0.551 *** 0.517 *** STAT6 0.293 *** 0.318 *** 0.265 *** 0.284 *** STAT5A 0.53 *** 0.495 *** 0.549 *** 0.491 *** IL13 0.079 0.072 0.032 0.479 0.152 ** 0.083 0.072 Page 10 of 14 Shen et al. Discussionh The JAK1/STAT​ signalling pathway, as a stimulant that is intimately related to the physiological function of interferon, plays a significant role in cell growth, differen- tiation, immune regulation and other aspects [11, 25, 26]. The exhaustive function of JAK1 in NSCLC has not yet been clarified. Here, we report the expression pro- file of JAK1 and its association with prognosis and immune infiltration in NSCLC. We found that JAK1 was expressed at low levels in NSCLC, and its expression level was positively correlated with the prognosis of NSCLC, especially in LUAD. Interest- ingly, JAK1 overexpression was associated with preferable survival in males, stage N2 patients and patients without distant metastasis. In addition, increased levels of JAK1 expression are associated with undesirable survival in patients with earlier stages (stage 1 and N0), suggesting that early-stage NSCLC patients with JAK1 overexpres- sion may have a bleak prognosis. Moreover, diverse immune infiltration cells and gene sets were positively correlated with JAK1 expression level. Hence, to the best of our knowledge, our study is the first to reveal the potential mechanism by which JAK1 functions in the TME and acts as a prognostic biomarker of NSCLC. The TME plays a crucial role in the gene expression and clinical efficacy of tumour tissues, which are prerequisites and guarantees tumour immune escape [27]. The TME refers to the sum of various immune-related factors, mainly consisting of immune cells and immune-related molecules. In our study, we found that JAK1 expression was significantly positively correlated with the infiltration of various immune cells (mono- cytes, neutrophils, B cells, dendritic cells, TAMs) in LUAD and LUSC. Presently, the antitumour function of manifold cells has been extensively recognized, especially ­CD8+ T cells [28], whose number reflects the immune system’s ability to kill tumour cells to some extent. Moreover, ­CD8+ T cell density was positively correlated with the efficacy of immune checkpoint inhibitors (ICIs) in NSCLC and melanoma [29, 30]. This finding may provide an early indication for the efficacy of immunotherapy for NSCLC. Another significant part of our study is that diverse gene sets were positively corre- lated with JAK1 expression levels. First, M1 macrophage-related gene markers, such as IRF5 and NOS2, and the gene marker CD163 of M2 macrophages were strongly corre- lated with JAK1 expression. These findings suggested that JAK1 may play a role in regu- lating TAM polarization in the TME. Correlations between JAK1 expression and immune gene markers These results indicated that LAYN may regulate DCs to play a major role in the TME. Regretfully, nearly all of the gene markers of NK cells had no correlation with JAK1 expression levels. Fur- thermore, we investigated the relationship between JAK1 expression and gene sets of Tregs and T cell exhaustion. All gene sets suggested a positive correlation with JAK1 Shen et al. BMC Bioinformatics (2021) 22:471 Page 11 of 14 expression. Finally, immune checkpoints such as PD-1, CTLA4, LAG3 and TIM3 were strongly connected with the level of JAK1 expression, which suggested that JAK1 may play a role in immunotherapy for NSCLC. Further molecular biology experiment ver- ification is needed. Discussionh Second, overexpression of JAK1 is associated with a variety of T helper cells (Th1, Th2). This intense correlation may indicate that JAK1 regulates T cell function in the immune microenvironment of NSCLC. Third, our study showed a significant correlation between Treg activation (FOXP3, STAT5B, TGFB1, CCR8, CD25 in LUAD and LUSC) and induced T cell exhaustion (PD-1, CTLA-4, TIM-3 in LUAD and LUSC) and JAK1 overexpression. PD-1 (programmed death receptor 1) is a vital immunosuppressive molecule expressed on the surface of T cells that regulates the immune system and promotes tolerance by downregulating the immune system’s Shen et al. BMC Bioinformatics (2021) 22:471 Page 12 of 14 response to human cells and by suppressing the inflammatory activity of T cells [31]. Additionally, CTLA-4 and Tim-3 are expressed on regulatory T cells and exhausted T cells as crucial receptor proteins, respectively [32, 33], and both are significantly posi- tively correlated with JAK1 expression. These results suggest that JAK1 plays a potential role in recruiting immune-infiltrating cells in the TME of NSCLC. i Recent studies provide possible mechanisms which explains why JAK1 overexpression correlates with immune infiltration and superior prognosis. Previous studies have shown that JAK1 overexpression can lead to the activation of downstream interferon-stimulated genes, which can eventually exert a range of antitumour effects [34, 35]. These include increased antigen presentation by inducing proteasome subunits, activating transport- ers associated with antigen processing (TAP), stimulating major histocompatibility com- plex (MHC) molecules to be involved in antigen recognition and promoting chemokine production to exploit a first-hand antitumour role [36]. Remarkably, numerous studies have revealed that loss-of-function JAK1 mutations are insinuative of immune evasion [11, 37, 38]. Research by Shin et al. [35] showed that JAK1 mutations could induce pri- mary resistance to PD-1 inhibitors in melanoma and colon cancer patients. Rodig et al. [39] also indicated that loss of JAK1 caused perinatal death in mice. Luo et al. [40] have shown that the response of melanoma to PD-L1 inhibitor immunotherapy requires JAK1 signaling, which may be related to its potentiated IFN-γ response in vivo and in vitro. Besides, researchers also point out that human melanoma cell lines are insensitive to interferon (IFN)-induced antitumor effects after JAK1/2 knockout [41]. Consequently, JAK1 may regulate immune-related pathways that affect the prognosis and immune infil- trates of NSCLC. Concrete mechanisms have yet to be explored. However, the shortcomings of our descriptive study should be noted. Discussionh First, the sequencing data and tumour tissue chips are based on a variety of platforms and data- bases, and systematic errors and bias are inevitable. Second, our study analysed only JAK1 expression and immune cell infiltration using a variety of databases, which still needs to be verified by specific in vitro experiments. Finally, the precise regulatory path- way of JAK1 in the TME of NSCLC still needs to be further explored. In summary, the elevated expression of JAK1 is associated with superior prognosis and abundant immune cell infiltration in NSCLC. These findings may lay the foundation for immunotherapy for NSCLC. pp y The online version contains supplementary material available at https://​doi.​org/​10.​1186/​s12859-​021-​04379-y. Additional file 1: Table S1. Association between JAK1 expression and prognosis with different clinicopathological features of NSCLC by Kaplan-Meier plotter (specific survival data). Fig. S1. Correlation between JAK1 expression level and immune cell infiltration in LUSC from the TISIDB web portal (501 samples). (A-H) JAK1 expression had no signifi- cant correlation with infiltrating levels of Act_CD4 and was significantly positively correlated with infiltrating levels of Act_DCs, iDCs, neutrophils, NK cells, pDCs, Tcm_CD4 and Tem_CD8. LUSC, lung squamous cell carcinoma; Act_CD4, activated CD4 T cells; Act_DCs, activated dendritic cells; iDCs, immature dendritic cells; NK cells, natural killer cells; pDCs, plasmacytoid dendritic cells; Tcm_CD4, central memory CD4 cells; Tem_CD8, effector memory CD8 cells. A P value less than 0.05 indicated statistical significance. Supplementary Information y The online version contains supplementary material available at https://​doi.​org/​10.​1186/​s12859-​021-​04379-y. Acknowledgements Not applicable. Acknowledgements Not applicable. Page 13 of 14 Shen et al. BMC Bioinformatics (2021) 22:471 Shen et al. BMC Bioinformatics Availability of data and materials All data generated or analysed during this study are included in this article. Funding g This work was supported by Anhui Provincial Special Project of Central Government Guiding Local Science and Technol- ogy Development of China (Grant No. 201907d07050001); Funding of “Peak” Training Program for Scientific Research of Yijishan Hospital, Wannan Medical College (Grant No. GF2019J03). Authors’ contributions KS and YW wrote the main manuscript text. WL, TL and YS mainly involved in the design of articles and financial support. XJ, ZL, PZ, XW, and MF help with the making of charts and diagrams for the article. All authors read and approved the final manuscript. Author details 1 1 Department of Critical Care Medicine, The First Affiliated Hospital of Wannan Medical College (Yijishan Hospital of Wan- nan Medical College), Wuhu 241000, China. 2 Department of Respiratory Medicine, The First Affiliated Hospital of Wannan Medical College (Yijishan Hospital of Wannan Medical College), Wuhu 241000, China. 3 Department of Respiratory Medicine, Jinling Hospital, Nanjing University School of Medicine, Nanjing 210002, China. 4 Department of Radiology Medicine, The No. 2 People’s Hospital, Hefei 230000, China. Received: 24 March 2021 Accepted: 17 September 2021 Received: 24 March 2021 Accepted: 17 September 2021 Received: 24 March 2021 Accepted: 17 September 2021 References 1. William CB, Caroline C, Timothy RC, et al. Lung cancer incidence and mortality with extended follow-up in the 1. William CB, Caroline C, Timothy RC, et al. Lung cancer incidence and national lung screening trial. 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Aerosol microphysics simulations of the Mt.~Pinatubo eruption with the UM-UKCA composition-climate model
Atmospheric chemistry and physics
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cc-by
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Aerosol microphysics simulations of the Mt. Pinatubo eruption with the UM-UKCA composition-climate model S. S. Dhomse1, K. M. Emmerson2, G. W. Mann1,3, N. Bellouin4, K. S. Carslaw1, M. P. Chipperfield1, R. Hommel5,*, N. L. Abraham3,5, P. Telford3,5, P. Braesicke3,5,**, M. Dalvi3,6, C. E. Johnson6, F. O’Connor6, O. Morgenstern7, J. A. Pyle3,5, T. Deshler8, J. M. Zawodny9, and L. W. Thomason9 4Department of Meteorology, University of Reading, Reading, UK 5Department of Chemistry, University of Cambridge, Cambridge, UK 6 7National Institute of Water and Atmospheric Research (NIWA), Lauder, New Zealand 8University of Wyoming, Wyoming, USA 7National Institute of Water and Atmospheric Research (NIWA), Lauder, New Zealand 8University of Wyoming, Wyoming, USA 9NASA Langley Research Center, Hampton, Virginia, USA **now at: IMK-ASF Karlsruhe Institute of Technology, Karlsruhe, Germany **now at: IMK-ASF Karlsruhe Institute of Technology, Karlsruhe, Germany Correspondence to: S. S. Dhomse (s.s.dhomse@leeds.ac.uk) Received: 15 December 2013 – Published in Atmos. Chem. Phys. Discuss.: 28 January 2014 Revised: 16 September 2014 – Accepted: 16 September 2014 – Published: 24 October 2014 Received: 15 December 2013 – Published in Atmos. Chem. Phys. Discuss.: 28 January 2014 Revised: 16 September 2014 – Accepted: 16 September 2014 – Published: 24 October 2014 Abstract. We use a stratosphere–troposphere composition– climate model with interactive sulfur chemistry and aerosol microphysics, to investigate the effect of the 1991 Mount Pinatubo eruption on stratospheric aerosol properties. Satel- lite measurements indicate that shortly after the eruption, between 14 and 23 Tg of SO2 (7 to 11.5 Tg of sulfur) was present in the tropical stratosphere. Best estimates of the peak global stratospheric aerosol burden are in the range 19 to 26 Tg, or 3.7 to 6.7 Tg of sulfur assuming a composition of between 59 and 77 % H2SO4. In light of this large uncer- tainty range, we performed two main simulations with 10 and 20 Tg of SO2 injected into the tropical lower stratosphere. Simulated stratospheric aerosol properties through the 1991 to 1995 period are compared against a range of available satellite and in situ measurements. Stratospheric aerosol opti- cal depth (sAOD) and effective radius from both simulations show good qualitative agreement with the observations, with the timing of peak sAOD and decay timescale matching well with the observations in the tropics and mid-latitudes. Aerosol microphysics simulations of the Mt. Pinatubo eruption with the UM-UKCA composition-climate model How- ever, injecting 20 Tg gives a factor of 2 too high stratospheric aerosol mass burden compared to the satellite data, with con- sequent strong high biases in simulated sAOD and surface area density, with the 10 Tg injection in much better agree- ment. Our model cannot explain the large fraction of the in- jected sulfur that the satellite-derived SO2 and aerosol bur- dens indicate was removed within the first few months after the eruption. We suggest that either there is an additional al- ternative loss pathway for the SO2 not included in our model (e.g. via accommodation into ash or ice in the volcanic cloud) or that a larger proportion of the injected sulfur was removed via cross-tropopause transport than in our simulations. We also critically evaluate the simulated evolution of the particle size distribution, comparing in detail to balloon- borne optical particle counter (OPC) measurements from Laramie, Wyoming, USA (41◦N). Overall, the model cap- tures remarkably well the complex variations in particle con- centration profiles across the different OPC size channels. However, for the 19 to 27 km injection height-range used here, both runs have a modest high bias in the lowermost stratosphere for the finest particles (radii less than 250 nm), and the decay timescale is longer in the model for these parti- cles, with a much later return to background conditions. Also, Atmos. Chem. Phys., 14, 11221–11246, 2014 www.atmos-chem-phys.net/14/11221/2014/ doi:10.5194/acp-14-11221-2014 © Author(s) 2014. CC Attribution 3.0 License. Aerosol microphysics simulations of the Mt. Pinatubo eruption with the UM-UKCA composition-climate model S. S. Dhomse1, K. M. Emmerson2, G. W. Mann1,3, N. Bellouin4, K. S. Carslaw1, M. P. Chipperfield1, R. Hommel5,*, N. L. Abraham3,5, P. Telford3,5, P. Braesicke3,5,**, M. Dalvi3,6, C. E. Johnson6, F. O’Connor6, O. Morgenstern7, J. A. Pyle3,5, T. Deshler8, J. M. Zawodny9, and L. W. Thomason9 1School of Earth and Environment, University of Leeds LS2 9JT, UK 2CSIRO Marine and Atmospheric Research, Aspendale, Victoria 3195, Australia 3National Centre for Atmospheric Science (NCAS-Climate), UK 4Department of Meteorology, University of Reading, Reading, UK 5Department of Chemistry, University of Cambridge, Cambridge, UK 6Met Office, Exeter, UK 7National Institute of Water and Atmospheric Research (NIWA), Lauder, New Zealand 8University of Wyoming, Wyoming, USA 9NASA Langley Research Center, Hampton, Virginia, USA *now at: IUP, University of Bremen, Bremen, Germany **now at: IMK-ASF Karlsruhe Institute of Technology, Karlsruhe, Germany Correspondence to: S. S. Dhomse (s.s.dhomse@leeds.ac.uk) Received: 15 December 2013 – Published in Atmos. Chem. Phys. Discuss.: 28 January 2014 Revised: 16 September 2014 – Accepted: 16 September 2014 – Published: 24 October 2014 Atmos. Chem. Phys., 14, 11221–11246, 2014 www.atmos-chem-phys.net/14/11221/2014/ doi:10.5194/acp-14-11221-2014 © Author(s) 2014. CC Attribution 3.0 License. 1 Introduction Volcanic eruptions can have significant impacts on atmo- spheric composition and climate (e.g. McCormick et al., 1995; Robock, 2000). Powerful explosive eruptions can in- ject large amounts of SO2, ash, water vapour and various other chemical species directly into the stratosphere. Vol- canic SO2 injected into the stratosphere is chemically con- verted to sulfuric acid vapour over a timescale of days to months, causing substantial new particle formation and aerosol growth by condensation. Volcanic enhancements of the stratospheric aerosol can be long lasting, with optically active particle concentrations remaining substantially en- hanced for several years in the case of tropical eruptions (Deshler et al., 2003). The perturbed stratospheric aerosol al- ters the Earth’s radiative balance with increased albedo via enhanced back-scattering of solar radiation, cooling the sur- face and increased absorption of terrestrial long-wave radi- ation, warming the stratosphere (Labitzke and McCormick, 1992). The relative magnitude of these short-wave and long- wave radiative effects are strongly influenced by the aerosol particle size distribution (Lacis et al., 1992; Hansen et al., 1992). We use the stratosphere–troposphere composition-climate model UM-UKCA (Unified Model – UK Chemistry and Aerosol) to simulate stratospheric aerosol interactively. The model includes the GLOMAP-mode aerosol scheme and calculates aerosol optical properties online and consistently with the 3-D evolution of the particle size distribution, as driven by the underlying microphysical processes. We use the 1991 Mt Pinatubo eruption as a test case to examine simulated aerosol properties comparing to a range of satel- lite and in situ observations covering the background, vol- canic perturbation and decay periods. Pinatubo erupted in the Philippines (15.1◦N, 120.4◦E) on 15 June 1991 and was the largest tropical eruption since Krakatoa 1883. Based on column SO2 mass loadings derived from ultraviolet ra- diation measurements from the Total Ozone Monitoring Spectrometer (TOMS) instrument, Bluth et al. (1992) esti- mated that the eruption injected approximately 20 Tg into the tropical stratosphere. Guo et al. (2004a) re-evaluated the post-Pinatubo TOMS data, and also analysed measurements from the Television Infra-red Observation Satellite Vertical Sounder (TOVS), finding total SO2 released to be in the range 14 to 23 Tg (7 to 11.5 Tg of sulfur). The long-wave radiative heating induced by the thicker aerosol layer also modifies the stratospheric circulation (e.g. S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 11222 whereas the 10 Tg run compared best to the satellite measure- ments, a significant low bias is apparent in the coarser size channels in the volcanically perturbed lower stratosphere. Overall, our results suggest that, with appropriate calibration, aerosol microphysics models are capable of capturing the observed variation in particle size distribution in the strato- sphere across both volcanically perturbed and quiescent con- ditions. Furthermore, additional sensitivity simulations sug- gest that predictions with the models are robust to uncertain- ties in sub-grid particle formation and nucleation rates in the stratosphere. spheric aerosol, stratospheric aerosols are treated separately. Some models impose volcanic forcings and heating rates (e.g. Stenchikov et al., 1998) or base these on prescribed time-varying aerosol optical depth climatologies (such as Sato et al., 1993). None of the CMIP5 climate models are able to capture the complex dynamical changes associated with large tropical eruptions (Driscoll et al., 2012). There is now an established group of composition–climate models (CCMs) which simulate stratospheric chemistry with inter- active ozone radiative effects (e.g. SPARC, 2010), but few include prognostic treatment of stratospheric aerosol. Even relatively modest changes in stratospheric aerosol can exert a significant radiative forcing (e.g. Solomon et al., 2011) and expected future changes in stratospheric circulation further motivate the need for interactive stratospheric aerosol in cli- mate models. spheric aerosol, stratospheric aerosols are treated separately. Some models impose volcanic forcings and heating rates (e.g. Stenchikov et al., 1998) or base these on prescribed time-varying aerosol optical depth climatologies (such as Sato et al., 1993). None of the CMIP5 climate models are able to capture the complex dynamical changes associated with large tropical eruptions (Driscoll et al., 2012). There is now an established group of composition–climate models (CCMs) which simulate stratospheric chemistry with inter- active ozone radiative effects (e.g. SPARC, 2010), but few include prognostic treatment of stratospheric aerosol. Even relatively modest changes in stratospheric aerosol can exert a significant radiative forcing (e.g. Solomon et al., 2011) and expected future changes in stratospheric circulation further motivate the need for interactive stratospheric aerosol in cli- mate models. S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 11223 S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM a sectional aerosol microphysics module with injection alti- tude between 15.5 and 27 km, English et al. (2013) achieved good agreement with SAGE- and AVHRR-observed sAOD in NH mid-latitudes for the first 12 months after the eruption but too rapid decay in sAOD through later months. They also compared model aerosol effective radius (Reff) evolution against observations from SAGE II and in situ measurements (for e.g. Russell et al., 1996; Bauman et al., 2003), finding that peak values in the model NH tropical stratosphere oc- curred earlier than in the observations. Some of these model– observation biases in earlier studies may be linked with the transport-related issues (e.g. the lack of a quasi-biennial os- cillation, QBO) in the underlying GCM, whereas some may be linked to the simplified treatment of the particle size distri- bution. Other causes such as interactions with ash, or missing minor eruptions such as Mount Hudson in Chile (Septem- ber 1991) have also been suggested. into a peak aerosol sulfur burden uncertainty range of 3.7 to 6.7 Tg of sulfur. Taken together, these findings suggest that a large proportion of the sulfur was removed from the stratosphere within the first few months after the eruption, with potential loss pathways involving sedimentation, cross- tropopause transport out of the stratosphere (Deshler, 2008) or enhanced removal via interactions with ash or ice in the Pinatubo cloud (Guo et al., 2004b). Monthly balloon sound- ings of total and size-resolved particle concentrations carried out at Laramie, Wyoming (e.g. Deshler, 1994) showed that although substantially enhanced particle concentrations were detected in the lowermost stratosphere by mid-July, the main part of the volcanic plume was only transported to NH mid- latitudes several months later. There have been many previous global modelling stud- ies to investigate the evolution of the stratospheric aerosol following the Pinatubo eruption. However, most have used aerosol schemes that simulate only the evolution of aerosol mass, prescribing a fixed particle size distribution for sedi- mentation and radiative effects (e.g. Timmreck et al., 1999; Oman et al., 2006; Aquila et al., 2012). However, size- resolved stratospheric aerosol modules which include micro- physical processes such as new particle formation, coagula- tion and condensation have also been developed. S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM The first Pinatubo aerosol microphysics simulations were carried out in 2-D models (Bekki and Pyle, 1994; Weisenstein et al., 1997) with single-moment sectional schemes where mass in numerous size bins is transported. More recently, several 3- D general circulation models (GCMs) with aerosol micro- physics schemes have also been developed, to predict sedi- mentation and changes in radiative properties in conjunction with an evolving stratospheric particle size distribution (e.g. Timmreck, 2001; Toohey et al., 2011; English et al., 2012, 2013). Although they have near-global spatial extent, satellite measurements of sAOD and Reff constrain only integrated stratospheric aerosol properties over the full particle size range. Balloon-borne measurements (e.g. Deshler, 1994) en- able a closer examination of the particle size distribution, but are available at only a small number of sites. Here, we use both satellite and balloon-borne measure- ments to evaluate the UM-UKCA simulated stratospheric aerosol properties, and seek to provide a wider set of obser- vational constraints for the models and to better understand how the stratospheric aerosol layer was perturbed. In Sect. 2 we describe the model, including the experimental set-up and the developments to the aerosol and chemistry schemes which extend its applicability to both stratospheric and tro- pospheric conditions. Section 3 describes the measurements that are used to evaluate the model. Results and discussion about potential causes of model–observation biases are pre- sented in Sect. 4 and 5, respectively. Summary and major conclusions are presented in Sect. 6. Although there have been this development of more so- phisticated models, most of these studies evaluated their simulations against a limited set of observational data sets, primarily aerosol optical depths (AODs) derived using Ad- vanced Very High Resolution Radiometer (AVHRR) and SAGE II measurements. Using a mass-based prognostic stratospheric aerosol module in a middle-atmosphere ver- sion of the ECHAM4 climate model, Timmreck et al. (1999) showed that the two distinct maxima in AOD apparent in AVHRR- and SAGE-II-based stratospheric aerosol optical depth (sAOD) could be simulated, but the model failed to simulate the observed slow sAOD decay in the tropics af- ter the peak. Similarly, Pitari and Mancini (2002) used a GCM coupled to a global chemistry transport model with interactive aerosol microphysics, and could simulate SH sAOD reasonable well, but NH and tropical sAOD was bi- ased low. Aquila et al. (2012) used a GCM with a mass- based aerosol scheme radiatively coupled to the model dy- namics. S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM They found that simulated sAOD was higher than both AVHRR and SAGE II during the first few months, but showed very good agreement during the later phase. Using 1 Introduction Young et al., 1994), leading to indirect radiative effects via dynamical changes in ozone and meridional transport, with important implications for surface climate (Robock and Mao, 1992; Graf et al., 1993). Volcanically increased aerosol sur- face area density (SAD) can also accelerate heterogeneous chemistry perturbing stratospheric NOy species, halogens and ozone (e.g. Solomon et al., 1996). Quantifying the net impact from these direct and indirect radiative effects is very important to better understand volcanic influences within the historical climate records. g g g Assuming a 50 % conversion of SO2 to sulfuric acid by July and using an assumed size distribution and composition to convert the Stratospheric Aerosol and Gas Experiment II (SAGE II) satellite measurements of aerosol extinction, Mc- Cormick and Veiga (1992) estimated that the total global aerosol loading was increased by 20 to 30 Tg. Baran and Foot (1994) used infrared satellite measurements from the High- resolution Infrared Radiation Sounder (HIRS) instrument to derive a time series of the global stratospheric aerosol mass loading, finding a peak of 21 Tg in September 1991 with val- ues in excess of 15 Tg persisting until November 1992 and much earlier and steeper decay in the tropics than in Northern Hemisphere (NH) mid-latitudes. Lambert et al. (1993) found a peak aerosol loading of 19 to 26 Tg from the Improved Stratospheric and Mesospheric Sounder (ISAMS) measure- ments, which assuming the aerosol composition ranges from 59 to 77 % sulfuric acid (Grainger et al., 1993), translates There is an increasing recognition that having a good rep- resentation of stratospheric processes is important for climate projections (e.g. Scaife et al., 2012). However, whereas most coupled atmosphere–ocean climate models (e.g. Jones et al., 2011) that carried out historical integrations for CMIP5 (Tay- lor et al., 2012) included a prognostic treatment of tropo- Atmos. Chem. Phys., 14, 11221–11246, 2014 www.atmos-chem-phys.net/14/11221/2014/ 2.2 The aerosol microphysics module adapted for the stratosphere In order to simulate stratospheric aerosol precursor gas phase species, we have extended the existing UM-UKCA stratospheric chemistry scheme to also include sulfur chem- istry (see Sect. 2.1). The coupling to the GLOMAP-mode aerosol microphysics module (Mann et al., 2010), and its adaptation for stratospheric conditions, is described in Sect. 2.2. Surface emissions of NOx, CO and HCHO are from the RCP 4.5 scenario. Lower boundary conditions are ap- plied for CH4, N2O, CFC-11 (CFCl3) and CFC-12 (CF2Cl2) according to WMO (2011). Heterogeneous chemical reac- tions use time-varying prescribed aerosol surface area den- sity produced for the SPARC Assessment of the Strato- spheric Aerosols Properties report (SPARC, 2006). We in- clude surface and elevated emissions of anthropogenic SO2 from Lamarque et al. (2010) with also a 3-D source from passively degassing volcanoes from Andres and Kasgnoc (1998). DMS emissions are simulated interactively using a seawater concentration climatology of Kettle and Andreae (2000) with the sea–air exchange function of Liss and Mer- livat (1986). For OCS, which has a tropospheric lifetime of about 2 years (Montzka et al., 2007), we do not include an emissions source, but instead apply a fixed lower boundary condition of 550 pptm. The GLOMAP aerosol microphysics module was initially developed as a component of the TOMCAT 3-D offline Chemical Transport Model (Chipperfield, 2006) with both 2-moment sectional (Spracklen et al., 2005) and 2-moment modal versions (Mann et al., 2010) available. The computa- tionally faster modal scheme (GLOMAP-mode) was specif- ically designed for longer integrations within UM-UKCA and applies the same aerosol microphysics representations as the sectional scheme but with the size distribution pa- rameterised into seven log-normal modes, being similar in framework to that used in ECHAM-HAM (e.g. Stier et al., 2005). The GLOMAP-mode scheme produces aerosol prop- erties in good agreement with the more sophisticated sec- tional scheme under most tropospheric conditions (Mann et al., 2012). Since this study investigates the evolution of the strato- spheric aerosol layer after Pinatubo, we use only the four soluble modes and treat only sulfate and sea salt components – the latter included to give reasonable representation of tro- pospheric aerosol optical properties. For this work, the model approaches for water uptake, particle density, vapour conden- sation and new particle formation have been adapted to be applicable across stratospheric and tropospheric conditions. In the following sections, we briefly describe these updates. 2.2.1 Water uptake In the standard version of GLOMAP-mode described by Mann et al. (2010), water uptake is calculated using ZSR (Zdanovskii, 1948; Stokes and Robinson, 1966), which is not applicable in stratosphere conditions. At pressures below 150 hPa we therefore instead use the expression of Carslaw et al. (1995) to provide the aerosol water content. At 225 K and 101 hPa, the composition of the solution is 74.5 % H2SO4 and 25.5 % water, approximating the 75 % weight fraction assumed in some studies (e.g. Stenchikov et al., 1998; Oman et al., 2006). 2.1 Stratospheric chemistry extended to include the sulfur cycle The existing UM-UKCA stratospheric chemistry scheme (Morgenstern et al., 2009) covers the oxidation of CH4 and CO, with chlorine and bromine chemistry and their interac- tion with HOx, NOx and Ox cycles including heterogeneous reactions on polar stratospheric clouds (PSCs) and liquid sul- fate aerosols (Chipperfield and Pyle, 1998). Here, we have extended the scheme to also include a stratospheric aerosol precursor chemistry scheme (Weisenstein et al., 1997) with updates to reaction rates from Sander et al. (2006), see Ta- ble 1. The added chemistry includes the steady background source of SO2 from OCS, which principally maintains the stratospheric aerosol during volcanically quiescent periods (e.g. Carslaw and Kärcher, 2006). Also included are photol- ysis reactions for H2SO4 and SO3, which occur above about 30 km and lead to a reservoir of SO2 building up during po- lar winter, enabling new particle formation in the polar lower stratosphere during spring (Mills et al., 2005). The chemistry is integrated with the ASAD chemical integration package (Carver et al., 1997) with the Newton–Raphson sparse matrix solver from Wild et al. (2000). Photolysis rates are calculated using the FAST-JX online photolysis (Neu et al., 2007) fol- lowing the implementation described in Telford et al. (2013). The cross-section of H2SO4 is assumed analogous to the cross-section of HCl (× 0.016) following the method of Bekki and Pyle (1992). Aqueous sulfate production in (tropo- spheric) liquid clouds is also passed to the GLOMAP module for growth of accumulation and coarse soluble particles. 2.2.2 Particle density As composition of the aqueous sulfuric acid solution droplets also affects their density, we modified GLOMAP-mode for the stratosphere. For pressures lower than 150 hPa, density values for each mode are replaced with values from a look- up table based on the measurements of Martin et al. (2000) as a function of the sulfuric acid weight-fraction. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 2.2 The aerosol microphysics module adapted for the stratosphere S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 11224 2 Model description We use the stratospheric chemistry configuration of UM- UKCA (Morgenstern et al., 2009) which here is an extended configuration of the high-top version of the third-generation Hadley Centre Global Environmental Model (HadGEM3) (Hewitt et al., 2011) as used by Braesicke et al. (2013) and Telford et al. (2013). Although UM-UKCA can be run with fully coupled aerosol–chemistry dynamics with online ra- diative effects from the simulated aerosol, O3, CH4, N2O and other gases, here only the simulated O3 is radiatively coupled. Sea-surface temperature and sea-ice fields are pre- scribed from the AMIP time-varying data set (Hurrell et al., 2008). The simulations are carried out at N48 horizontal res- olution (2.5◦and 3.75◦in latitude and longitude) with 60 ver- tical hybrid-height levels from the surface to 84 km. Atmos. Chem. Phys., 14, 11221–11246, 2014 www.atmos-chem-phys.net/14/11221/2014/ 2.2.3 Condensation and vapour pressure of H2SO4 In all previous versions of the GLOMAP aerosol mod- ule, gas-to-particle transfer of H2SO4 occurs assuming zero vapour pressure, i.e. the transfer is represented as a conden- sation process. Although this approach is entirely appropriate Atmos. Chem. Phys., 14, 11221–11246, 2014 www.atmos-chem-phys.net/14/11221/2014/ S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 1 Table 1. Additional sulfur chemistry reactions and rates within UM-UKCA, W = Weisenstein et al. (1997), JPL = Sander et al. ( K03 = Kreidenweis et al. (2003). Rate Reference DMS + O(3P) →SO2 + 2CH3O2 1.3 × 10−11 exp(410/T ) W, JPL DMS + OH →SO2 + CH3O2 + HCHO 1.2 × 10−11 exp(−260/T ) W DMS + OH →SO2 + CH3O2 3.04 × 10−12 exp(350/T ) · (γ/1 + γ ) W γ = 5.53 × 10−31 exp(7460/T ) × [O2] DMS + NO3 →SO2 + HNO3 + CH3O2 + HCHO 1.9 × 10−13 exp(500/T ) W OCS + O(3P) →CO + SO2 2.1 × 10−11 exp(−2200/T ) W, JPL OCS + OH →CO2 + SO2 1.1 × 10−13 exp(−1200/T ) W, JPL SO2 + OH + M →SO3 + HO2 k(T ) = A 1+B × 0.6(1+(logB)2)−1 W A = 3.0 × 10−31 × (300/T )3.3 B = A/1.5 × 10−12 SO2 + O3 →SO3 3.0 × 10−12 exp(−7000/T ) W, JPL SO3 + H2O →H2SO4 8.5 × 10−41 exp(6540/T ) · [H2O] JPL SO2 + H2O2 aqueous → SO4 K03 OCS + hν →CO + SO2 Photolysis W H2SO4 + hν →SO3 + H2O Photolysis W SO3 + hν →SO2 + O(3P) Photolysis W 11225 S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM Table 1. Additional sulfur chemistry reactions and rates within UM-UKCA, W = Weisenstein et al. (1997), JPL = Sander et al. (2006), K03 = Kreidenweis et al. (2003). Table 1. Additional sulfur chemistry reactions and rates within UM-UKCA, W = Weisenstein et al. (1997), JPL = Sander et al. (2006), K03 = Kreidenweis et al (2003) Table 1. Additional sulfur chemistry reactions and rates within UM-UKCA, W = Weisenstein et al. (1997), JPL = Sander et al. (2006), K03 = Kreidenweis et al. (2003). nen and Kulmala (2002) to convert the cluster nucleation rate from Vehkamäki et al. (2002) into an “apparent nucleation rate” at 3 nm. The nucleation rate is set to zero in subsatu- rated conditions. 2.2.5 Size distribution Balloon-borne optical particle counter and condensation nu- cleus counter measurements in the mid-latitude stratosphere in the 1990s (e.g. Deshler et al., 2003) suggest a bimodal size distribution with the first mode at about 50–150 nm radius with geometric standard deviation (σg) between 1.6 and 1.8 and a larger much narrower mode (σg ∼1.2) at around 300–800 nm radius that is weak in volcanically qui- escent conditions but much stronger (in number) following the Pinatubo eruption (e.g. Carslaw and Kärcher, 2006). For example Deshler et al. (2003) show that in March 1993 (21 months after the Pinatubo eruption), in the NH mid- latitude lower stratosphere there was a 6 km layer (12–18 km) with the number concentration of particles with radii larger than 500 nm greater than 1 cm−3. Such coarse particles have grown from their original size of around 1 nm due to coagu- lation and gas-to-particle transfer of sulfuric acid. Modal mi- crophysics schemes such as GLOMAP-mode represent this condensational and coagulative growth, but must use a tech- nique referred to as “mode-merging” (e.g. Binkowski and Roselle, 2003) to transfer particles to adjacent larger modes following strong growth. In the case of a large volcanic erup- tion, the mode-merging may transfer particles first from the nucleation mode to the soluble Aitken mode, and follow- ing further growth up to the soluble accumulation mode and then to the soluble coarse mode. In each case, when particles 2.2.3 Condensation and vapour pressure of H2SO4 in tropospheric conditions, above ∼25–30 km, the vapour pressure of H2SO4 (pH2SO4) becomes significant as the tem- perature increases in the stratosphere and above ∼35 km the sulfuric acid droplets rapidly evaporate (Hamill et al., 1997; Hommel et al., 2011). We therefore now calculate pH2SO4 online in the model following Kulmala and Laaksonen (1990) and the conden- sation rates are calculated consistently with the difference between the vapour pressure and the gas phase partial pres- sure. We also apply a simple approach to particle evaporation whereby if the ambient gas phase H2SO4 partial pressure is less than pH2SO4, the number concentration for all modes is reduced at a fast decay rate of 50 % per condensation time step, which corresponds to an e-folding timescale of around 17 min. 2.2.4 New particle formation Previous versions of GLOMAP (e.g. Mann et al., 2010) formed new H2SO4–H2O particles based on the Kulmala et al. (1998) parameterisation for binary homogeneous nu- cleation. This is only applicable at temperatures in the range 233–298 K. Vehkamäki et al. (2002) suggested that condi- tions for nucleation are also favourable at ∼200 K in the up- per tropical troposphere and they updated the Kulmala et al. (1998) parameterisation to be applicable down to lower tem- peratures and humidities. To allow GLOMAP-mode to be ap- plied in both tropospheric and stratospheric conditions, we have incorporated the Vehkamäki et al. (2002) parameteri- sation, and used it within the recommended ranges of tem- perature (190 to 305 K) and H2SO4 concentration (104 to 1011 cm−3). Note that we also use the expression of Kermi- Atmos. Chem. Phys., 14, 11221–11246, 2014 Atmos. Chem. Phys., 14, 11221–11246, 2014 S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM are received from the adjacent smaller mode, the transferred number and mass is added to that existing in the mode, with the mean size reformulated reformulated consistently with the σg value for the mode. The spin-up run started from zero aerosol and gas phase sul- fur species, with other gases initialised from the UM-UKCA REF-C1 integration from the SPARC Lifetimes of Strato- spheric Ozone-Depleting Substances, Their Replacements, and Related Species report (SPARC, 2013), representative of 1990 conditions. g Kokkola et al. (2009) compared size distributions simu- lated by a modal and three sectional schemes in a box model. While the four models agreed well in background strato- spheric conditions, in volcanically perturbed conditions, the size distributions were found to be better represented with narrower mode widths. In particular, with the original coarse mode σg of 2.0, they found the modal scheme overpre- dicted the Reff compared to a reference sectional scheme with a large number of bins. Niemeier et al. (2009) used an improved version of the same modal microphysics scheme whereby σg for the accumulation soluble mode was reduced to 1.2 and the coarse mode was deactivated. To assess the robustness of the model to uncertainties in particle formation processes, which are known to be consid- erable uncertain in the stratosphere, we also carried out two sensitivity simulations with are as run B_Control10, but we switch off the sub-grid particle source (primary sulfate emis- sion) from Pinatubo (D_noPrimary10) and reduce the nucle- ation rate (E_ScaledStNuc10) in the stratosphere by a factor of 2 (by multiplying it with 0.01). Here we are applying the modal GLOMAP scheme to vol- canically perturbed stratospheric conditions, and also using the same modes to represent tropospheric aerosol. In the tro- posphere, the coarse soluble mode in GLOMAP-mode al- most exclusively contains sea-salt, and the scheme follows Wilson et al. (2001) and Vignati et al. (2004) in using a value of 2.0 for σg in this mode, which are based on values given in D’Almeida et al. (1991). 3 Measurements To evaluate the UM-UKCA simulations, we use measure- ments from the SAGE II instrument (McCormick and Veiga, 1992), which was launched on the Earth Radiation Budget Satellite (ERBS) in 1984. SAGE II was a seven-channel sun photometer operated in solar occultation mode with a verti- cal resolution of about 0.5 km. Spectral windows were cen- tred at 385, 448, 453, 525, 600, 940 and 1020 nm. For eval- uating the model stratospheric aerosol optical depth, we use the gap-filled SAGE II (V6.2) product (Hamill et al., 2006) produced for ASAP (Assessment of Stratospheric Aerosol Properties SPARC, 2006). Simulated aerosol extinction is compared against the recently updated version (V7.0) of the SAGE II data (Damadeo et al., 2013). We also com- pare to the SAGE-derived SAD product (Thomason et al., 1997) that is obtained from http://www.sparc-climate.org/ data-center/data-access/asap/. Simulated SAD is also com- pared against the recently available SAD data (Arfeuille et al., 2013) which was created using SAGE II V7.0 data, and is provided for the Chemistry Climate Model Initiative (CCMI) simulations. Further evaluation of the post-Pinatubo simulated sAOD evolution was carried out by comparing to that measured by the Advanced Very High Resolution Radiometer (AVHRR/2), which was onboard on the Na- tional Oceanic and Atmospheric Administration (NOAA/11) satellite. For details see http://www.nsof.class.noaa.gov/ release/data_available/avhrr/index.htm. The AVHRR instru- ment measures the reflectance of the Earth in five spectral bands centred around 0.6, 0.9, 3.5, 11 and 12 µm. To ensure the size distribution and vertical profile of the simulated coarse sea-salt particles is retained as evaluated in previous model versions (Mann et al., 2012), we retain the σg value of 2.0 for the coarse soluble mode. However, we now de-activate mode-merging between the accumulation and coarse soluble modes, which allows the accumulation soluble mode to continue to grow larger than 1 micron diam- eter in strongly perturbed conditions. We also retain the σg value of 1.4 for the soluble accumulation mode in GLOMAP- mode, as reduced by Mann et al. (2012) from the value of 1.59 used in Mann et al. (2010), to better compare with size distributions simulated by the sectional scheme and from ob- servations. The σg=1.59 values for the nucleation and Aitken modes are also retained. www.atmos-chem-phys.net/14/11221/2014/ 11226 S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM Table 2. Microphysical parameter settings used in model simulations. Run SO2 Nucleation Sub-grid primary injection sulphate emission A_Control20 20 Tg (19–27 km) Standard Yes B_Control10 10 Tg (19–27 km) Standard Yes C_noPinatubo No Standard Yes D_noPrimary10 10 Tg (19–27 km) Standard No E_ScaledStNuc10 10 Tg (19–27 km) ×0.01 in stratosphere Yes coarse regions of the size spectrum. The OPC is a light counter to derive integrated size distribution from measured aerosol scattering in the forward direction. The standard OPC design gives integral number concentrations larger than 150 nm and 250 nm radius, and has been used in balloon sounding measurements of stratospheric aerosol since 1963 (Rosen, 1964), also giving important information about the stratospheric aerosol changes induced by the 1963 Mt Agung (Rosen, 1964), 1980 Mt St Helen’s (Hofmann and Rosen, 1982) and 1982 El Chichón (Hofmann and Rosen, 1984) eruptions. Deshler et al. (1992) present the measurements taken in July and August 1991, with most balloon flights using this original two-channel OPC. An enhanced OPC, us- ing an increased scattering angle, measured concentrations in eight size channels for radii larger than 150 nm to around 10 microns. The eight-channel OPC had been developed shortly before the eruption, and became the default measuring sys- tem a few months after the eruption (Deshler et al., 1993). The measurement capabilities were later further enhanced to measure up to 12 size ranges (see Deshler et al., 2003). ing the evolution of the background as well as volcanically enhanced stratospheric aerosol layer. Stronger BD circula- tion leads to more rapid transport of air masses (and chem- ical species) from the tropics to high latitudes (e.g, We- ber et al. 2003; Dhomse et al. 2006). This circulation also affects aerosol removal from the stratosphere (e.g. Desh- ler, 2008) via stratosphere–troposphere exchange (STE, e.g. Holton et al. 1995). However, the strength of the BD circu- lation is also coupled with the phases of the QBO via the Holton–Tan mechanism (Holton and Tan, 1980). Using satellite observations, Trepte and Hitchman (1992) showed the importance of the QBO phase in determining the initial dispersion of the Pinatubo plume. 4 Results Stratospheric aerosol sizes and concentrations are influenced by dynamical, chemical and microphysical processes. For example, background aerosol are formed by homogeneous nucleation of H2SO4 and H2O, with H2SO4 concentrations affected by oxidation of OCS and SO2. Microphysical pro- cesses such as nucleation, condensational growth, coagu- lation and sedimentation along with large-scale poleward transport timescales affect stratospheric lifetimes of these aerosol. To ensure the model is fully evaluated, it is neces- sary to evaluate the model against a range of aerosol proper- ties, but it is also important to assess stratospheric circulation in the model and assess the evolution of key precursor gases which influence the aerosol. A common metric used to assess stratospheric transport in CCMs (e.g. Strahan et al., 2011) is the mean stratospheric age-of-air. Figure 1c shows the latitude and altitude distri- bution of the model zonal-mean age-of-air (for 1991–2000) and Fig. 1d compares the model age of air at 50 hPa against that derived from aircraft observations of the long-lived tro- pospheric source gases SF6 and CO2 (Hall et al., 1999). The values from other CCMs participating in the recent SPARC Lifetime Assessment (e.g. Chipperfield et al., 2014) are also shown for reference. In the tropics, the model age-of-air agrees well with the observations, but at mid- and high lat- itudes there is a low bias compared to the observations; up to 1 yr too young air at high latitudes. The low bias in mid- latitude age-of-air indicates that the model may have too rapid meridional poleward transport and/or stronger STE. In S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM For the simulations presented here, the model is initialised such that the lower stratospheric winds are in the easterly phase of the QBO at the time of the eruption, as observed. Figure 1a shows the time evolution of the model monthly and zonal-mean zonal wind in the tropics (15◦S–15◦N) against those from the ERA interim re-analysis from 1990 until 1995 (Fig. 1b, Dee et al., 2011). As in ERA interim, the model begins an easterly QBO phase in mid-1991, although the model easterlies are weaker than in ERA-interim in the lower stratosphere for the first 6 months after the eruption. Also, the model easterly QBO phase begins slightly later than in ERA-interim, continu- ing until around September 1993 (at 30 hPa), compared to around January 1993 in the re-analysis. The semi-annual os- cillation in the tropical middle and upper stratosphere is also well represented in the model. 4.1 Stratospheric dynamics in the UM-UKCA One of the most important factors controlling stratospheric aerosol is the stratospheric transport which is determined by the strength of the stratospheric Brewer–Dobson (BD) cir- culation. This circulation plays a crucial role in determin- Atmos. Chem. Phys., 14, 11221–11246, 2014 Atmos. Chem. Phys., 14, 11221–11246, 2014 2.3 Experimental setup For this study, we carried out several 5-year model integra- tions, as summarised in Table 2. In addition to a background run (C_noPinatubo) without any Pinatubo emission, two ref- erence simulations were carried out with 20 (A_Control20) and 10 Tg (B_Control10) of SO2 injected into the tropical stratosphere on 15 June 1991 between 19 and 27 km. To en- sure we closely match the initial spatial distribution of the aerosol cloud, we inject the SO2 across the eight model grid boxes between 0–20◦N along 120.5◦E. We emit 3 % of the SO2 mass from Pinatubo directly as sulfuric acid particles (assumed to form at the sub-grid scale) with half emitted with assumed geometric mean radius of 15 and 40 nm as in Spracklen et al. (2005). For all the simulations the entire set of tracers were initialised from fields after 8 years spin-up. To examine the simulated particle size distribution that un- derpins the model aerosol optical properties, we also com- pare to profile measurements of size-resolved number con- centration made at Laramie, USA (Deshler et al., 2003). The balloon-borne system includes a condensation nucleus counter (CNC) to measure the concentrations of particles larger than 10 nm and an optical particle counter (OPC, Deshler et al., 1992) to measure size-resolved particle con- centrations in several size ranges in the accumulation and www.atmos-chem-phys.net/14/11221/2014/ Atmos. Chem. Phys., 14, 11221–11246, 2014 11227 www.atmos-chem-phys.net/14/11221/2014/ S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM Figure 1. (a) Model-simulated tropical (15◦S–15◦N) mean monthly mean zonal wind (m s−1, QBO propagation). (b) Same as (a) but from ERA-interim reanalysis data (Dee et al., 2011). (c) Zonal mean age-of-air (years, mean 1991–2000), and (d) mean age-of-air (1991–2000) comparison at 50 hPa. Triangles and filled circles show estimated age-of-air from CO2 and SF6 (Hall et al., 1999). Mean age-of-air from various CCMs which participated in SPARC Lifetime Assessment are shown with yellow lines and the one from this study is shown with the red line. Figure 1. (a) Model-simulated tropical (15◦S–15◦N) mean monthly mean zonal wind (m s−1, QBO propagation). (b) Same as (a) but from ERA-interim reanalysis data (Dee et al., 2011). (c) Zonal mean age-of-air (years, mean 1991–2000), and (d) mean age-of-air (1991–2000) comparison at 50 hPa. Triangles and filled circles show estimated age-of-air from CO2 and SF6 (Hall et al., 1999). Mean age-of-air from various CCMs which participated in SPARC Lifetime Assessment are shown with yellow lines and the one from this study is shown with the red line. Figure 2. Time series of the global burden (in Tg of sulfur) of SO2 (red), total sulfur (includes both SO2 and aerosol, black), aerosol sulfur (dark blue) for runs A_Control20 (solid lines), B_Control10 (dashed lines), and C_noPinatubo (dotted lines). Integrated aerosol sulfur burdens in the UTS and lower–middle troposphere (deter- mined by above or below 400 hPa) are also shown with green and aqua lines, respectively. The stratospheric aerosol burden derived by Baran and Foot (1994) using HIRS measurements is shown by the green line with filled circles, assuming 75% sulphuric acid by weight. turn, such a mixing can cause too fast removal of aerosol from the stratosphere into the mid- and high-latitude tropo- sphere, and should be considered when drawing inference from the evaluation of the model post-Pinatubo stratospheric aerosol decay. www.atmos-chem-phys.net/14/11221/2014/ 11228 S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM McCormick and Veiga (1992) derived an approximate aerosol sulfur bur- den assuming a 50 % conversion from SO2 to H2SO4 by the end of July, which corresponds to an e-folding timescale of 43 days. Oman et al. (2006) found an SO2 e-folding conver- sion timescale of 35 days in their model, which used fixed OH concentrations. We note however that in the first month of the eruption there is much slower conversion to aerosol of the volcanic emitted SO2, compared to the timescale over 60 days. For example, at day 200 (34 days after the eruption) there is ∼5.6 Tg of sulfur in the form of SO2, which gives an e-folding timescale of 59 days. Bekki (1995) found that oxi- dant concentrations can be strongly depleted after very large volcanic eruptions, and in their Pinatubo simulation Bekki and Pyle (1994), found a timescale of 40 days. volume mixing ratio (pptv) d) NH mid-lat Oct 91 10-2 100 102 104 106 volume mixing ratio (pptv) 10 20 30 40 50 Height [km] c) tropics Oct 91 10-2 100 102 104 106 volume mixing ratio (pptv) 10 20 30 40 50 Height [km] c) tropics Oct 91 d) NH mid-lat Oct 91 ht [km] ht [km] Figure 3. Volume mixing ratios of various sulfur containing species Figure 3. Volume mixing ratios of various sulfur containing species (pptv) in the tropics (20◦S–20◦N, left) and NH mid-latitudes (35– 60◦N, right) during July 1991 (top) and October 1991 (bottom). Gas phase and particle phase H2SO4 ratios are shown with green and black lines, respectively. OCS and SO2 are shown with blue and purple lines, respectively. Runs A_Control20 and C_noPinatubo are shown with solid and dashed lines, respectively. Also, A_Control20 has a peak stratospheric aerosol burden of 9.3 Tg at about day 260, but the aerosol burden from B_Control10 peaks around a month earlier, at around day 230, with 5.25 Tg of sulfur, which closely matches the HIRS observations. For A_Control20 and B_Control10, we find around 6.1 Tg and 3.5 Tg of sulfur by June 1992 (12 months after the eruption, day 530, day 530, first vertical dashed line in Fig. 2), suggesting e-folding timescales of 19 and 24 months, respectively. The shorter removal timescale for the 20 Tg run is likely due to the particles growing to larger sizes compared to the 10 Tg run (e.g. as seen in Fig. S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 11229 11229 a) tropics July 91 10-2 100 102 104 106 volume mixing ratio (pptv) 10 20 30 40 50 Height [km] b) NH mid-lat July 91 10-2 100 102 104 106 volume mixing ratio (pptv) 10 20 30 40 50 Height [km] SO2 OCS H2SO4(g) H2SO4(p) c) tropics Oct 91 10-2 100 102 104 106 volume mixing ratio (pptv) 10 20 30 40 50 Height [km] d) NH mid-lat Oct 91 10-2 100 102 104 106 volume mixing ratio (pptv) 10 20 30 40 50 Height [km] Figure 3. Volume mixing ratios of various sulfur containing species (pptv) in the tropics (20◦S–20◦N, left) and NH mid-latitudes (35– 60◦N, right) during July 1991 (top) and October 1991 (bottom). Gas phase and particle phase H2SO4 ratios are shown with green and black lines, respectively. OCS and SO2 are shown with blue and purple lines, respectively. Runs A_Control20 and C_noPinatubo are shown with solid and dashed lines, respectively. a) tropics July 91 10-2 100 102 104 106 volume mixing ratio (pptv) 10 20 30 40 50 Height [km] (0.15 Tg S) found by Hommel et al. (2011). Tropospheric aerosol burdens are also higher than other models (e.g. Tex- tor et al., 2006) at around 1.25 Tg S on the annual mean. b) NH mid-lat July 91 10-2 100 102 104 106 volume mixing ratio (pptv) 10 20 30 40 50 Height [km] SO2 OCS H2SO4(g) H2SO4(p) g For run A_Control20, the global column SO2 burden decays from an immediate post-eruption peak of 10.3 Tg to around 2.0,Tg S SO2 burden on day 226 (60 days af- ter the eruption). Subtracting the 0.3 Tg SO2 mass from B_Control10 (which is all in the troposphere), gives 1.7 Tg S, indicating that 8.3 of the emitted 10 Tg S emitted as SO2 has been chemically converted to sulfuric acid over that period. We therefore estimate the e-folding timescale for conversion of SO2 into sulfuric acid aerosol as 60 divided by ln(10/1.7) which is 35 days, which agrees closely with most previous studies. For example Bluth et al. (1992) derived an e-folding timescale of 35 days from the TOMS satellite SO2 measure- ments, but present this as a tentative estimate. S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 8), and therefore sedimenting faster, moving to altitudes closer to the tropopause, where removal from the stratosphere is more effective. We note that, despite the close agreement be- tween the A_Control10 and the HIRS-derived burdens, the timescale estimates are considerably longer than values cited in the literature which range from around 12 to 14 months (e.g. see Baran and Foot, 1994 and Bluth et al., 1997). In Fig. 2, we also show a time series of the stratospheric aerosol sulfur burden derived from HIRS measurements by Baran and Foot (1994), assuming a composition of 75% sul- phuric acid by weight. For run A_Control20, we find the peak in UTS aerosol sulfur burden occurs 3 months after the eruption in September, in agreement with the timing derived from HIRS. However, the stratospheric aerosol sulfur bur- den from A_Control20 is much higher than the observations, with a maximum of 9.3 Tg of sulfur (37 Tg aerosol mass as- suming 75 % sulfuric acid composition), substantially higher than the 5.4 Tg of sulfur (21.6 Tg of aerosol) from Baran and Foot (1994). Based on ISAMS measurements, Lambert et al. (1993) estimated the post-Pinatubo peak stratospheric aerosol burden as between 19 and 26 Tg (4.75 to 6.5 Tg of sulfur). Since A_Control20 gives much too much sulfur in the stratospheric aerosol compared to both of these estimates, we carried out a second control simulation – B_Control10 with 10 Tg of SO2 (dashed line in Fig. 2). 4.3 Perturbation in sulfur species Figure 3 shows vertical profiles of mixing ratios of the three key gas phase sulfur species OCS, SO2, H2SO4, and of sulfuric acid in the particle phase (P-H2SO4), from runs A_Control20 and C_noPinatubo. The left and right panels are for mean profiles in the tropics (20◦S–20◦N) and NH mid-latitudes (35–60◦N), respectively with the top and bottom rows indicating the means for July 1991 and The stratospheric aerosol sulfur burden from B_Control10 is in good agreement with the values derived from HIRS through the second half of 1991 and the whole of 1992. How- ever, the HIRS measurements suggest a return to approxi- mately background stratospheric aerosol levels by the mid- dle of 1993, while the model aerosol shows much slower de- cay, even showing modest enhancement at the end of 1994. Atmos. Chem. Phys., 14, 11221–11246, 2014 4.2 Global burden and e-folding timescale Figure 2 shows the January 1991 to December 1994 time evolution of the daily total global column mass burden of sulfur in the gas phase (as SO2, red) and in the aerosol particle phase (blue) from runs A_Control20 (solid line), B_Control10 (dashed line) and C_noPinatubo (dotted line). Separate lines indicating the upper tropospheric and strato- sphere (UTS) aerosol sulfur burden (above 400 hPa, green lines) and that in the lower–middle troposphere (below 400 hPa, aqua lines) are also shown. From the no-Pinatubo run C_noPinatubo, the global SO2 and aerosol sulfur burdens are mostly in the troposphere, and their time series are dominated by anthropogenic emission sources, which are mainly in NH mid-latitudes. Photochemistry is strongest during summer, with higher oxidants then causing efficient conversion of SO2 to aerosol sulfate. Only 10 % of this background total sulfur burden is in the form of SO2 during the NH sum- mer, compared to around 50 % during winter. We find 30– 40 % of the total aerosol sulfur burden (around 0.5 Tg S) is in the stratosphere, which is considerably higher than the 17 % Figure 2. Time series of the global burden (in Tg of sulfur) of SO2 Figure 2. Time series of the global burden (in Tg of sulfur) of SO2 (red), total sulfur (includes both SO2 and aerosol, black), aerosol sulfur (dark blue) for runs A_Control20 (solid lines), B_Control10 (dashed lines), and C_noPinatubo (dotted lines). Integrated aerosol sulfur burdens in the UTS and lower–middle troposphere (deter- mined by above or below 400 hPa) are also shown with green and aqua lines, respectively. The stratospheric aerosol burden derived by Baran and Foot (1994) using HIRS measurements is shown by the green line with filled circles, assuming 75% sulphuric acid by weight. www.atmos-chem-phys.net/14/11221/2014/ Atmos. Chem. Phys., 14, 11221–11246, 2014 4.4 Stratospheric aerosol optical depth (sAOD) comparison 3a) show large changes in concentrations of SO2 and P- H2SO4 (between 20 and 30 km) relative to run C_noPinatubo increasing by factors 103–104 and factor 102, respectively. The enhanced P-H2SO4 profile indicates that much of the SO2 has already been oxidised and condensed into the par- ticle phase. By contrast, the NH mid-latitude July profiles show that the Pinatubo plume has not yet been transported, with SO2 and aerosol H2SO4 still at quiescent concentrations over almost the entire stratosphere, although some perturba- tion can be observed in the lowermost stratosphere and up- permost troposphere. It is notable that balloon-borne particle concentration soundings at Laramie (41◦N) in July 1991 al- ready show some enhanced layers between 15–18 km (Desh- ler et al., 1992) which corresponds well with the altitude of the SO2 and P-H2SO4 enhancement seen in the July-mean NH mid-latitude profiles. g However, consistent with Fig. 2 (more aerosol loading than estimated by Baran and Foot, 1994) the simulated sAOD in A_Control20 is much larger than both sets of observations. The sAOD distribution in B_Control10 is in better agree- ment with the satellite measurements, comparing well to both satellite measurements in mid- and high latitudes. Compar- ing to the observed sAOD enhancements in the SH, both model simulations are also in quite good qualitative agree- ment. However, in the tropics the sAOD in B_Control10 is still about 50 % larger than that derived from AVHRR, and a factor of 2 larger than SAGE II. Possible causes for these biases are discussed later in this section. The October mean SO2 profile is still strongly enhanced (factor 100) in the tropics with the P-H2SO4 enhancement only slightly higher than in July but over a much deeper layer. This tropical enhancement in both SO2 and P-H2SO4 propagates up to about 40 km, and above that only the SO2 profiles show differences between runs A_Control20 and C_noPinatubo. It is interesting that the October 1991 trop- ical gas phase H2SO4 profile from run A_Control20 actually shows lower values than in run C_noPinatubo in the main part of the plume (15–30 km), due to the condensation sink to aerosol being so much stronger. By contrast, above 30 km the increase in vapour pressure shuts off the condensation sink leading to the H2SO4 vapour concentrations being higher than quiescent at those altitudes. 4.4 Stratospheric aerosol optical depth (sAOD) comparison October 1991, selected to correspond to the 15–45 day post- eruption period when the SO2 is oxidised to H2SO4 vapour, and approximately when the peak global aerosol burden oc- curs in the model. The profile of OCS shows the expected shape, being constant in the troposphere and then reducing with increasing altitude in the stratosphere as it is photol- ysed. The SO2 profile from run C_noPinatubo shows a sharp reduction with height across the tropopause but then reaches a minimum and begins to increase with height to a local maximum at 30 km corresponding to where the source from OCS photolysis is largest. Below 30 km the sulfuric acid vapour follows a similar shape as SO2 (but at lower concen- trations) but above that altitude continues to increase up to about 40 km. Below 35 km, the vertical profile of P-H2SO4 is approximately constant in the tropics in these quiescent conditions, but has a slight decrease with altitude. In the upper-middle stratosphere rapidly evaporating particles re- lease their H2SO4 to the gas phase causing a sharp reduction in P-H2SO4 about 40 km. Figures 4a and b show the time evolution of the model mid-visible sAOD from runs A_Control20 and B_Control10 while Fig. 4c and d show the mid-visible sAOD measured by SAGE II and derived from AVHRR. Since AVHRR is a nadir viewing instrument, in Fig. 4d we have subtracted monthly-mean total AODs for the year prior to the eruption, matching the procedure recommended by Long and Stowe (1994) and used by Aquila et al. (2012). Note that the SAGE- II-derived sAOD is much lower than AVHRR in the trop- ics during the very high loading period after Pinatubo due to the measured extinction saturating at values above about 0.01 km−1 (Hamill et al., 2006). In both the A_Control20 and B_Control10 runs there is good qualitative agreement with the satellite regarding spatial and temporal distribution. For example, there is high sAOD after the eruption centred around the equator with peak sAOD in September 1991 in both model simulations and in the two satellite data sets. However, the model feature is narrower, confined between 10◦N and 10◦S. Another well-captured feature in the model is that there is no significant enhancement of sAOD in NH mid- and high latitudes until October 1991. In the tropics, the July profiles from run A_Control20 (Fig. 4.4 Stratospheric aerosol optical depth (sAOD) comparison The October 1991 NH mid- latitude SO2 and P-H2SO4 profiles show only moderate en- hancement, suggesting that the easterly phase of the QBO has prevented transport of Pinatubo-enhanced air masses. S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 4.4 Stratospheric aerosol optical depth (sAOD) comparison 11230 Atmos. Chem. Phys., 14, 11221–11246, 2014 S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 11231 Figure 4. Time series of model-simulated zonal mean sAOD at 525 nm (calculated by integrating the extinction above the tropopause) for runs (a) A_Control20 and (b) B_Control10. (c) and (d) show the sAOD measured by SAGE II (525 nm) and derived from AVHRR (600 nm) measurements. AVHRR sAOD is derived as the difference from the background total AOD from the 2 years before the eruption (Long and Stowe, 1994). Figure 4. Time series of model-simulated zonal mean sAOD at 525 nm (calculated by integrating the extinction above the tropopause) for runs (a) A_Control20 and (b) B_Control10. (c) and (d) show the sAOD measured by SAGE II (525 nm) and derived from AVHRR (600 nm) measurements. AVHRR sAOD is derived as the difference from the background total AOD from the 2 years before the eruption (Long and Stowe, 1994). average) particles sedimenting more slowly. Larger model high bias is seen for simulated tropical extinctions at 32 km, for both the runs (A_Control20 and B_Control10) that may indicate that the upper altitude used for SO2 injection was too high. At 32 km, the modelled extinction is slightly larger than SAGE II and, although peaks and troughs are mostly similar to the satellite measurements, the model variability is less than in the observations. We note again that the simulations presented here do not include the dynamical effects of aerosol-induced radiative heating. Such a radiative heating is known to cause increased tropical upwelling, which would cause greater dilution, could alter horizontal transport through the subtropical barrier and may also alter microphysical processes such as evaporation and coagulation. tropics (20◦S–20◦N). We choose these altitudes to allow comparison with the evaluation presented in Weisenstein et al. (2006, Fig. 6.20) for other stratospheric aerosol models. We compare extinction in the mid-visible (left panels) as well as the near-infrared (right panels). Here we use the updated v7.0 SAGE II data set and the profiles shown are averages between 20◦S and 20◦N. Monthly mean observed values are calculated based on both sunrise and sunset profiles. p At 20 and 25 km, both runs (A_Control20 and B_Control10) capture the general evolution of the trop- ical mid-visible extinction (Fig. 4.5 Extinction comparison Extinction profile measurements from SAGE II between July and September show (e.g. McCormick et al., 1995) that trans- port to the SH occurred mostly above about 24 km altitude. Aquila et al. (2012) highlighted the importance of resolving the enhanced tropical upwelling which occurred due to the long-wave absorption by the relatively larger stratospheric aerosol after the Pinatubo eruption. As explained in Sect. 2, in these simulations we do not radiatively couple the simu- lated aerosol with the model dynamics, and yet we capture quite well the SH post-Pinatubo sAOD evolution. We note that Aquila et al. (2012) do not include evaporation of sulfu- ric acid in their model, which could play an important role in influencing transport to SH mid-latitudes. Figure 5 shows a time series of aerosol extinction from the three model simulations (runs A_Control20, B_Control10, C_noPinatubo) and SAGE II at 32, 25 and 20 km in the Atmos. Chem. Phys., 14, 11221–11246, 2014 www.atmos-chem-phys.net/14/11221/2014/ S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 5), with the magnitude and timing of peak values, and the decay timescale, agreeing well with SAGE II. However, before the eruption (background conditions), modelled extinctions have a moderate low bias of 20–50 % at these levels. For the tropical mid-visible ex- tinction time series, run B_Control10 is in better agreement with the observations than A_Control20, which tends to be high biased (consistent with the sAOD and aerosol mass high biases seen in Figs. 2 and 4 respectively). However, against the tropical near-infrared extinction, run A_Control20 is in better agreement, with run B_Control10 generally showing modest low bias, although still in reasonable agreement. At 25 km, and for both wavelengths, the model tropical extinction peaks in August 1991, whereas in the satellite measurements, values plateau for 2–3 months before the decay period begins. In the model, the decay is fastest in the first 6–8 months after the peak value, with an approximately constant e-folding timescale from mid-1992 onwards. The faster decay in the early phase may be due to the shift in size distribution which produced larger particles at this time. Faster sedimentation would remove larger particles during this initial period, with the remaining (smaller on Figure 6 shows a similar analysis to Fig. 5, but for NH mid-latitudes (35–60◦N), again to compare against mod- els shown in Weisenstein et al. (2006). At 20 km, there is very good agreement between modelled and SAGE II ex- tinctions at both mid-visible and near-infrared wavelengths. Similarly good agreement is also observed at 25 km. How- ever, during 1993, SAGE II measurements show a signifi- cant decrease at both wavelengths which is not captured by the model. At 32 km the modelled extinction enhancement is slightly larger than the observations. We also note that whereas in the tropics the model and observations showed a faster decay phase in the first 6–8 months after the peak aerosol loading compared to the later phase, in NH mid- latitudes, both model and observations have constant ex- ponential decay timescale throughout the post-eruption pe- riod. For this latitude band, the model shows much better agreement with SAGE II measurements than seen in any of Atmos. Chem. Phys., 14, 11221–11246, 2014 www.atmos-chem-phys.net/14/11221/2014/ Atmos. Chem. Phys., 14, 11221–11246, 2014 11232 S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 10-6 10-5 10-4 10-3 10-2 10-1 ext [km-1] SAGE II (525nm) A_Control20 B_Control10 C_noPinatubo Tropics (550nm) 32km 10-6 10-5 10-4 10-3 10-2 10-1 Tropics (1020nm) 32km 10-6 10-5 10-4 10-3 10-2 10-1 ext [km-1] 25km 10-6 10-5 10-4 10-3 10-2 10-1 25km 1990 1991 1992 1993 1994 1995 1996 10-6 10-5 10-4 10-3 10-2 10-1 ext [km-1] 20km 1990 1991 1992 1993 1994 1995 1996 10-6 10-5 10-4 10-3 10-2 10-1 20km Figure 5. Comparison between modelled and SAGE II (V7.0) retrieved extinction at 525 nm (left) and 1020 nm (right) in the tropics (20◦S– 20◦N) for 20 km (bottom), 25 km (middle) and 32 km (top). Extinctions from runs A_Control20, B_Control10 and C_noPinatubo are shown with red, orange and blue lines, respectively. The vertical black lines show the range of plus or minus one standard deviation over the individual measurements used in the calculation of the monthly mean. 10-6 10-5 10-4 10-3 10-2 10-1 Tropics (1020nm) 32km 10-6 10-5 10-4 10-3 10-2 10-1 25km 1990 1991 1992 1993 1994 1995 1996 10-6 10-5 10-4 10-3 10-2 10-1 20km Tropics (1020nm) Figure 5. Comparison between modelled and SAGE II (V7.0) retrieved extinction at 525 nm (left) and 1020 nm (right) in the tropics (20◦S– 20◦N) for 20 km (bottom), 25 km (middle) and 32 km (top). Extinctions from runs A_Control20, B_Control10 and C_noPinatubo are shown with red, orange and blue lines, respectively. The vertical black lines show the range of plus or minus one standard deviation over the individual measurements used in the calculation of the monthly mean. 10-6 10-5 10-4 10-3 10-2 10-1 ext [km-1] SAGE II (525nm) A_Control20 B_Control10 C_noPinatubo NH mid-lat (550nm) 32km 10-6 10-5 10-4 10-3 10-2 10-1 NH mid-lat (1020nm) 32km 10-6 10-5 10-4 10-3 10-2 10-1 ext [km-1] 25km 10-6 10-5 10-4 10-3 10-2 10-1 25km 1990 1991 1992 1993 1994 1995 1996 10-6 10-5 10-4 10-3 10-2 10-1 ext [km-1] 20km 1990 1991 1992 1993 1994 1995 1996 10-6 10-5 10-4 10-3 10-2 10-1 20km Figure 6. Same as Fig. 5 but for NH mid-latitudes (35–65◦N). 10-6 10-5 10-4 10-3 10-2 10-1 NH mid-lat (1020nm) 32km 10-6 10-5 10-4 10-3 10-2 10-1 25km 1990 1991 1992 1993 1994 1995 1996 10-6 10-5 10-4 10-3 10-2 10-1 20km Figure 6. Same as Fig. www.atmos-chem-phys.net/14/11221/2014/ 5 but for NH mid-latitudes (35–65◦N). portion of aerosol is removed in the tropics in the 20 Tg run than in 10 Tg run (likely related to stronger sedimentation). As we also saw in the tropics, in the NH mid-latitudes, at portion of aerosol is removed in the tropics in the 20 Tg run than in 10 Tg run (likely related to stronger sedimentation). As we also saw in the tropics, in the NH mid-latitudes, at the simulations submitted for the Pinatubo intercomparison in SPARC (2006). Interestingly, differences between runs A_Control20 and B_Control10 are much smaller at this lati- tude band than in the tropics (Fig. 5), suggesting a larger pro- Atmos. Chem. Phys., 14, 11221–11246, 2014 www.atmos-chem-phys.net/14/11221/2014/ S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 11233 1 Figure 7. Comparison between zonal mean modelled (run A_Control20) and satellite-derived V1 and V2 SAD (µm2 cm−3) from SPARC (2006) and Arfeuille et al. (2013), respectively, for various months before and after the eruption. one can see weak meridional transport to NH and SH sub- tropics at about 20–22 km. Young et al. (1994) showed that including the aerosol radiative effects on the model dynamics broadens the latitudinal extent of the Pinatubo cloud which, in our simulations would improve agreement with the satel- lite observations. And as mentioned earlier, such a heating can alter local circulation and may partially explain the SAD high biases seen here. By January 1992, the model high bias has reduced to a factor of 2, and the model shows meridional transport to NH mid-latitudes in the lowermost stratosphere, also seen in the observations. However, the satellite-derived SAD suggests meridional transport also occurs to the SH, but at slightly higher altitudes. By May 1992, high biases in modelled SAD are much smaller and the general latitu- dinal and altitudinal distribution is still in good qualitative agreement with the observations, aside from the continued low bias in the SH. Also, as observed in Fig. 2 (younger age- of-air), in the lowermost stratosphere, the model seems to have too much diffusion near the tropopause. Hence the dis- tinct cross-tropopause gradients seen in satellite data are not seen in our simulations. www.atmos-chem-phys.net/14/11221/2014/ While interpreting the model–observation SAD discrep- ancies, one should consider how the satellite SAD product is derived from the SAGE I, SAGE II, SAM II (Stratospheric Aerosol Instrument II) and SME (Solar Mesosphere Ex- plorer) measurements. As noted earlier, the extinction mea- sured by the SAGE and SAM instruments has an upper limit of 0.01 km−1, above which the atmosphere is effectively opaque to the instruments (Hamill et al., 2006). During the peak aerosol loading period, when the model SAD is a factor of 2 high biased, it is apparent (for example in Fig. 5) that the SAGE II 525 nm and 1020 nm extinctions in the tropi- cal lower stratosphere are saturating at the upper limit value, with actual extinction values likely to have been higher. The late-1991 to 1992 period was flagged as missing data in the original SAGE II extinction data set. The data gaps during that period were addressed by Hamill et al. (2006), who used lidar data from two tropical sites (Camaguey, Cuba and Mauna Loa, Hawaii) and two mid-latitude sites (lidar from NASA Langley, Virginia, USA and backscatter sonde from Lauder, New Zealand), to fill the missing data. Figure 7. Comparison between zonal mean modelled (run A_Control20) and satellite-derived V1 and V2 SAD (µm2 cm−3) from SPARC (2006) and Arfeuille et al. (2013), respectively, for various months before and after the eruption. 20 km, the 1020 nm extinction from run A_Control20 shows better agreement with SAGE II than B_Control10. 4.6 Surface area density comparison Another important issue to consider with the SAGE-II- derived SAD product is that, even outside the gap-filled part of the data set, particles smaller than 50 nm are essentially invisible to the satellite and there is little sensitivity to parti- cles smaller than 100 nm. For example, Reeves et al. (2008) derived extinction, SAD and volume concentration from air- craft measurements of the aerosol particle size distribution (in quiescent conditions) and compared to SAGE II prod- ucts. They found that the aircraft measured SAD was a fac- tor 1.5–3 higher than the SAGE-II-derived values, whereas volume concentrations were only 35 % higher.The apparent SAD high bias in the model may therefore partly be caused by the contribution from smaller particles which are not ob- servable from the SAGE II instrument Figure 7 compares the vertical and latitudinal distribution of zonal mean SAD from run A_Control20 against two ver- sions of the satellite-derived SAD data set, for 4 selected months between May 1991 and May 1992. Before the erup- tion (May 1991), the model captures the global SAD distri- bution reasonably well compared to the SAGE-derived data sets, although model values are higher in the upper tropo- sphere and lower stratosphere (UTLS) region. For Septem- ber 1991 (3 months after the eruption), although the simu- lated SAD distribution broadly matches the observed shape, it is up to a factor 2 to 3 high in the tropics. Also, the model Pinatubo-enhanced SAD plume is strongly confined to the tropical pipe, whereas in the satellite-derived SAD (Fig. 7d) 4.8 Particle size distribution However, above 25 km the model is not able to simulate steeper decreases observed in vertical profiles of N150 and N250. Intriguingly, in November 1991 (Fig. 9d) run A_Control20 predicts only slightly higher N150 and N250 than run B_Control10, with particle concentrations at larger sizes showing a much larger relative enhancement in the 20 Tg run than the 10 Tg run. The size distribution simulated by the model is generally in good agreement with the ob- servations, although in August and September 1991, for the peak at about 18 km, the model N550 are low biased com- pared to the observations, and there is a general overpredic- tion of N5 in this initial post-eruption phase. The low bias in the larger sizes could be related to the high bias in the smallest sizes, with the condensation sink being shared out across a larger number of particles leading to reduced parti- cle growth. Another possibility is that there is faster STE as observed in the age-of-air comparison (Fig. 1c and d). Obser- vations also show approximately constant N5 between 20 and 30 km, but run A_Control20 (B_Control10) have elevated Figure 8 shows the evolution of the model zonal-mean Reff at 20 and 25 km from runs A_Control20 and B_Control10 compared to that derived by Bauman et al. (2003) from the SAGE II and CLAES satellite measurements. The gen- eral spatial and temporal evolution of the model Reff is in good qualitative agreement with the observations in both runs, with values at 20 km larger than at 25 km, likely due to sedimentation. In the tropics, at both altitudes, the ob- servations suggest that, whereas sAOD and extinction are decaying by November or December 1991 (Figs. 4 and 5), the effective radius peaks several months later (early 1992) with only a slow decay beginning later in 1992. By contrast, in NH mid-latitudes, the observations suggest that the de- cay in effective radius is slightly earlier and occurs faster. Both simulations capture the timing of these Reff peaks well, although at 25 km, the model peak is later than observed, matching the timing at 20 km. Effective radius values are always higher in the tropics than at mid-latitudes, a feature that is consistent between the model and observations. 4.8 Particle size distribution Reff = Pm i=1 Nir3 gi exp  9/2(lnσi)2 Pm i=1 Nir2 gi exp  2(lnσi)2 . (1) To give a stronger observational constraint on the simulated size distribution, we compare the model against balloon- borne CNC and OPC measurements made at Laramie, Wyoming, USA (41◦N, see Sect. 3). Figures 9 and 10 com- pare model profiles of size-resolved number concentrations (larger than a given particle diameter) against those mea- sured by the CNC and OPC. In each case we are com- paring a monthly-mean size-resolved particle concentration from the model to a single balloon sounding. Note also that whereas the number concentration profiles for particles larger than 5 nm, 150 nm and 250 nm are exactly as measured by the OPC, for the larger size channels we have interpolated the observations (linearly in log N vs. log R space) onto reg- ular Dp > 550 nm, 750 nm and 1000 nm size channels from the irregular size thresholds given in the individual sounding data files. (1) The two gap-filled SAGE/SAM extinction data products provide 3-D time-varying volume concentration and SAD which together give Reff throughout the Pinatubo period. This record therefore has the potential to provide informa- tion on how the particle size distribution in the stratosphere was perturbed by the eruption. However, again, when com- paring the model to the satellite Reff, the limitations associ- ated with the derived product need to be considered. In par- ticular, because of the “blind spot” associated with particles smaller than 50–100 nm, Hamill et al. (2006) state that since the derived SAD may have an inherent low bias (whereas the derived volume density will be less affected) the derived Reff may overestimate the true value. Figure 9 shows the observed (plus signs) profile evolution of the particle size distribution through August to Novem- ber 1991, for the period after the Pinatubo plume was first detected at Laramie on 16 July (Deshler et al., 1992). In August and September 1991, both runs A_Control20 and B_Control10 show elevated values of N150 and N250 be- tween 14 and 20 km, whereas at higher altitudes (above 25 km) the profile remains close to background values (not shown). The region with elevated N150 and N250 profiles matches reasonably well with the observations, and indicates efficient transport of air from the tropics in the lowermost stratosphere. 4.7 Effective radius comparison tudes and before the eruption, which suggests that it is not associated with sedimentation, since that would be expected to occur mostly during the highest loading period. There ap- pears to be a more persistent bias in simulated particle size distribution, but it is unclear whether the model has too many small particles, or too few large particles. Another product derived from the gap-filled satellite extinc- tion record, that can be used to assess the evolution of the stratospheric aerosol properties following the Pinatubo erup- tion, is Reff, defined as the ratio of the third and second inte- gral moments in radius, and which for multimodal distribu- tion can be represented as (Russell et al., 1996, Eq. 6) www.atmos-chem-phys.net/14/11221/2014/ Atmos. Chem. Phys., 14, 11221–11246, 2014 11234 4.8 Particle size distribution How- ever, although Reff from run A_Control20 is slightly larger than from B_Control10, modelled values are up to 30–40 % smaller than those derived from the satellite, with maximum model Reff of around 0.4 and 0.35 µm, compared to around 0.6 µm from the satellites. At 20 km (Fig. 8b), despite combining the two sets of satel- lite products, there is no observational constraint on the trop- ical Reff between approximately June 1991 and August 1992, but the overall shape suggests the Reff was likely even larger than 0.6 µm during that period. The model low bias in Reff is apparent at about the same extent at all latitudes and alti- Atmos. Chem. Phys., 14, 11221–11246, 2014 www.atmos-chem-phys.net/14/11221/2014/ S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 11235 Figure 8. Satellite-derived (shaded, from Bauman et al., 2003) and modelled (contours) effective radii (Reff) in µm at 25 and 20 km from runs A_Control20 (panels a and b) and B_Control10 (panels c and d). Figure 8. Satellite-derived (shaded, from Bauman et al., 2003) and modelled (contours) effective radii (Reff) in µm at 25 and 20 km from runs A_Control20 (panels a and b) and B_Control10 (panels c and d). longer-timescale evolution of the size distribution through the Pinatubo period, comparing soundings in March 1992, 1993 and 1994 (Fig. 10b, c and d). Before the eruption, the observations show that N5 decreases with increasing altitude between 12 and 23 km, whereas N150 and N250 show a much slower decrease with height. This feature is well captured by the model with N5 and N150, in excellent agreement with the observations in this altitude range, although N250 has a slight low bias. Between 25 and 30 km, the observed N5 profile shows a layer of enhanced concentrations, by around a fac- tor of 10 compared to a continuation of the decrease seen at lower altitudes. This layer indicates a source of freshly nucle- ated particles which have not grown to larger sizes. The simu- lated N5 profile also shows this feature, but the enhancement of particle concentrations is much stronger in the model, and extends to lower altitudes, down to around 20km. Gas phase sulfuric acid concentrations are known to increase rapidly with height in this region from balloon-borne ion mass spec- trometer measurements (e.g. Arnold et al., 1981). These ele- vated concentrations of gas phase H2SO4 have been shown to cause significant nucleation in the middle stratosphere (Hom- mel et al., 2011) which is almost certainly the cause of this feature. The high bias in the model N5 profile in this en- hanced layer likely indicates that nucleation is too strong in the model. The overpredicted nucleation rate in these vol- canically quiescent conditions may be a result of gas phase concentrations of H2SO4 being too high in the model. Pos- sible explanations might be that the model does not include the sink for gas phase H2SO4 provided by meteoric debris, N5 between 20 and 25 km. 5 Discussion In Sect. 4.2 we found that injecting 20 Tg SO2 into the tropi- cal stratosphere substantially overestimates the stratospheric aerosol sulfur burden, with a 10 Tg SO2 injection in much better agreement with observations. Most previous modelling studies of the Pinatubo eruption have also tended to inject 20 Tg of SO2, and we show here that the high bias in our model is also found in other studies. Oman et al. (2006) and English et al. (2013) found peak stratospheric sulfuric acid aerosol burdens of 27 and 24 Tg respectively, translating to 36 and 32 Tg aerosol mass assuming 75 % weight sulfuric acid, similar to our 37 Tg peak value. Niemeier et al. (2009) injected 17 Tg of SO2, and their 30 Tg peak stratospheric aerosol burden also agrees with our simulation, accounting proportionally for the reduced sulfur source. We note also that Niemeier et al. (2009) and English et al. (2013) have presented the HIRS stratospheric aerosol burden time series from Baran and Foot (1994) assuming the mass burden is for sulfuric acid, without accounting for the fraction of water content implicit in those values. In March 1993 (Fig. 10c), the observations show clear separation between N5 and N150, although N150 and N250 are close together. This indicates the formation of a bimodal size distribution consisting of an external mixture of particles which have grown to larger sizes following condensation af- ter oxidation of volcanic SO2 and a separate sub-population of particles less influenced by the eruption. Observed pro- files of N550, N750 and N1000 show peak values at around 12km at this time, much lower altitudes than at March 1992 (Fig. 10b). It is interesting that the March 1993 N550 and N750 profiles are higher in the 10–15 km region than in March 1992, likely indicating that, although slow at these particle sizes, sedimentation is transporting the particles to lower altitudes over these longer timescales. The model cap- tures the observed size distribution fairly well, with N250 in quite good agreement with the measurements. However, the model N150 profile has a high bias of around a factor of 2 dur- ing March 1993 and is still together with the N5 profile be- tween 15 and 20 km. S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 11236 shown to be important by Saunders et al. (2012) and Brühl et al. (2013) or underestimated H2SO4 photolysis rates in our simulations. lated profiles not capturing the increase in particles larger than N550 in the lowermost stratosphere. By March 1994 (Fig. 10d) the OPC measurements show that there has been a general decay in all size channels towards background con- ditions. The model N150 high bias seen in March 1993 has worsened with the decay rate at these channels slower than in the observations. In the N550, N750 and N1000 channels, the model continues to have a low bias in both simulations. It is notable that throughout the period, the model N150 and N250 profiles are remarkably similar between the A_Control20 and B_Control10 simulations, with much larger differences in the coarser sized particles. In March 1992 (Fig. 10b), 9 months after the eruption, the observed particle concentration profiles show major en- hancements throughout the upper troposphere and lower stratosphere (10 to 25 km), for size channels 150 nm and larger. By contrast, N5 shows a slight decrease compared to March 1991, and is only marginally higher than N150 and N250 for this month, suggesting that a large propor- tion of the particles have grown to sizes larger than 250 nm. The enhanced profiles of N550, N750 and N1000 are approx- imately constant between 15 and 20 km with a fast decrease at higher altitudes. Model run A_Control20 (solid line) cap- tures this volcanically enhanced particle size distribution re- markably well, with good qualitative and quantitative agree- ment across all the size channels in the main part of the plume. Run B_Control10 (dashed line) also captures well the N5, N150 and N250 profiles, but is low biased in the larger size channels. Despite generally very good agreement with the Laramie OPC data at this time, in the lowermost stratosphere and upper troposphere (between 10 and 15 km), both model runs show a high bias in N150 and N250. We saw from the previous comparisons that run A_Control20 has too high a burden in the stratospheric aerosol compared to the HIRS and ISAMS satellite measurements (Fig. 2) and that it is strongly biased high in aerosol optical depth against the SAGE II and AVHRR data (Fig. 4). S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM The comparisons to the OPC data sug- gest that the high sAOD bias originates from the overpre- dicted particle concentrations in the 150 to 550 nm radius range in the lowermost stratosphere, with coarser particles in that part of the atmosphere in reasonable agreement (run A_Control20) or showing low bias (run B_Control10). S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM The observations show that con- centrations of particles at 150 nm and larger reduce sharply above 25 km, whereas the model profiles show only moder- ate decline. This suggests that the simple approach to particle evaporation may need improving. N5 between 20 and 25 km. The observations show that con- centrations of particles at 150 nm and larger reduce sharply above 25 km, whereas the model profiles show only moder- ate decline. This suggests that the simple approach to particle evaporation may need improving. p y p g For November 1991, the run A_Control20 shows enhance- ment up to 25 km for all the particle size thresholds, with the coarse mode higher than the run C_noPinatubo in the low- ermost stratosphere (not shown) and the shape of the verti- cal profile for each channel compares well with the obser- vations. The model also shows an enhanced layer of N5, N150 and N250 at about 35km, suggesting transport of the Pinatubo plume to mid-latitudes throughout the lower and middle stratosphere. In both the model and observations, in these initial months, there is a layer where the N5 and N150 lines come together, reflecting that few particles remain smaller than 150 nm and indicating that particle growth at these sizes is strongest in that part of the stratosphere. We note however that in the observations this confluence occurs at around 20 km, whereas in the model this occurs around 16–17 km. This discrepancy in altitude could be reflecting the chosen injection height range, or be related to transport deficiencies in the model, and the general good qualitative agreement with the observations suggests that the modal ap- proach to aerosol dynamics is capturing the evolution of the size distribution rather well. In order to evaluate the model size distribution profile in quiescent conditions, we compare to the Laramie balloon measurements in March 1991 (Fig. 10a). We then probe the Atmos. Chem. Phys., 14, 11221–11246, 2014 www.atmos-chem-phys.net/14/11221/2014/ S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 11237 Figure 9. August, September, October, November 1991 profiles of size-resolved number concentrations of particles (cm−3) with radii larger than 5, 150, 250, 550, 750 and 1000 nm from Laramie (41.3◦N, 105.5◦W) are shown with plus (+) symbol. Solid and dashed lines show aerosol profiles from the runs A_Control20 and B_Control10, respectively, highlighting the region where the model predicts perturbation in the aerosol profiles. Horizontal coloured lines represent standard deviations (1σ) in number concentrations for a given month calculated from daily values for run A_Control20. Figure 9. August, September, October, November 1991 profiles of size-resolved number concentrations of particles (cm−3) with radii larger than 5, 150, 250, 550, 750 and 1000 nm from Laramie (41.3◦N, 105.5◦W) are shown with plus (+) symbol. Solid and dashed lines show aerosol profiles from the runs A_Control20 and B_Control10, respectively, highlighting the region where the model predicts perturbation in the aerosol profiles. Horizontal coloured lines represent standard deviations (1σ) in number concentrations for a given month calculated from daily values for run A_Control20. Figure 10. Same as Fig. 9 but for March 1991, March 1992, March 1993 and March 1994. Figure 10. Same as Fig. 9 but for March 1991, March 1992, March 1993 and March 1994. the scattering efficiency of of incoming solar radiation. These altered radiative effects illustrate the importance of resolv- ing aerosol particle size changes and subsequent feedback on the scattering efficiency of of incoming solar radiation. These altered radiative effects illustrate the importance of resolv- ing aerosol particle size changes and subsequent feedback on a larger Reff will have caused important changes in the radia- tive properties of the stratospheric aerosol, with significant absorption of outgoing terrestrial radiation and a decrease in a larger Reff will have caused important changes in the radia- tive properties of the stratospheric aerosol, with significant absorption of outgoing terrestrial radiation and a decrease in Atmos. Chem. Phys., 14, 11221–11246, 2014 5 Discussion Also, simulated particle concentrations in the larger size channels have a strong low bias of around a factor of 10 (run A_Control20) or 20 (run B_Control10) in the lowermost stratosphere at this time, with the simu- For our 10 Tg Pinatubo simulation, we found generally good agreement with observed sAOD (section 4.4), extinc- tion (section 4.5), and SAD (section 4.6). Our 20 Tg simu- lation gives consistently too high sAOD in the tropics, mid- latitudes and polar regions, whereas in most of the previous studies mentioned above, reasonable agreement is found in peak AOD, despite the high bias in stratospheric aerosol bur- den. We note however that there is a considerable diversity in the injection height-range, latitudinal spread and duration of the volcanic source used in these different model experi- ments. The comparisons against the balloon measurements (Figs. 9 and 10) show that our model captures well the gen- eral evolution of the particle size distribution in the strato- sphere through the Pinatubo period. The observations indi- cate how the huge injection of SO2 led after the eruption to the growth of some particles to sizes larger than 1 µm at peak loading (e.g. Fig. 10b), with a long-lasting perturbation to concentrations larger than 150 nm causing a complex evo- lution of Reff (Fig. 8). The shift in the size distribution to Atmos. Chem. Phys., 14, 11221–11246, 2014 www.atmos-chem-phys.net/14/11221/2014/ S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM Also, our model has too young age-of-air in mid-latitudes (see Fig. 1d) which may also be affecting the simulated transport and particle size evolution. Finally, we also note that nucleation rates at the very low humidity and temperature conditions in the strato- sphere are known to be highly uncertain. The Vehkamäki et al. (2002) parameterisation used in this paper is the best available for stratospheric conditions, but is essentially an extrapolation from laboratory measurements at much higher temperatures and humidities, based on classical nucleation theory. Figure 11 suggests that, following Pinatubo, strong nu- cleation occurred throughout the injection height range of 19–27 km for around 6 weeks after the eruption. Although twice as much SO2 is injected in A_Control20 than in B_Control10, the nucleation rates in the two runs are simi- lar for July 1991. This could possibly be indicative of a de- pletion of oxidants which is limiting SO2 oxidation at this time, although an alternative explanation might be that there is much more surface area in the 20 Tg injection run to act as a condensation sink for sulfuric acid vapour. Nucleation rates then reduce in magnitude through August and Septem- ber as the emitted SO2 is completely converted to sulfuric acid and there is a substantial surface area to provide a con- densation sink of H2SO4. By October 1991, nucleation rates in A_Control20 and B_Control10 have returned to similar values to those found in the quiescent C_noPinatubo simu- lation. Following the eruption of Mount Pinatubo, the bal- loon observations at Laramie indicate that, by March 1992 (e.g. Fig. 10b), N150 is increased by a factor of 8, whereas N5 has already returned to pre-eruption values, consistent with the reduced nucleation rate seen here. As a consequence, N5 and N150 profiles are separated by only a few tens of percent, indicating that the majority of particles in the lower strato- sphere have grown larger than 150 nm at that time. This fea- ture was well captured by the model in runs A_Control20 and B_Control10 with the N5, N150 and N250 profiles being remarkably similar between the two runs. Our study is the first to fully examine the variation in sim- ulated particle size distribution through the Pinatubo post- eruption period, and we therefore choose to document the nu- cleation rate occurring in our simulations. S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 11238 dynamics in stratospheric CCMs, and we aim to include and assess the impact of these feedbacks in a future study (Mann et al., in prep., 2014). cleated particles grow to sizes larger than 150 nm, with most remaining at smaller sizes. We note however that nucleation can be seen in the middle stratosphere at SH mid-latitudes in the volcanically quiescent C_noPinatubo September 1991 monthly-mean, indicating the occurrence of nucleation in springtime, as seen in the McMurdo OPC record (Campbell and Deshler, 2014). Note that the mechanism here is that par- ticle evaporation and subsequent photolysis of sulfuric acid leads to a reservoir of SO2 building up during polar winter, which leads to new particle formation in polar spring (Mills et al., 2005). This is the same mechanism that is leading to the layer of elevated N5 at 25–30 km in the March Laramie profiles (see Fig. 10). p p Our simulations here indicate that the model is capable of capturing the main features of the observed evolution of the particle size distribution very well, with particularly good agreement with the measurements in the most perturbed post- eruption period through to mid-1992. However, Fig. 10c and d suggest that the decay phase is not well captured, with N150 reducing much more slowly than the measurements and the return to a background size distribution occurs much later in the model. We have seen that simulated particle concen- trations in the 5–250 nm size range, whilst agreeing well in background conditions, have moderate high bias in the first year after the eruption, with the bias worsening as the model decays too slowly in the subsequent period. There are sev- eral possible causes for this model size distribution bias. It could be that the simplified modal representation of aerosol dynamics may be only partly capturing the different parti- cle growth and removal rates across the particle size range. However, it is also worth noting that the largest biases oc- curred in the lowermost stratosphere and upper troposphere where STE-related processes may not be well captured in our low-resolution GCM. Another related issue is that we again note that these simulations do not include the coupling to dy- namics which would increase the altitude of the aerosol layer and reduce concentrations in the lower part of the plume, where the high bias is mostly evident. S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 11239 Figure 11. Modelled nucleation rates (cm3 s−1) from runs A_Control20 (left), B_Control10 (middle), and C_noPinatubo (right) for (top to bottom) July, August, September and October 1991. Figure 11. Modelled nucleation rates (cm3 s−1) from runs A_Control20 (left), B_Control10 (middle), and C_noPinatubo (right) for (top to bottom) July, August, September and October 1991. cesses are highly uncertain in the stratosphere and the two additional simulations essentially test how robust the model is to changes in the model physics. In the first 12 months after the eruption the tropical and global sAOD is around 80 % higher in run A_Control20 than B_Control10, but in the second half of 1992 the difference in sAOD between the two control runs reduces to only around 10 %. In run D_noPrimary10, sAOD is only very slightly lower than in B_Control10, suggesting that including the source of pri- mary particles has only a minor impact on the aerosol evo- lution post-Pinatubo. The factor-100 reduced nucleation run E_ScaledStNuc10 causes a prolonged peak in tropical mid- visible AOD, with values around 10 % higher during Septem- ber 1991, with E_ScaledStNuc10 continuing to have AOD around 5 % higher than B_Control10 through the remainder of the simulation. Intriguingly, the impact of the nucleation rate reduction on N150 is, in the first 6 months after the erup- tion, to reduce N150, which is opposite to the slight increase in mid-visible AOD. This likely is due to a reduced num- ber of smaller particles growing to radii larger than 150 nm, with the sAOD increase caused by larger particles which will part of the plume (28 to 30 km). However, the accumulation mode SAD fraction (Fig. 12c) shows that even during the early phase of eruption total SAD is primarily determined by these larger particles in the lower–middle stratosphere. At a later stage (December 1991, not shown), the contribution from nucleation and Aitken mode is insignificant and, as ex- pected, the accumulation mode then contributes the vast ma- jority of the SAD. We note that in Fig. 7 the model shows highest biases in simulated SAD against the observations during the first few months after the eruption. Figure 13 compares tropical (panel a) and global (panel b) mid-visible sAOD and N150 evolution from the three main simulations A_Control20, B_Control10 and C_noPinatubo against the satellite observations from AVHRR. S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM Figure 11 shows, for runs A_Control20, B_Control10 and C_noPinatubo, the zonal-mean nucleation rate against latitude and altitude for monthly means through August to October 1991. In volcani- cally quiescent conditions (C_noPinatubo), the model has nucleation occurring mainly in the tropical upper troposphere with negligible new particle formation in the stratosphere. Note that the observed and simulated lower stratospheric N5 and N150 profiles at Laramie in March 1991 (Fig. 10a) are in very good agreement, and Fig. 11 indicates that these strato- spheric particles were actually formed in the tropical upper troposphere, consistent with the stratospheric aerosol lifecy- cle described by Hamill et al. (1997). The observations at Laramie indicate that only a small proportion of these nu- Since uptake of reactive gases is dependent on particle size, accounting for the shift in size distribution may also be important for better quantification of the influence that volcanically enhanced aerosol has on stratospheric ozone through accelerated heterogeneous chemistry. We therefore investigate the evolution of the SAD distribution across the three stratospheric aerosol modes (Fig. 12) from July 1991, 15–45 days after the eruption (panels a to d) and in October 1991, when aerosol loading was close to its peak (panels e to h). Nucleation mode particles are always smaller than 10 nm, so even during July 1991, when substantial nucleation is oc- curring (Fig. 11), their contribution to total SAD is at most only around 10 %. However, although the Aitken mode par- ticles are smaller than 100 nm, during the early part of the eruption they contribute significantly to SAD in the upper Atmos. Chem. Phys., 14, 11221–11246, 2014 www.atmos-chem-phys.net/14/11221/2014/ S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM We also compare time series of simulated N150 at 18 and 22 km al- titude against the long time series OPC measurements from Laramie. Also presented in Fig. 13 are results from two additional 10 Tg simulations, designed to test the sensitiv- ity of the model predictions to sub-grid particle formation (run D_noPrimary10) and with much reduced new particle formation rate (run E_ScaledStNuc10). Both of these pro- Atmos. Chem. Phys., 14, 11221–11246, 2014 www.atmos-chem-phys.net/14/11221/2014/ S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 11240 S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM a) AOD - 20oS-20oN 1991 1992 1993 1994 1995 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 stratospheric AOD b) AOD - 60oS-60oN 1991 1992 1993 1994 1995 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 stratospheric AOD OBS. (AVHRR) A_Control20 B_Control10 C_noPinatubo D_noPrimary10 E_ScaledStNuc10 c) N150 at 18km 1991 1992 1993 1994 1995 0 5 10 15 20 N150 conc. (cm-3) d) N150 at 22km 1991 1992 1993 1994 1995 0 5 10 15 20 N150 conc. (cm-3) Figure 13. (a) Mean tropical (20◦S, 20◦N) and (b) near-global (60◦S, 60◦N) sAOD from AVHRR (550 nm) and various model simulations (525 nm). (c) and (d) show comparison between mod- elled and observed N150 time series from Laramie (shown with + sign), at 18 km and 22 km, respectively. a) AOD - 20oS-20oN 1991 1992 1993 1994 1995 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 stratospheric AOD Figure 12. Percentage zonal mean surface area densities in nucle- ation, Aitken and accumulation modes from run A_Control20 for July 1991 (panels a, b, c) and December 1991 (panels e, f, g). Total SAD for July 1991 and December 1991 (µm2 cm−3) are shown in panels (d) and (h), respectively. b) AOD - 60oS-60oN 1991 1992 1993 1994 1995 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 stratospheric AOD OBS. (AVHRR) A_Control20 B_Control10 C_noPinatubo D_noPrimary10 E_ScaledStNuc10 d) N150 at 22km c) N150 at 18km 1991 1992 1993 1994 1995 0 5 10 15 20 N150 conc. (cm-3) d) N150 at 22km 1991 1992 1993 1994 1995 0 5 10 15 20 N150 conc. (cm-3) Figure 13. (a) Mean tropical (20◦S, 20◦N) and (b) near-global (60◦S, 60◦N) sAOD from AVHRR (550 nm) and various model simulations (525 nm). (c) and (d) show comparison between mod- elled and observed N150 time series from Laramie (shown with + sign), at 18 km and 22 km, respectively. 6 Summary and conclusions We have extended the UM-UKCA composition-climate model to incorporate stratospheric sulfur chemistry and up- dated the process descriptions in the GLOMAP aerosol mi- crophysics module to be applicable for both tropospheric and stratospheric conditions. Using stratospheric aerosol changes after the Mt Pinatubo eruption as a test case, we have evalu- ated simulated aerosol properties against a wide range of ob- servations in both quiescent and volcanically perturbed con- ditions. The improvements to the model enable a prognos- tic treatment of stratospheric aerosol with dynamically vary- ing particle size distribution alongside stratospheric transport and chemistry up to a model top of 80 km. Figure 12. Percentage zonal mean surface area densities in nucle- ation, Aitken and accumulation modes from run A_Control20 for July 1991 (panels a, b, c) and December 1991 (panels e, f, g). Total SAD for July 1991 and December 1991 (µm2 cm−3) are shown in panels (d) and (h), respectively. In general, the model captures the observed distribution and evolution of stratospheric aerosol properties well, in both quiescent and volcanically perturbed conditions. For the Pinatubo test case, the timing of the peak in global aerosol mass and decay timescale are captured well compared to val- ues derived from HIRS satellite measurements (Baran et al., 1993). However, our control simulation, with a 20 Tg emis- sion of SO2 produces much too high a burden of aerosol sul- fur compared to the HIRS measurements, whereas a 10 Tg injection is in good agreement. The 10 Tg run also com- pares better to the magnitude of the enhanced sAOD distri- bution seen in SAGE II and AVHRR, and captures well the transport to the Southern Hemisphere. Modelled extinction in the tropical and NH mid-latitude lower stratosphere shows good agreement with SAGE V7 data in both the mid-visible and near-infrared. However, for the first 6 months after the have received more gas-to-particle transfer of sulfuric acid, enhancing condensational growth and increasing their scat- tering efficiency. In summary, however, although these mi- crophysical sensitivities are interesting, the results suggest a low sensitivity to uncertainties in the nucleation rate, and to model treatment of sub-grid particle formation. The low sensitivity gives additional credibility to the aerosol micro- physics models, suggesting the models are robust to known uncertainties in some processes in stratospheric conditions. have received more gas-to-particle transfer of sulfuric acid, enhancing condensational growth and increasing their scat- tering efficiency. S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM Comparing the evolution and altitude of the high biases in extinction to those seen in the OPC profile measurements of the size distribution suggest that the main source of the biases is in particles in the 150 to 550 nm size range in the lowermost stratosphere. The discrepancy could be related to the modal aerosol dynamics failing to capture the differential growth across the particle size range. However, an alternative explanation could be that too young age-of-air and too rapid STE is affecting the simulated stratospheric aerosol evolu- tion. We also note that enhanced upwelling from radiative heating of the enhanced aerosol layer, not included in these uncoupled simulations, will also change the vertical distri- bution and transport of the aerosol. It is worth noting that in radiatively coupled simulations, we expect that increased tropical upwelling would dilute the lower part of the plume, decreasing particle concentrations in the lowermost strato- sphere. eruption simulated sAOD and SAD are larger than the satel- lite measurements, and the model enhancement in Reff is too low (e.g. compared to Bauman et al., 2003). Lack of radia- tive coupling in these simulations is likely to be the dominant contribution to the high AOD, since radiative heating of the Pinatubo cloud in the tropical lower stratosphere is known to have enhanced upwelling and reduces aerosol optical depth in model simulations (e.g. Young et al., 1994). To better understand how the particle size distribution was perturbed during the Pinatubo eruption, we have compared against mid-latitude balloon-borne measurements during that time period, allowing a strong observational constraint on concentrations of particles larger than 5, 150, 250, 550, 750 and 1000 nm. Although there have been many model studies covering the Pinatubo period over the 22 yr since the erup- tion, to our knowledge, this is the first time the full profile of a simulated size distribution in a global model has been compared to these measurements in volcanically perturbed conditions. Overall, the general good agreement with the size dis- tribution measurements from Laramie and the global effec- tive radius evolution from satellite suggest that the modal aerosol microphysics module used in our CCM is capable of representing the variation in particle size distribution in the strongly volcanically perturbed post-Pinatubo period. The sensitivity simulations also suggest that such simulated per- turbations to stratospheric aerosol properties are robust to known uncertainties in nucleation rate and sub-grid parti- cle formation. 6 Summary and conclusions In summary, however, although these mi- crophysical sensitivities are interesting, the results suggest a low sensitivity to uncertainties in the nucleation rate, and to model treatment of sub-grid particle formation. The low sensitivity gives additional credibility to the aerosol micro- physics models, suggesting the models are robust to known uncertainties in some processes in stratospheric conditions. Atmos. Chem. Phys., 14, 11221–11246, 2014 www.atmos-chem-phys.net/14/11221/2014/ 11241 S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM Our results underline the importance of bet- ter constraining transport and growth of Aitken-mode-sized particles in the first few months after the eruption to improve prediction of volcanic impacts on climate with stratospheric aerosol microphysics models. The findings highlight the need for a coordinated set of experiments to inter-compare and evaluate current global stratospheric aerosol models against the wide set of observations available through the Pinatubo- perturbed period. The model finds that in the volcanically quiescent strato- sphere, nucleation occurs only during polar spring, with stratospheric particles at Laramie mostly originating from the tropical upper troposphere. In such background conditions the model agrees very well with size distribution observed at Laramie, with only a small proportion of these nucleated particles have grown to 150 nm by coagulation and conden- sation, with N5 larger than N150 by around a factor of 10. We have investigated the impact on the size distribution of 20 and 10 Tg tropical injections of SO2 from Pinatubo. g p j 2 In the first 2 months after the eruption, nucleation is found to occur throughout the volcanic plume and the large in- jection of SO2 leads to strong growth of these particles to- gether with growth of older particles formed in the tropi- cal upper troposphere. Comparing the 10 and 20 Tg control simulations, we find much larger relative difference between concentrations of particles larger than 550 nm between runs A_Control20 and B_Control10 than in the smaller particles between 150 and 550 nm, which may be indicative of two types of volcanically enhanced particles. Overall the simu- lated profile of the particle size distribution agrees remark- ably well with the observations, capturing most of the com- plex shape of the concentration profiles in the different size channels. However, the decay timescale for N150 is slower in the model than the observations, which leads to an initially modest high bias increasing to around a factor of two by mid- 1993 (e.g. Fig. 13), with the return to a background size dis- tribution occurring much later in the model. The spatial and temporal evolution of the Reff in the lower stratosphere seen by satellite (Bauman et al., 2003) is also well captured by the model, albeit with a low bias in size compared to the mea- surements. www.atmos-chem-phys.net/14/11221/2014/ Atmos. Chem. Phys., 14, 11221–11246, 2014 References Carslaw, K. and Kärcher, B.: Stratospheric aerosol processes. In: Thomason L. and Peter Th. (Eds.) 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RH was partly funded by German Federal Ministry of Education and Research (BMBF) project ROSA (reference code 01LG1212A) within the ROMIC (Role Of the Middle Atmosphere in Climate) research programme. We would also like to thank James Keeble for his help during model development. We acknowledge use of the MONSooN system, a collaborative facility supplied under the Joint Weather and Climate Research Programme, which is a strategic partnership between the UK Met Office and the Natural Environment Research Council. This work has been also supported by NIWA as part of its Government-funded, core research programme. The in situ measurements were supported by the US National Science Foundation. Current measurements are supported under grant number ATM-0437406. Acknowledgements. We would like to thank P. B. Russell for effective radius data. This work was supported by the UK Natural Environment Research Council (NERC grants NE/E005659/1 and NE/E017150/1). GWM and KSC received funding from the National Centre for Atmospheric Science, one of the NERC research centres. GWM received EU funding from the European Research Council (ERC) under Seventh Framework Programme (FP7) consortium projects MACC and MACC-II (grant agreements 218793 and 283576 respectively). NB, MD, CEJ, FOC were supported as part the UK Integrated Climate Programme funded by the Department for Energy and Climate Change (DECC) and De- partment for Environment Food and Rural Affairs – DECC/Defra (GA01101). RH was partly funded by German Federal Ministry of Education and Research (BMBF) project ROSA (reference code 01LG1212A) within the ROMIC (Role Of the Middle Atmosphere in Climate) research programme. We would also like to thank James Keeble for his help during model development. S. S. 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Ghan, S., Ginoux, P., Gong, S., Grini, A., Hendricks, J., Horowitz, L., Huang, P., Isaksen, I., Iversen, I., Kloster, S., Solomon, S., Portmann, R. W., Garcia, R. R., Thomason, L. W., Poole, L. R., and McCormick, M. P.: The role of aerosol varia- tions in anthropogenic ozone depletion at northern midlatitudes, J. Geophys. Res.-Atmos., 101, 6713–6727, 1996. Koch, D., Kirkevåg, A., Kristjansson, J. E., Krol, M., Lauer, A., Solomon, S., Daniel, J., Neely, R., Vernier, J.-P., Dutton, E., and Thomason, L.: The persistently variable background strato- spheric aerosol layer and global climate change, Science, 333, 866–870, 2011. S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM Res., 69, 4673–4676, 1964. Russell, P., Livingston, J., Pueschel, R., Bauman, J., Pollack, J., Brooks, S., Hamill, P., Thomason, L., Stowe, L., and Deshler, T.: Global to microscale evolution of the Pinatubo volcanic aerosol derived from diverse measurements and analyses, J. Geophys. Res., 101, 18745–18763, 1996. Mann, G. W., Carslaw, K. S., Ridley, D. A., Spracklen, D. V., Pringle, K. J., Merikanto, J., Korhonen, H., Schwarz, J. P., Lee, L. A., Manktelow, P. T., Woodhouse, M. T., Schmidt, A., Breider, T. J., Emmerson, K. M., Reddington, C. L., Chipper- field, M. P., and Pickering, S. J.: Intercomparison of modal and sectional aerosol microphysics representations within the same 3-D global chemical transport model, Atmos. Chem. Phys., 12, 4449–4476, doi:10.5194/acp-12-4449-2012, 2012. Sander, S., Friedl, R., Golden, D., Kurylo, M., Huie, R., Orkin, V., Moortgat, G., Wine, P., Ravishankara, A., Kolb, C., Molina, M., Finlayson-Pitts, B., Huie, R., Orkin, V. L., and Keller-Rudek, H.: Chemical kinetics and photochemical data for use in atmospheric studies, NASA Panel for Data Evaluation, Evaluation number 15, Atmos. Chem. Phys., 14, 11221–11246, 2014 www.atmos-chem-phys.net/14/11221/2014/ S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM Weber, M., Dhomse, S., Wittrock, F., Richter, A., Sinnhuber, B. M., and Burrows, J. P.: Dynamical control of NH and SH win- ter/spring total ozone from GOME observations in 1995-2002, Geophys. Res. Lett., 30, 2003. Strahan, S. E., Douglass, A. R., Stolarski, R. S., Akiyoshi, H., Bekki, S., Braesicke, P., Butchart, N., Chipperfield, M. P., Cugnet, D., Dhomse, S., Frith, S. M., Gettelman, A., Hardi- man, S. C., Kinnison, D. E., Lamarque, J. F., Mancini, E., Marc- hand, M., Michou, M., Morgenstern, O., Nakamura, T., Olivie, D., Pawson, S., Pitari, G., Plummer, D. A., Pyle, J. A., Scinocca, J. F., Shepherd, T. G., Shibata, K., Smale, D., Teyssedre, H., Tian, W., and Yamashita, Y.: Using transport diagnostics to understand chemistry climate model ozone simulations, J. Geophys. Res.- Atmos., 116, D17302, doi:doi:10.1029/2010JD015360, 2011. Weisenstein, D., Bekki, S., Mills, M., Pitari, G., and Timmreck, C.: Modeling of stratospheric aerosols, edited by: Thomason, L. and Peter, Th., SPARC Assessment of Stratospheric Aerosol Prop- erties (ASAP), 219–272, SPARC Report No. 4, World Climate Research Programme, 2006. Weisenstein, D. K., Yue, G. K., Ko, M. K. W., Sze, N. D., Ro- driguez, J. M., and Scott, C. J.: A two-dimensional model of sul- fur species and aerosols, J. Geophys. Res.-Atmos., 102, 13019– 13035, 1997. Atmos. Chem. Phys., 14, 11221–11246, 2014 Atmos. Chem. Phys., 14, 11221–11246, 2014 www.atmos-chem-phys.net/14/11221/2014/ S. S. Dhomse et al.: Simulation of Pinatubo aerosol in a CCM 11246 Wild, O., Zhu, X., and Prather, M. J.: Fast-J: Accurate simulation of in-and below-cloud photolysis in tropospheric chemical mod- els, J. Atmos. Chem., 37, 245–282, 2000. Young, R. E., Houben, H., and Toon, O. B.: Radiatively forced dis- persion of the Mt. Pinatubo volcanic cloud and induced tempera- ture perturbations in the stratosphere during the first few months following the eruption, Geophys. Res. Lett., 21, 369–372, 1994. Wilson, J., Cuvelier, C., and Raes, F.: A modeling study of global mixed aerosol fields, J. Geophys. Res.-Atmos., 106, 34081– 34108, 2001. Zdanovskii, A.: New methods for calculating solubilities of elec- trolytes in multicomponent systems, Zh. Fiz. Khim, 22, 1475– 1485, 1948. WMO: Scientific Assessment of Ozone Depletion: 2010, Global Ozone Research and Monitoring Project Report 52, World Mete- orological Organization, Geneva, 2011. Atmos. Chem. Phys., 14, 11221–11246, 2014 www.atmos-chem-phys.net/14/11221/2014/
https://openalex.org/W3204775518
https://comum.rcaap.pt/bitstream/10400.26/40992/1/Microbiological%20evaluation%20in%20oral%20health%20units.pdf
English
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Microbiological evaluation in oral health units: detection of antibiotic resistant bacteria
Annals of medicine (Helsinki)/Annals of medicine
2,021
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1,520
Annals of Medicine ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/iann20 Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=iann20 Annals of Medicine Annals of Medicine Acknowledgements The authors acknowledge funding from FCT projects – PTDC/BIM-MEC/6631/2014 and Egas Moniz, Cooperativa de Ensino Superior CRL. DOI: 10.1080/07853890.2021.1895585 Inês Gama & Helena Barroso Inês Gama & Helena Barroso To cite this article: Inês Gama & Helena Barroso (2021) Microbiological evaluation in oral health units: detection of antibiotic resistant bacteria, Annals of Medicine, 53:sup1, S95-S96, DOI: 10.1080/07853890.2021.1895586 To link to this article: https://doi.org/10.1080/07853890.2021.1895586 © 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group Published online: 28 Sep 2021. Submit your article to this journal Article views: 41 View related articles View Crossmark data © 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group Published online: 28 Sep 2021. Submit your article to this journal Article views: 41 View related articles View Crossmark data S95 ANNALS OF MEDICINE S9 ANNALS OF MEDICINE Figure 1. Viability of cells treated with docetaxel for 48 h. Untreated cells are considered to have 100% of viability. Values are pre- sent has average ± SEM of three independent MTT assays. *p-value <.05 in relation to untreated cells within the same cell line. cells treated with docetaxel for 48 h Untreated cells are considered to have 100% of viability Values are pre- Figure 1. Viability of cells treated with docetaxel for 48 h. Untreated cells are considered to have 100% of viability. Values are pre- sent has average ± SEM of three independent MTT assays. *p-value <.05 in relation to untreated cells within the same cell line. ABSTRACT Introduction: The environment in oral health care units can represent an important source of transmission of infections, which can be acquired through aerosols, bleeding, saliva and respiratory secretions [1]. It is increasingly important to pre- vent cross-infection in dental clinics [2]. Resistance to antibiotics is a serious public health problem. Presence of resistant microorganisms in health care units is a worrying reality, but little is known about oral health care units [3]. The aim of this study was the detection of microorganisms resistant to antibiotics at the Clınica Dentaria Egas Moniz, in the dentist’s chair, trays and lamp handles. y p Materials and methods: Environmental samples were collected at the dental clinic with a swab. Sampling was made at trays, chairs and lamp handles, at the end of the appointments. All samples were inoculated in Trypticase soy agar (TSA), Mannitol salt agar (MSA) and MacConkey agar. All the bacteria that grew in MSA and were mannitol positive were inocu- lated in chromogenic agar, because we wanted to detect Methicillin resistant Staphylococcus aureus (MRSA). g g , p y ( ) Results: Of the 123 samples obtained in 41 working stations, only two (1.6%) lactose negative bacteria were found. One was isolated from a tray and the other from a lamp handle, in two different working stations. We found 51 mannitol positive Staphylococcus samples (41.5%), were isolated from 36 different working stations, being 14 samples identified as MRSA (11.4%). These MRSA were isolated from 13 different working stations. In our study, we cannot identify if there was a preferential location for the presence of MRSA, but we found it mainly at trays and dentist’s chairs. We found 51 mannitol positive Staphylococcus samples (41.5%), were isolated from 36 different working stations, being 14 samples identified as MRSA (11.4%). These MRSA were isolated from 13 different working stations. In our study, we cannot identify if there was a preferential location for the presence of MRSA, but we found it mainly at trays and dentist’s chairs. Discussion and conclusions: There was a low contamination by Enterobacteriaceae. However, a percentage of MRSA iso- lation of 11.4% was obtained. There are few similar studies. Although, in a study where 95 surfaces from 7 different uni- versity dental clinics were evaluated, 8 MRSA were found, which corresponds to 8.4%. Comparing to our study, we obtained a slightly higher percentage. In^es Gamaa and Helena Barrosoa,b aInstituto Universitario Egas Moniz (IUEM), Caparica, Portugal; bCentro de Investigac¸~ao Interdisciplinar Egas Moniz (CiiEM), IUEM; Cooperativa de Ensino Superior, C.R.L., Caparica, Portugal ABSTRACT Discussion and conclusions: There was a low contamination by Enterobacteriaceae. However, a percentage of MRSA iso- lation of 11.4% was obtained. There are few similar studies. Although, in a study where 95 surfaces from 7 different uni- versity dental clinics were evaluated, 8 MRSA were found, which corresponds to 8.4%. Comparing to our study, we obtained a slightly higher percentage. g y g p g These results demonstrate that oral health care units are also sites of contamination, where bacteria with resistance to g y g p g These results demonstrate that oral health care units are also sites of contamination, where bacteria with resistance to S96 ABSTRACTS antibiotics can circulate. They also reinforce the need for good hygiene and disinfection of the site between appointments. Acknowledgements The authors are grateful to ESSEM, for the support given on the Pedagogical Simulacrum. No funding to declare. Acknowledgements The authors acknowledge funding from Egas Moniz, CRL. [1] Pozzoli SML, Cecılio LCO. Sobre o cuidar e o ser cuidado na atenc¸~ao domiciliar. Saude Debate. 2017;41(115):1116–1129. [2] Dias C. Oxigenoterapia de longa durac¸~ao no domicılio. Med Interna. 1999;6(4):270–275. ABSTRACT ABSTRACT Introduction: Oxygen therapy is applied in a variety of clinical situations where patients have low levels of oxygen in the blood and is part of one of the procedures in nursing care. Knowing that home care is more beneficial for the patient, because it promotes a faster recovery and guarantees comfort and safety, preserving its autonomy [1], it is pertinent to train (enable) both patients and informal caregivers (e.g. family, friends), with the correct knowledge (empowerment) and procedures to fol- low. Nurses have a fundamental role in the training of patients and/or informal caregivers, knowing their difficulties and real- ities, thus enabling caregivers to be autonomous in-home care [2–4]. This work presents an original educational tool that uses pictograms to teach and empower patients and/or caregivers to use oxygen therapy materials correctly at home. Materials and methods: An Educational Tool for Health purposes was originally designed using pictograms, which illus- trates/anticipates the therapeutic procedures, destined for patients who need oxygen therapy with regularity. The instru- ment is made up of a set of pictograms that complement the clinical nursing procedure on oxygen therapy (teaching step by step the therapeutic) and a good practice manual (helps to understand oxygen therapy and serves as a record and monitoring of the difficulties experienced). Results: The use of pedagogic tools for health using pictograms can be a strategy to improve interpersonal communica- tion between nurse and the patient and/or informal caregiver [5]. They can also be used as complementary learning tools, because of their universal character, the pictograms constitute an effective way to overcome individual constraints, such as literacy level, familiarity with the images, interpretation, and perception of what is being pictorially represented [6]. The suggested good practice manual is interactive and dynamic, enabling both patient and informal caregiver to focus and actively participate. y p p Discussion and conclusions: The Strategies for Health Education are increasingly relevant on what concerns the change in health behaviour. Pedagogical materials of this nature facilitate the understanding and use of therapeutic procedures used daily, allowing to reduce the excessive dependence of professionals and health services and are fundamental contri- butions for a greater autonomy and adherence to the health behaviours of the patient and/or informal caregiver. CONTACT Ana Cristina Neves anaccseven@gmail.com The authors are grateful to ESSEM, for the support given on the Pedagogical Simulacrum. No funding to declare. A teaching tool for nursing procedure with oxygen therapy Maria Rodrigueza, Catia Balsinhaa, Nataliya Garciaa, Valeria Vicentea and Ana Cristina Nev aEscola Superior de Saúde Egas Moniz (ESSEM), Egas Moniz Cooperativa de Ensino Superior, Caparica, Portugal; Investigação Interdisciplinar Egas Moniz (CiiEM), Egas Moniz Cooperativa de Ensino Superior, Caparica, Portugal CONTACT Ana Cristina Neves anaccseven@gmail.com References [1] Al-Marzooq F, Bayat SA, Sayyar F, et al. Can probiotic cleaning solutions replace chemical disinfectants in dental clinics? Eur J Dentistry. 2018;12:532–539. [2] Silva CR Jorge AOCJ Evaluation of surface disinfectants utilized in dentistry Pesqui Odontol Bras 2002;16(2):107–114 [1] Al-Marzooq F, Bayat SA, Sayyar F, et al. Can probiotic cleaning solutions replace chemical disinfe Eur J Dentistry. 2018;12:532–539. [2] Silva CR, Jorge AOCJ. Evaluation of surface disinfectants utilized in dentistry. Pesqui Odontol Bras. 2002;16(2):107–114. [3] Roberts MC, Soge OO, Horst JA, et al. Methicillin-resistant Staphylococcus aureus from dental school clinic surfaces and students. Am J Infect Control. 2011;39(8):628–632. [ ] , g y q ; ( ) [3] Roberts MC, Soge OO, Horst JA, et al. Methicillin-resistant Staphylococcus aureus from dental school clinic surfaces and students. Am J Infect Control. 2011;39(8):628–632. [3] Roberts MC, Soge OO, Horst JA, et al. Methicillin-resistant Staphylococcus aureus from dental sc students. Am J Infect Control. 2011;39(8):628–632. DOI: 10.1080/07853890.2021.1895586 References [1] Pozzoli SML, Cecılio LCO. Sobre o cuidar e o ser cuidado na atenc¸ao domiciliar. Saude Debate. 2017;41(115):1116 1129. [2] Dias C. Oxigenoterapia de longa durac¸~ao no domicılio. Med Interna. 1999;6(4):270–275.
https://openalex.org/W4235135380
https://journals.vgtu.lt/index.php/JEELM/article/download/7929/6870
English
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APPLICATION OF BACKWARD AIR MASS TRAJECTORY ANALYSIS IN EVALUATING AIRBORNE POLLEN DISPERSION
Journal of environmental engineering and landscape management
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1. Introduction identify the beginning of bloom of the plant that is chang- ing year after year and is partially determined by mete- orological factors in the period of blossom formation [5]. Investigaions into the dynamics of airborne birch (Betula L.) pollen are performed in many European states [1–4]. The attention of aerobiologists, botanists, planters, clinicists to this biological kind of air pollution has ap- peared because of large amounts of pollen dispersed during bloom period and decreasing life quality of the part of human population. It has been estimated that most often in spring the allergy caused by birch pollen is diagnosed [5]. Using special methods, it has been shown that people are sensitive even to small amounts of pollen: people experi- ence unpleasant sensations when the number of birch pol- len in 24 hours reaches 35 grains/m³, for people ill in asthma the process of illness becomes more complicated when 68–81 grains/m³ is recorded, 81–252 grains/m³ causes conjunctivitis and/or rhinitis [6]. The periods of birch pollination at different aerobiology stations in Lithuania were evaluated [7], but the influence of long- range transport on pollen dynamics was not shown. Performing the forecast, the necessary evaluation of interaction between airborne pollen concentration and environmental factors is conducted [9]. It is shown that warm and wet weather influences bigger airborne birch pollen concentrations [2], and comparing pollen dynam- ics with temperature (minimum and/or maximum) strong positive correlation is observed, while comparing it with rainfall negative correlation occurs [4]. It has been esti- mated that airborne birch pollen concentration signifi- cantly increases when anticyclone is dominating, relative humidity falls below 70 %, and the temperature reaches 6–11 °C [6]. Thus, meteorological conditions and weather types have a vital influence not only on pollen formation, but also on the release of pollen from anthers and airborne concentration. To investigate the movement of airborne pollen aerobiologists use backward air mass trajectories [9]. Using this method, long-range transport and the move- ment of airborne pollen is investigated. Airborne pollen dispersion may be forecast having evaluated biological features of a plant, climate factors and circulation of the atmosphere. The situation analysis becomes more complicated because of the lack of unani- mous opinion on birch reproduction cycle. In some au- thors’ opinion, the triennial rhythm is characteristic of birch [8] maintaining synchrony above large territories; in other authors’ opinion, the rhythm is biennial (Spieksma et al., 2003). APPLICATION OF BACKWARD AIR MASS TRAJECTORY ANALYSIS IN EVALUATING AIRBORNE POLLEN DISPERSION Ingrida Šaulienė, Laura Veriankaitė Dept of Environmental Sciences, Šiauliai University, Vilniaus g. 88, LT-76285 Šiauliai, Lithuania. E-mail: oikos@fm.su.lt Submitted 30 Mar 2006; accepted 15 Apr 2006 Ingrida Šaulienė, Laura Veriankaitė Abstract. To investigate airborne pollen movement aerobiologists use backward air mass trajectories. In the present paper the peculiarities of airborne birch pollen dispersion are analysed. In 2005 at Šiauliai Aerobiology Station pollen was re- corded using Hirst-type spore trap. The situation of birch bloom in neighbouring European countries was evaluated ac- cording to the European Aeroallergen Network database. It was generalized and used to prove long-range pollen transport. Air mass trajectories were calculated according to the HYSPLIT 4 model the trajectory drawing principle of which is based on the integration of the position of air mass with regard to time. The present study has shown that backward air mass trajectory analysis may be applied in investigating relationship among pollen dispersion, meteorological conditions and air mass transport. It is estimated that at Šiauliai Aerobiology Station the dynamics of pollen concentration is determi- nated by local flora and airborne pollen long-range transport. The evaluation of pollen dispersion by the backward air mass trajectory method presented in the paper established precondition to look for possibilities of application of the HYSPLIT 4 model to prepare the forecasts of pollen dispersion. Keywords: pollen, meteorological conditions, long-range transport, backward air mass trajectories, birch pollination, air pollution. 113 113 ISSN 1648–6897 print ISSN 1822–4199 online JOURNAL OF ENVIRONMENTAL ENGINEERING AND LANDSCAPE MANAGEMENT 2006, Vol XIV, No 3, 113–120 2006, Vol XIV, No 3, 113–120 2.3. Backward trajectory analysis Air mass trajectories were calculated according to HYSPLIT 4 model [12] the trajectory drawing principle of which is based on the integration of the position of air mass with regard to time. Since air mass is transported with the help of the wind, its passive transport may be calculated evaluating three-dimensional means of speed vector when the particles are in the primary position ( )t P and the first approximate position ( ) dt t P + ′ . Speed vectors are interpolated with regard both to space and time. Using the HYSPLIT 4 model the first approximate position is calculated: The study of airborne pollen dynamics was con- ducted choosing 3 days the peaks of which were the most distinct during the entire period of birch pollination: on 3, 13 and 21 May (Fig 2). On 3 and 21 May at Šiauliai Aerobiological Station peaks of pollen concentration atypical of morning hours were recorded. On May 13 it was the middle of pollina- tion season, but abundant rainfall produced atypical con- ditions for pollen dispersion. Having established presumption about the dispersion of pollen of a nonlocal origin, backward air mass trajectory analysis to perform the case evaluation was used. ( ) dt t P + ′ = ( ) ( ) dt t, P V t P + . (1) (1) While the last position of air particles: While the last position of air particles: While the last position of air particles: p p ( ) dt t P + = ( ) ( ) ( ) [ ] dt dt t P V t P V t P + ′ + + , , 5,0 . (2) p p ( ) dt t P + = ( ) ( ) ( ) [ ] dt dt t P V t P V t P + ′ + + , , 5,0 . (2) p p ( ) dt t P + = ( ) ( ) ( ) [ ] dt dt t P V t P V t P + ′ + + , , 5,0 . (2) Thus, backward air mass trajectories may be integra- ted and presented graphically. Backward air mass trajectories were calculated for every day analysed (at midnight, at 6 a.m., at noon, at 6 p.m. according to local time) at three different heights: 1500 m, 1000 m, 500 m. 2.3. Backward trajectory analysis The following heights were chosen because to identify air mass trajectories it is rec- ommended to choose heights with the pressure interval of 950–10 mb. It is suggested that the use of pressures less than 950 mb often results in parcels going into the ground [9]. 2.1. Object Birch forests in the whole Šiauliai region cover about 29 % (61 300 ha) of the forest area and make up 15 % of the Lithuanian territory [10]. In Šiauliai city surroundings birch is in the second place according to number and makes up about 32 % of the forest area. 3.1. Blooming season of birch genus (Betula L.) in Šiauliai city in 2005 For the case study, data about the amount of air- borne birch pollen are obtained from Šiauliai Aerobi- ological Station. Pollen was recorded using the Hirst-type spore trap raised 18 m above the ground and localized in the western part of the city (55°56'96 N, 23°16'95 E). Samples were prepared, and the data were processed according to a standard method [11]. Birch pollen counts were recorded every 2 hours and expressed as the number of grains counted per transect (field of view of 0,55 mm diameter). Each transect represented 0,001 % of the total volume of sampled air. The blooming season of the plants of birch genus in 2005 lasted for 27 calendar days. During these days 5 concentration peaks dangerous to human health were recorded: May 3(397 grains/m³), May 6 (212 grains/m³), May 8 (205 grains/m³), May 13–15 (560–286 grains/m³) and May 21 (347 grains/m³) (Fig 1). Fig 1. Birch pollen season in Šiauliai in 2005 The situation of birch bloom in May in neighbouring European countries was evaluated referring to the Euro- pean Aeroallergen Network (EAN) database. It was gen- eralized and used to prove long-range pollen transport with the help of air mass. 2. Materials and methods With the help of the HYSPLIT 4 model the rainfall in the way of air masses was evaluated, the data were taken from the National Weather Service's NCEP (the National Weather Service's National Centers for Envi- ronmental Prediction) database. 2.2. Meteorological data The meteorological data for Šiauliai city were pro- vided by Šiauliai Meteorological Station of Hydrometeo- rological Service of Lithuania at the Ministry of Environment. The station is in the eastern part of the city about 3 km from Šiauliai Aerobiological Station. Fig 1. Birch pollen season in Šiauliai in 2005 Having evaluated the meteorological situation dur- ing the period of birch pollination it was identified that conditions to release pollen from anthers were sufficient: relative humidity during the season fluctuated from 53– 88 %, average weather temperature in 24 hours was from 7,5–20,5 °C, average wind speed in 24 hours reached 1,4–4,8 m/s, the sunniness was changing at an interval of 1,3–15,2 hours, the rainfall was 0,6–11,6 mm. 1. Introduction For the forecasts, it is not less important to The aim of the present study is to establish relation- ship of pollen dispersion, meteorological conditions and air mass transport using a selected case for modeling. Such a study helps to forecast probability for pollen of a nonlocal origin to get to the territory of Lithuania and the dynamics of biological pollution in the atmosphere. 3.4. Pollen dispersion and backward air mass trajectory analysis on 21 May 2005 On 21 May 2005 the peak of birch pollen concentra- tion (Fig 1), recorded at Šiauliai Aerobiological Station was evaluated as noncharacteristic of the period because at the end of pollination season such an emission from local flora was impossible. The analysis of the aerobiolo- gical preparations of the 24 hours analysed (Fig 2) high- lighted the pollen peak of 6 a.m.–8 a.m. showing probabi- lity of pollen of a nonlocal origin. Analysis of backward 3.2. Pollen dispersion and backward air mass trajectory analysis on 3 May 2005 The HYSPLIT 4 model showed that on May 13 air mass came from the northern part of Europe: 500 m above the ground air mass retained similar direction during 24 hours, while at a height of 1000 m and 1500 m it went from the North East to the North where exceptionally large amounts of birch pollen were recorded. A part of pollen could have settled toge- ther with rainfall (especially in morning hours), and the evening pollen peak occurred due to dried and airborne spread local pollen (Fig 2). The situation analysis allows to establish presumption that at Šiauliai Aerobiological Station pollen concentration in the morning of May 13 increased due to long-range transport of particles, and in the evening – due to the emission from the local flora. Using the HYSPLIT 4 model and the EAN database it was revealed that on 2 May very small amounts of birch pollen were recorded in Latvia, and air mass cros- sed this region during night hours, therefore, it could not bring pollen from this European region. Having evaluated that maximum pollen concentrations were recorded from midnight to 6 a.m. and on a bigger part of the day on May 2 local air mass was dominating, therefore, increased amounts of pollen were related to blooming of local plants in the central and southern parts of Lithuania. Du- ring later hours of May 3 little amounts of airborne pollen recorded may be related to an unfavourable meteorologi- cal situation in Šiauliai city (a high relative humidity, rainfall, a low pressure) preventing to spread pollen and an unfavourable air mass circulation because at the after- noon hours air mass came from the central and northern parts of Europe. Although, according to the EAN data, in Denmark and Poland much pollen was recorded these days, however, having evaluated meteorological condi- tions with the help of the model, it is possible to establish presumption that pollen may have been washed by rain- fall that occurred in the way of air mass. 3.2. Pollen dispersion and backward air mass trajectory analysis on 3 May 2005 During 24 hours of analysis the pollen peak was re- corded at night hours (Fig 2). Having evaluated pollen recorded during 24 hours it was estimated that 67 % of the total amount was recorded from midnight to 6 a.m.). I. Šaulienė, L. Veriankaitė / JOURNAL OF ENVIRONMENTAL ENGINEERING AND LANDSCAPE MANAGEMENT – 2006, Vol XIV, No 3, 113–120 115 0 20 40 60 80 100 120 140 160 180 200 00–02 02–04 04–06 06–08 08–10 10–12 12–14 14–16 16–18 18–20 20–22 22–24 Pollen concentration (grains/m3) 3 May 13 May 21 May hours Fig 2. Birch pollination on 3, 13 and 21 May Fig 2. Birch pollination on 3, 13 and 21 May The biggest pollen concentration that day was from 4 a.m. to 6 a.m. (184,11 grains/m³) and significantly dec- reased in the afternoon hours. The meteorological situa- tion of 24 hours was unfavourable for pollen dispersion (Table 1). An average temperature reached 8,8 °C, the rainfall (from 5:28 to 11:06) was to 1,1 mm, the day was cloudy, the variable wind speed reached 2,8 m/s. Knowing that for the release of pollen from anthers a dry and sunny weather is essential, the problem of the situa- tion analysis occurs. To evaluate the changes of pollen concentration of that day, analysis of air mass transport was conducted (Figs 3–6). decreasing towards the end of the day. The total rainfall amount reached 11,6 mm, its biggest part fell until 11 a.m. Other meteorological conditions (Table 2) were unfavourable for the release of pollen, but airborne pollen concentration under such conditions reached a maximum of the entire season (560 grains/m³). Local pollen (of plants from Šiauliai surroundings) was wet and heavy and could not spread, so it was possible to establish presump- tion that the part of pollen could be brought together with air mass (Figs 7–10) from neighbouring regions. Having evaluated plant pollination in Europe (according to the EAN database) it was estimated that in Latvia and in the southern part of Finland big amounts of birch pollen were recorded, while in the southern and central parts of Fin- land birch bloom was over. I. Šaulienė, L. Veriankaitė / JOURNAL OF ENVIRONMENTAL ENGINEERING AND LANDSCAPE MANAGEMENT – 2006, Vol XIV, No 3, 113–120 116 I. Šaulienė, L. Veriankaitė / JOURNAL OF ENVIRONMENTAL ENGINEERING AND LANDSCAPE MANAGEMENT – 2006, Vol XIV, No 3, 113–120 116 116 Table 1. Meteorological data recorded on May 3, 2005 Time Surface level pressure mb Average tem- perature, °C Minimal tem- perature, °C Wind speed, m/s Wind direc- tion, degrees Relative humidity, % 24 1006,6 8,5 8,4 2 20 62 6 1004,0 9 8,2 3 180 71 12 999,6 7,9 7,7 3 160 82 18 997,7 8,7 8,6 3 180 92 NOAA HYSPLIT MODEL Backward trajectories ending at 21 UTC 2 May 2005 FNL Meteoroogical Data Fig 3. Backward trajectory analysis (24 hours) at three al- titudes, start of run at 00:00, May 3, 2005 NOAA HYSPLIT MODEL Backward trajectories ending at 3 UTC 3 May 2005 FNL Meteoroogical Data NOAA HYSPLIT MODEL Backward trajectories ending at 9 UTC 3 May 2005 FNL Meteoroogical Data Fig 5. Backward trajectory analysis (24 hours) at three al- titudes, start of run at 12:00, May 3, 2005 NOAA HYSPLIT MODEL Backward trajectories ending at 15 UTC 3 May 2005 FNL Meteoroogical Data Table 1. Meteorological data recorded on May 3, 2005 Time Surface level pressure mb Average tem- perature, °C Minimal tem- perature, °C Wind speed, m/s Wind direc- tion, degrees Relative humidity, % 24 1006,6 8,5 8,4 2 20 62 6 1004,0 9 8,2 3 180 71 12 999,6 7,9 7,7 3 160 82 18 997,7 8,7 8,6 3 180 92 Table 1. Meteorological data recorded on May 3, 2005 Time Surface level pressure mb Average tem- perature, °C Minimal tem- perature, °C Wind speed, m/s Wind direc- tion, degrees Relative humidity, % 24 1006,6 8,5 8,4 2 20 62 6 1004,0 9 8,2 3 180 71 12 999,6 7,9 7,7 3 160 82 18 997,7 8,7 8,6 3 180 92 Table 1. Meteorological data recorded on May 3, 2005 18 997,7 8,7 8,6 3 180 92 NOAA HYSPLIT MODEL Backward trajectories ending at 21 UTC 2 May 2005 FNL Meteoroogical Data Fig 3. Backward trajectory analysis (24 hours) at three al- titudes, start of run at 00:00, May 3, 2005 NOAA HYSPLIT MODEL Backward trajectories ending at 3 UTC 3 May 2005 FNL Meteoroogical Data Fig 4. Backward trajectory analysis (24 hours) at three al- titudes, start of run at 6:00, May 3, 2005 NOAA HYSPLIT MODEL Backward trajectories ending at 9 UTC 3 May 2005 FNL Meteoroogical Data Fig 5. 3.3. Pollen dispersion and backward air mass trajectory analysis on 13 May 2005 The 24 hours analysed were distinguished by an abundant rainfall that began at night and was gradually I. Šaulienė, L. Veriankaitė / JOURNAL OF ENVIRONMENTAL ENGINEERING AND LANDSCAPE MANAGEMENT – 2006, Vol XIV, No 3, 113–120 116 Veriankaitė / JOURNAL OF ENVIRONMENTAL ENGINEERING AND LANDSCAPE MANAGEMENT – 2006, Vol XIV, No 3, 113–120 117 Table 2. Meteorological data recorded on May 13, 2005 Time Surface level pressure mb Average temperature, °C Minimal temperature, °C Wind speed, m/s Wind direc- tion, degrees Relative humidity, % 24 1002,4 6,4 5,9 5 360 91 6 1000,0 6,8 6,1 5 360 93 12 1000,5 10,5 7,9 6 340 64 18 1001,6 12,9 11,4 5 340 55 NOAA HYSPLIT MODEL Backward trajectories ending at 21 UTC 12 May 2005 FNL Meteoroogical Data Fig 7. Backward trajectory analysis (24 hours) at three al- titudes, start of run at 00:00, May 13, 2005 NOAA HYSPLIT MODEL Backward trajectories ending at 3 UTC 13 May 2005 FNL Meteoroogical Data NOAA HYSPLIT MODEL Backward trajectories ending at 9 UTC 13 May 2005 FNL Meteoroogical Data Fig 9. Backward trajectory analysis (24 hours) at three al- titudes, start of run at 12:00, May 13, 2005 NOAA HYSPLIT MODEL Backward trajectories ending at 15 UTC 13 May 2005 FNL Meteoroogical Data Table 2. Meteorological data recorded on May 13, 2005 Time Surface level pressure mb Average temperature, °C Minimal temperature, °C Wind speed, m/s Wind direc- tion, degrees Relative humidity, % 24 1002,4 6,4 5,9 5 360 91 6 1000,0 6,8 6,1 5 360 93 12 1000,5 10,5 7,9 6 340 64 18 1001,6 12,9 11,4 5 340 55 Table 2. Meteorological data recorded on May 13, 2005 Time Surface level pressure mb Average temperature, °C Minimal temperature, °C Wind speed, m/s Wind direc- tion, degrees Relative humidity, % 24 1002,4 6,4 5,9 5 360 91 6 1000,0 6,8 6,1 5 360 93 12 1000,5 10,5 7,9 6 340 64 18 1001,6 12,9 11,4 5 340 55 Table 2. Meteorological data recorded on May 13, 2005 , , , NOAA HYSPLIT MODEL Backward trajectories ending at 21 UTC 12 May 2005 FNL Meteoroogical Data Fig 7. Backward trajectory analysis (24 hours) at three al- titudes, start of run at 00:00, May 13, 2005 NOAA HYSPLIT MODEL Backward trajectories ending at 3 UTC 13 May 2005 FNL Meteoroogical Data Fig 8. Backward trajectory analysis (24 hours) at three al- titudes, start of run at 6:00, May 13, 2005 NOAA HYSPLIT MODEL Backward trajectories ending at 9 UTC 13 May 2005 FNL Meteoroogical Data Fig 9. I. Šaulienė, L. Veriankaitė / JOURNAL OF ENVIRONMENTAL ENGINEERING AND LANDSCAPE MANAGEMENT – 2006, Vol XIV, No 3, 113–120 116 Backward trajectory analysis (24 hours) at three al- titudes, start of run at 12:00, May 3, 2005 NOAA HYSPLIT MODEL Backward trajectories ending at 15 UTC 3 May 2005 FNL Meteoroogical Data Fig 6. Backward trajectory analysis (24 hours) at three al- titudes, start of run 18:00 hours, May 3, 2005 NOAA HYSPLIT MODEL Backward trajectories ending at 21 UTC 2 May 2005 FNL Meteoroogical Data NOAA HYSPLIT MODEL Backward trajectories ending at 21 UTC 2 May 2005 FNL Meteoroogical Data NOAA HYSPLIT MODEL Backward trajectories ending at 9 UTC 3 May 2005 FNL Meteoroogical Data NOAA HYSPLIT MODEL Backward trajectories ending at 9 UTC 3 May 2005 FNL Meteoroogical Data NOAA HYSPLIT MODEL Backward trajectories ending at 9 UTC 3 May 2005 FNL Meteoroogical Data Fig 5. Backward trajectory analysis (24 hours) at three titudes, start of run at 12:00, May 3, 2005 NOAA HYSPLIT MODEL Backward trajectories ending at 21 UTC 2 May 2005 FNL Meteoroogical Data Fig 3. Backward trajectory analysis (24 hours) at three al- titudes, start of run at 00:00, May 3, 2005 Fig 5. Backward trajectory analysis (24 hours) at three al- titudes, start of run at 12:00, May 3, 2005 Fig 3. Backward trajectory analysis (24 hours) at three al- titudes, start of run at 00:00, May 3, 2005 NOAA HYSPLIT MODEL Backward trajectories ending at 15 UTC 3 May 2005 FNL Meteoroogical Data NOAA HYSPLIT MODEL Backward trajectories ending at 3 UTC 3 May 2005 FNL Meteoroogical Data Fig 4. Backward trajectory analysis (24 hours) at three al- titudes, start of run at 6:00, May 3, 2005 NOAA HYSPLIT MODEL Backward trajectories ending at 15 UTC 3 May 2005 FNL Meteoroogical Data Fig 6. Backward trajectory analysis (24 hours) at three a titudes, start of run 18:00 hours, May 3, 2005 NOAA HYSPLIT MODEL Backward trajectories ending at 3 UTC 3 May 2005 FNL Meteoroogical Data Fig 6. Backward trajectory analysis (24 hours) at three al- titudes, start of run 18:00 hours, May 3, 2005 Fig 6. Backward trajectory analysis (24 hours) at three al- titudes, start of run 18:00 hours, May 3, 2005 Fig 4. Backward trajectory analysis (24 hours) at three al- titudes, start of run at 6:00, May 3, 2005 Fig 4. Backward trajectory analysis (24 hours) at three al- titudes, start of run at 6:00, May 3, 2005 I. Šaulienė, L. I. Šaulienė, L. Veriankaitė / JOURNAL OF ENVIRONMENTAL ENGINEERING AND LANDSCAPE MANAGEMENT – 2006, Vol XIV, No 3, 113–120 116 Backward trajectory analysis (24 hours) at three al- titudes, start of run at 12:00, May 13, 2005 NOAA HYSPLIT MODEL Backward trajectories ending at 15 UTC 13 May 2005 FNL Meteoroogical Data Fig 10. Backward trajectory analysis (24 hours) at three altitudes, start of run at 18:00, May 13, 2005 NOAA HYSPLIT MODEL Backward trajectories ending at 21 UTC 12 May 2005 FNL Meteoroogical Data NOAA HYSPLIT MODEL Backward trajectories ending at 9 UTC 13 May 2005 FNL Meteoroogical Data Backward trajectories ending at 21 UTC 12 May 2005 FNL Meteoroogical Data Fig 7. Backward trajectory analysis (24 hours) at three al- titudes, start of run at 00:00, May 13, 2005 Backward trajectories ending at 9 UTC 13 May 2005 FNL Meteoroogical Data Fig 9. Backward trajectory analysis (24 hours) at three al titudes, start of run at 12:00, May 13, 2005 g FNL Meteoroogical Data Fig 7. Backward trajectory analysis (24 hours) at three al- titudes, start of run at 00:00, May 13, 2005 Fig 9. Backward trajectory analysis (24 hours) at three al- titudes, start of run at 12:00, May 13, 2005 NOAA HYSPLIT MODEL Backward trajectories ending at 3 UTC 13 May 2005 FNL Meteoroogical Data NOAA HYSPLIT MODEL Backward trajectories ending at 15 UTC 13 May 2005 FNL Meteoroogical Data NOAA HYSPLIT MODEL Backward trajectories ending at 3 UTC 13 May 2005 FNL Meteoroogical Data Fig 8. Backward trajectory analysis (24 hours) at three al titudes, start of run at 6:00, May 13, 2005 NOAA HYSPLIT MODEL Backward trajectories ending at 15 UTC 13 May 2005 FNL Meteoroogical Data Fig 10. Backward trajectory analysis (24 hours) at three altitudes, start of run at 18:00, May 13, 2005 Fig 8. Backward trajectory analysis (24 hours) at three al- titudes, start of run at 6:00, May 13, 2005 Fig 10. Backward trajectory analysis (24 hours) at three altitudes, start of run at 18:00, May 13, 2005 Fig 10. Backward trajectory analysis (24 hours) at three altitudes, start of run at 18:00, May 13, 2005 I. Šaulienė, L. Veriankaitė / JOURNAL OF ENVIRONMENTAL ENGINEERING AND LANDSCAPE MANAGEMENT – 2006, Vol XIV, No 3, 113–120 118 118 Table 3. I. Šaulienė, L. Veriankaitė / JOURNAL OF ENVIRONMENTAL ENGINEERING AND LANDSCAPE MANAGEMENT – 2006, Vol XIV, No 3, 113–120 116 Backward trajectory analysis (24 hours) at three altitudes, start of run at 18:00, May 21, 2005 O S O Backward trajectories ending at 15 UTC 21 May 2005 FNL M t i l D t j g FNL Meteoroogical Data The meteorological data of 24 hours (Table 3) were favourable for pollination: the day was sunny, a sudden change of relative humidity from 95 % to 33 %, quite a high pressure. Evaluating the air mass that reached Šiau- liai Aerobiological Station at a different time, the air mass movement of the same direction was stated: air mass moved from the southern part of Estonia through Russia, Latvia and northern Lithuania (Figs 11–12). On the way of the air mass there was no rainfall. According to the EAN data, in Latvia there was a large amount of birch pollen and it could get into the air mass moving towards Lithuania. A low morning temperature (Table 3) unfavourable for the pollination of local plants allows consideration of the probability of long-range transport, so it is possible to establish presumption that the birch pollen from southern Latvia could have been recorded at Šiauliai Aerobiological Station. I. Šaulienė, L. Veriankaitė / JOURNAL OF ENVIRONMENTAL ENGINEERING AND LANDSCAPE MANAGEMENT – 2006, Vol XIV, No 3, 113–120 116 Meteorological data recorded on May 21, 2005 Time Surface level pressure mb Average temperature, °C Minimal temperature, °C Wind speed, m/s Wind direction, degrees Relative humidity, % 24 1012,3 7 6,9 0 Tyla 73 6 1011,1 3,5 3,2 0 Tyla 95 12 1009,7 16,3 12,4 3 135 38 18 1006,1 19,3 18,1 3 90 33 Wind speed, m/s Wind direction, degrees Relative humidity, % 0 Tyla 73 0 Tyla 95 3 135 38 3 90 33 NOAA HYSPLIT MODEL Backward trajectories ending at 15 UTC 21 May 2005 FNL Meteoroogical Data Fig 12. Backward trajectory analysis (24 hours) at three altitudes, start of run at 18:00, May 21, 2005 corded on May 21, 2005 Average temperature, °C Minimal temperature, °C Wind speed, m/s Wind direction, degrees Relative humidity, % 7 6,9 0 Tyla 73 3,5 3,2 0 Tyla 95 16,3 12,4 3 135 38 19,3 18,1 3 90 33 erent height helped to explain concentration occurred at the a of 24 hours (Table 3) were the day was sunny, a sudden from 95 % to 33 %, quite a e air mass that reached Šiau- at a different time, the air me direction was stated: air hern part of Estonia through n Lithuania (Figs 11–12). On re was no rainfall. According there was a large amount of et into the air mass moving morning temperature (Table 3) ation of local plants allows ility of long-range transport, h presumption that the birch could have been recorded at on. that backward trajectory ana- ate specific pollen episodes. PLIT MODEL t 21 UTC 20 M 2005 NOAA HYSPLIT MODEL Backward trajectories ending at 15 UTC 21 May 2005 FNL Meteoroogical Data Fig 12. Backward trajectory analysis (24 hours) at three altitudes, start of run at 18:00, May 21, 2005 Table 3. Meteorological data recorded on May 21, 2005 NOAA HYSPLIT MODEL Backward trajectories ending at 15 UTC 21 May 2005 FNL Meteoroogical Data air mass trajectories at a different height helped to explain why the increase of pollen concentration occurred at the end of pollination season. NOAA HYSPLIT MODEL Backward trajectories ending at 15 UTC 21 May 2005 FNL Meteoroogical Data Fig 12. Backward trajectory analysis (24 hours) at three altitudes, start of run at 18:00, May 21, 2005 NOAA HYSPLIT MODEL Backward trajectories ending at 15 UTC 21 May 2005 FNL Meteoroogical Data Fig 12. References 1. Filon, F. L.; Bosco, A.; Berbina, P.; Sauli, M. L.; Longo, L. R. Betulaceae and Corylaceae in Trieste (NE- Italy): Aerobiological and clinical data. Aerobiologia, Vol 16, 2000, p. 87–91. 10. Kuliešis, A.; Kasperavičius, A.; Kulbokas, G.; Kval- kauskienė, M. Lietuvos nacionalinė miškų inventorizacija 1998–2002. Atrankos schema, metodai, rezultatai. Kaunas: Naujasis lankas, 2003. 256 p. 2. Corden, J.; Millington, W; Bailey, J.; Brookes, M.; Caul- ton, E.; Emberlin, J.; Mullins, J.; Simpson, C.; Wood, A. UK regional variations in Betula pollen (1993–1997). Aerobiologia, Vol 16, 2000, p. 227–232. 11. Mandrioli, P.; Comtois, P.; Levizzani, V. Methods in Aerobiology. Bologna: Pitagora Editrice, 1998. 262 p. 12. Draxler, R. R.; Rolph, G. D. HYSPLIT (HYbrid Single- Particle Lagrangian Integrated Trajectory) Model access via NOAA ARL READY Website (http://www.arl.noaa.gov/ready/hysplit4.html). NOAA Air Resources Laboratory, Silver Spring, MD, 2003. 3. Radišić, P.; Šikoparija, B. Betula spp. Pollen in the at- mosphere of Novi Sad (2000–2002). Aerobiologia, Vol 21, 2005, p. 63–67. 4. Mendez, J.; Comtois, P.; Iglesias, I. Betula pollen: One of the most important aeroallergens in Ourense, Spain. Aerobiological studies from 1993 to 2000. Aerobiologia, Vol 21, 2005, p. 115–123. 4. Discussion This study has shown that backward trajectory ana- lysis can be used to investigate specific pollen episodes. Fig 12. Backward trajectory analysis (24 hours) at three altitudes, start of run at 18:00, May 21, 2005 NOAA HYSPLIT MODEL Backward trajectories ending at 21 UTC 20 May 2005 FNL Meteoroogical Data Fig 11. Backward trajectory analysis (24 hours) at three altitudes, start of run at 00:00, May 21, 2005 NOAA HYSPLIT MODEL Backward trajectories ending at 21 UTC 20 May 2005 FNL Meteoroogical Data On May 3, 13, 21 the evaluation of the aerobiologi- cal situation was successful having used backward air mass trajectory analysis. Using the HYSPLIT 4 model, it is revealed why uncommonly large pollen concentrations were recorded. Possibility to use the EAN data allowed to establish presumption that under atypical conditions at Šiauliai Aerobiological Station the increase of pollen concentration may have been recorded not only because of the dynamics of the pollination of local flora, but also because of airborne pollen long-range transport. Evaluating the importance of air mass movement for long-range pollen transport, it is necessary to choose a correct model. Application of the HYSPLIT 4 model revealed the regularities of transport and may be succes- sfully used. Evaluation of pollen dispersion by the backward air mass trajectory method presented in the paper established the precondition to look for possibilities of application of the HYSPLIT 4 model to prepare the forecasts of pollen dispersion. Such a permanent control of pollen spread (together with evaluating the processes that determine long-range transport) is important for pa- tients with allergic illnesses, patient associations, doctors, pharmaceutical companies, organizers of international sport and leisure events, tourism business. Precise pollen Fig 11. Backward trajectory analysis (24 hours) at three altitudes, start of run at 00:00, May 21, 2005 I. Šaulienė, L. Veriankaitė / JOURNAL OF ENVIRONMENTAL ENGINEERING AND LANDSCAPE MANAGEMENT – 2 identification may decrease losses experienced because of mistakes in choosing medicine against a specific allergen that is usually of a nonlocal origin. 5. Latałowa, M.; Miętus, M.; Uruska, A. Seasonal variations in the atmospheric Betula pollen count in Gdańsk (south- ern Baltic coast) in relation to meteorological parameters. Aerobiologia, Vol 18, 2002, p. 33–43. 6. Laaidi, K. Predicting days of high allergenic risk during Betula pollination using weather types. International Journal of Biometeorology, Vol 45, 2001, p. 124–132. 5. Conclusions 1. It is shown that backward air mass analysis may be applied in investigating relationship among pollen dispersion, meteorological conditions and air mass trans- port. 1. It is shown that backward air mass analysis may be applied in investigating relationship among pollen dispersion, meteorological conditions and air mass trans- port. 7. Šaulienė, I.; Motiekaitytė, V. Pecularities of Aero- palinological Monitoring in Northern Lithuania. In: The 6th International Conference on Environmental Engineer- ing. Selected Papers, Vol 1 (May 26–27 2005, Vilnius, Lithuania). Vilnius: Technika, 2005, p. 252–256. p 2. It is shown that the HYSPLIT 4 model may be used to investigate long-range pollen transport. Š p 2. It is shown that the HYSPLIT 4 model may be used to investigate long-range pollen transport. 8. Jager, S.; Spieksma, F. Th. M.; Nolard, N. Fluctuations and trends in airborne concentrations of some abundant pollen types, monitored at Vienna, Leiden, and Brussels. Grana, Vol 30, 1991, p. 3009–312. g g g p p 3. It is revealed that at Šiauliai Aerobiological Sta- tion the dynamics of pollen concentration is conditioned by the local flora and airborne pollen long-range trans- port. 3. It is revealed that at Šiauliai Aerobiological Sta- tion the dynamics of pollen concentration is conditioned by the local flora and airborne pollen long-range trans- port. 9. Smith, M.; Emberlin, J.; Kress, A. Examing high magni- tude grass pollen episodes at Worcester, United Kingdom, using back-trajectory analysis. Aerobiologia, Vol 21, 2005, p 85–94. ОЦЕНКА ДИСПЕРСИИ ПЫЛЬЦЫ В АТМОСФЕРЕ ПО АНАЛИЗУ ТРАЕКТОРИЙ ОБРАТНЫХ ВОЗДУШНЫХ МАСС И. Шаулене, Л. Верянкайте S a n t r a u k a Žiedadulkių judėjimą atmosferoje aerobiologai tiria analizuodami atgalinių oro masių trajektorijas. Šiame straipsnyje gili- namasi į beržo žiedadulkių sklaidos atmosferoje ypatumus. Šiaulių aerobiologijos stotyje 2005 m. žiedadulkės buvo fik- suojamos Hirst tipo sporų gaudykle. Žiedadulkių judėjimas žydint beržams aplinkinėse Europos šalyse buvo įvertintas remiantis Europos aeroalergenų tinklo duomenų baze. Apibendrintais duomenimis pasinaudota, įrodant tolimąją žiedadulkių pernašą. Oro masių trajektorijos apskaičiuotos pagal HYSPLIT 4 modelį, trajektorijų brėžimo principą grindžiant oro masės padėties integravimu laiko atžvilgiu. Tyrimas parodė, kad atgalinių oro masių trajektorijų analizė gali būti taikoma tiriant sąsają tarp žiedadulkių dispersijos, meteorologinių sąlygų ir oro masių pernašos. Nustatyta, kad Šiaulių aerobiologijos stotyje žiedadulkių koncentracijos dinamiką lemia vietinė augalija ir žiedadulkių atmosferoje to- limosios pernašos. Straipsnyje pateiktas žiedadulkių dispersijos vertinimas atgalinių oro masių trajektorijų analizės me- todu suformavo prielaidas siekti modelio HYSPLIT 4 taikymo galimybių žiedadulkių sklaidai prognozuoti. Prasminiai žodžiai: žiedadulkės, meteorologinės sąlygos, tolimosios pernašos, atgalinės oro masių trajektorijos, beržo dulkėjimas, oro tarša. r of Ecology and Environmental Research, Šiauliai University. Research interests: meteorology, environmental re- erobiology. Р е з ю м е Р е з ю м е Для исследования пыльцы в атмосфере аэробиологи используют траектории обратных атмосферных масс. В статье анализируются особенности рассеянности берёзовой пыльцы в атмосфере в 2005 г. Пыльца фиксировалась с использованием ловушки спор типа Хирст. Ситуация цветения берёзы в соседних странах Европы была оценена согласно данным базы Европейской сети по аэроаллергенам. Данные обобщены, использованы, доказан дальний I. Šaulienė, L. Verinkaitė / JOURNAL OF ENVIRONMENTAL ENGINEERING AND LANDSCAPE MANAGEMENT – 2006, Vol XIV, No 3, 113–120 120 перенос пыльцы. Траектории обратных воздушных масс установлены согласно модели HYSPLIT 4. Исследования показали, что анализ обратных воздушных масс может быть использован для изучения связи между дисперсией пыльцы, метеоусловиями и переносом атмосферных масс. Установлено, что на аэробиологической станции города Шяуляй динамика пыльцы предопределена местной флорой и дальними переносами. Представленная оценка дисперсии пыльцы может служить основой при изучении возможности использования HYSPLIT 4 для прогнозов. Ключевые слова: пыльца, метеоусловия, дальний перенос, обратные траектории атмосферных масс, пыление берёзы, загрязнение воздуха. Ingrida ŠAULIENĖ. Dr, Assoc Prof and head of Dept of Environmental Research, Šiauliai University (ŠU), Vilniaus g. 88, LT-76285, Šiauliai, Lithuania. Ingrida ŠAULIENĖ. Dr, Assoc Prof and head of Dept of Environmental Research, Šiauliai University (ŠU), Vilniaus g. 88, LT-76285, Šiauliai, Lithuania. Doctor of Natural Sciences (botany), 2002. Probation: Poznan University (Poland), Certificate of Aerobiologist, 2003; In- stitute of Zoology and Botany, Estonian Agricultural University, 2003; Faculty of Geography and Earth Sciences, Univer- sity of Latvia, 2005. Membership: Lithuanian Association of Plant Physiology, Phenology Community of Lithuanian Association of Nature, International Association for Aerobiology, Nordic Aerobiology Federation. Publications: author of 11 research papers. Research interests: plant physiology (influence of environmental factors on plant regeneration proc- esses), aerobiology, environment quality. Laura VERIANKAITĖ. Master student, Dept of Hydrology and Climatology, Vilnius University (VU), M. K. Čiurlionio g. 21/27, LT-03101, Vilnius, Lithuania. Bachelor of Ecology and Environmental Research, Šiauliai University. Research interests: meteorology, environmental re- search, aerobiology.
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English
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Dynamic prediction of outcome for patients with severe aortic stenosis: application of joint models for longitudinal and time-to-event data
BMC cardiovascular disorders
2,015
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© 2015 Andrinopoulou et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Andrinopoulou et al. BMC Cardiovascular Disorders (2015) 15:28 DOI 10.1186/s12872-015-0035-z Andrinopoulou et al. BMC Cardiovascular Disorders (2015) 15:28 DOI 10.1186/s12872-015-0035-z Dynamic prediction of outcome for patients with severe aortic stenosis: application of joint models for longitudinal and time-to-event data Eleni-Rosalina Andrinopoulou1,4*, Dimitris Rizopoulos1, Marcel L. Geleijnse2, Emmanuel Lesaffre1,3, Ad J. J. C. Bogers4 and Johanna J. M. Takkenberg4 Abstract Background: Physicians utilize different types of information to predict patient prognosis. For example: confronted with a new patient suffering from severe aortic stenosis (AS), the cardiologist considers not only the severity of the AS but also patient characteristics, medical history, and markers such as BNP. Intuitively, doctors adjust their prediction of prognosis over time, with the change in clinical status, aortic valve area and BNP at each outpatient clinic visit. With the help of novel statistical approaches to model outcomes, it is now possible to construct dynamic event prediction models, employing longitudinal data such as AVA and BNP, and mimicking the dynamic adjustment of prognosis as employed intuitively by cardiologists. We illustrate dynamic prediction of patient survival and freedom from intervention, using baseline patient characteristics and longitudinal BNP data that are becoming available over time, from a cohort of patients with severe aortic stenosis. Methods: A 3-step approach was employed: (1) construction of a mixed-effects model to describe temporal BNP progression, (2) jointly modeling the mixed-effects model with time-to-event data (death and freedom from intervention), and (3) using the joint model to build subject-specific prediction risk models. The dataset used for this purpose includes 191 patients with severe aortic stenosis who were followed over a 3-year time period. Results: In the mixed-effects model BNP was significantly influenced by time, baseline patient age, gender, LV fractional ejection fraction and creatinine. Additionally, the joint model showed that an increasing BNP trend over time was found to be a significant predictor of death. Conclusions: By jointly modeling longitudinal data with time-to-event outcomes it is possible to construct individualized dynamic event prediction models that renew over time with accumulating evidence. It provides a potentially valuable evidence-based tool for everyday use in medical practice. ar disease, Brain natriuretic peptide, Aortic valve disease, Survival, Individualized prediction Keywords: Valvular disease, Brain natriuretic peptide, Aortic valve disease, Survival, Individualized p RESEARCH ARTICLE Open Access * Correspondence: e.andrinopoulou@erasmusmc.nl 1Department of Biostatistics, Erasmus MC, Rotterdam, The Netherlands 4Department of Cardiothoracic Surgery, Erasmus MC, Rotterdam, The Netherlands Full list of author information is available at the end of the article Background age and comorbidities, New York Heart Association (NYHA) functional class and patient history, in order to make an assessment of patient prognosis. Additionally, biomarkers such as brain natriuretic peptide (BNP) can be used to further assess AS severity and prognosis. A small AVA and a high BNP are both associated with a more severe disease and a worse outcome [1-3]. In clinical practice, physicians utilize different sources of information to predict patient prognosis. For example, in diagnosing a new patient with severe aortic stenosis (AS), a cardiologist considers not only the severity of the AS (for example through aortic valve area AVA mea- surement) but also patient characteristics such as patient Empirically, cardiologists adjust their prognosis over time at each outpatient clinic visit, with the change in functional class, AVA and BNP. Based on emerging evidence on determinants of the outcome in AS, and with the help of novel statistical approaches to model * Correspondence: e.andrinopoulou@erasmusmc.nl 1Department of Biostatistics, Erasmus MC, Rotterdam, The Netherlands 4Department of Cardiothoracic Surgery, Erasmus MC, Rotterdam, The Netherlands Full list of author information is available at the end of the article Page 2 of 8 Andrinopoulou et al. BMC Cardiovascular Disorders (2015) 15:28 outcomes, it is now possible to construct dynamic predic- tion models for patient outcome, employing repeatedly collected (longitudinal) data such as BNP, mimicking the dynamic adjustment of prognosis as employed intuitively by cardiologists at each outpatient clinic visit. and longitudinal BNP measurement, requires that we first describe the evolution of BNP over time, correcting for baseline variables. Second, we use this information in a time-to-event model. Finally, using the combined model, we perform dynamic event predictions. In the next para- graphs we describe in detail the statistical methods that were employed in this 3-step process, and the rationale be- hind these methods. This paper aims to illustrate the use of joint models of longitudinal and time-to-event data to dynamically pre- dict individualized event occurrence severe AS. For this purpose, data from a prospective cohort study of 191 pa- tients with severe AS is modeled to dynamically predict prognosis of two patients: Mr. Jones and Mr. Smith; who were recently diagnosed with severe AS. First, we fitted a mixed-effects model to describe the evolution of BNP over time. Patient dataset We used the patient dataset of a previously reported pro- spective cohort study of 191 adult patients, who were diag- nosed with severe aortic valve disease in seven cardiology clinics in the wider Rotterdam area between 2006 and 2009, and who were followed for 2 years [4]. Inclusion cri- teria were AVA ≤1 cm2, peak transaortic jet velocity (Vmax) ≥4 m/s, or aortic valve/left ventricular outflow tract velocity time integral ratio ≥4. The patients were followed clinically, including BNP measurements, and echocardio- graphically at baseline and then after 6, 12 and 24 months. Baseline patient characteristics are displayed in Table 1. In total 561 BNP measurements were collected over a 2-year period (mean 0.9 years; range 0–2.5 years). During the follow-up period, 15 % of the patients (N = 28) died and 48 % (N = 91) received an aortic valve replacement of trans- catheter aortic valve implantation. Second, to investigate the effect of the repeated BNP measurements on death and intervention probabilities, separate joint models of longitudinal and survival out- comes were constructed [5,6]. AVA, age, symptoms, gen- der, LV fraction and creatinine (all at baseline) were included as additional confounders. More details about the joint models are presented in the Appendix. The study protocol was approved by the medical ethics committee of Erasmus University Medical Center (MEC 2006–066); all patients provided written informed consent. Background Particularly, the model in- cluded time (years) and the baseline covariates: AVA (cm2), patient age (years), symptoms (yes/no), gender, transformed LV ejection fraction (%) and transformed creatinine (micromol/L). Transformation was done by dividing the values with the standard deviations of the specific covariates. Moreover, due to heterogeneity in the residuals plot the logarithmic scale of BNP was used. An advantage of the mixed-effects models is that they account for the positive correlation between the mea- surements that are observed within the same patient. For example, the values of BNP that are observed over time from the same patient are expected to be more cor- related than between patients. Moreover, these models account for the biological variability in the longitudinal outcome. Specifically, if we measure BNP twice a day, we may not obtain the same result. By taking this into account using the mixed-effects model, more reliable re- sults will be observed. Statistical methods Third, we considered the joint modeling framework and focused on the assessment of the predictive ability of our survival outcomes. Specifically, it was of interest to predict patient survival and aortic valve intervention- free for a new patient that has provided us with a set of BNP measurements and baseline characteristics, using the fitted joint model for all patients. Due to the fact that BNP is time-dependent and not constant between the visits and therefore providing longitudinal measure- ment up to a specific time, assumes survival up to this time, it was more relevant to calculate the probability of surviving a future time point, given that the patient was alive until his last follow-up visit [7,8]. Using this approach, we applied the resulting joint modeling framework to two hypothetical patients: Mr. Jones and Mr. Smith and pre- dicted their future survival and aortic valve intervention- free probabilities. Specifically, Mr. Jones is a 72 year old male, with creatinine value at baseline 92 micromol/L, AVA of 0.96 cm2, LV ejection fraction 61 % and BNP The development of a dynamic event prediction model that takes into account both baseline patient characteristics The development of a dynamic event prediction model that takes into account both baseline patient characteristics Table 1 Baseline patient characteristics All patients (Number = 191) Male gender (n, %) 118, 62 % Age in years (mean, sd) 72.6, 11.4 Symptomatic at study entry (n, %) 132, 69 % Smoking (n, %) 115, 60 % Hypertension (n, %) 100, 52 % Diabetes (n, %) 39, 20 % Dyslipidemia (n, %) 93, 49 % AVA in cm2 (mean, sd) 0.74, 0.27 LV ejection fraction in % (mean, sd) 61, 6.7 Creatinine in micromol/L (mean, sd) 89, 125 AVA = aortic valve area; LV = left ventricular Table 1 Baseline patient characteristics All patients (Number = 191) Table 1 Baseline patient characteristics Page 3 of 8 Page 3 of 8 Andrinopoulou et al. BMC Cardiovascular Disorders (2015) 15:28 Table 2 Coefficients, standard error of coefficients and p-values for the mixed-effects model describing the evolution of BNP over time Coef Se(coef) p-value (Intercept) 2.92 0.95 0.0025 Time (years) 0.23 0.04 <0.0001 AVA (cm2) −1.48 0.3 <0.0001 Age (years) 0.05 0.007 <0.0001 Symptoms 0.43 0.18 0.0188 Male gender −0.34 0.18 0.0607 *LV ejection fraction (%) −0.16 0.08 0.0486 *Creatinine (micromol/L) 0.4 0.09 <0.0001 AVA = aortic valve area; LV = left ventricular. Statistical methods * Trasnformed LV ejection fraction and Creatinine in the models Table 2 Coefficients, standard error of coefficients and p-values for the mixed-effects model describing the evolution of BNP over time 287, 1068 and 1070 pg/ml measured at 0, 0.9, 1.2 and 2 years. Furthermore, we performed internal validation using a bootstrapping procedure (size of 1000). Specifically, we focused on discrimination, that is, how well can the model discriminate between patients who are about to experience the event within a time frame after the last measurement, from patients that are going to surpass this time frame. Since the patients were visiting their physician approximately every half year, we set this time frame. In particularly, we relied on the receiver operating characteristic (ROC) approach to assess the predictive ability of the longitudinal biomarker BNP [7]. All analyses have been implemented in R-3.2.0, which can be downloaded as freeware at http://www.r-project.org, using the JM package [9]. values over time 64, 70, 72 and 78 pg/ml measured at 0.5, 0.9, 1.5 and 1.5 years. Moreover, he is asymptomatic at baseline. Additionally, Mr. Smith is a 79 year old male that has creatinine equal to 92 micromol/L, AVA equal to 0.61 cm2, LV ejection fraction equal to 61 % and he is symptomatic at baseline. Finally, his BNP values are 381, values over time 64, 70, 72 and 78 pg/ml measured at 0.5, 0.9, 1.5 and 1.5 years. Moreover, he is asymptomatic at baseline. Additionally, Mr. Smith is a 79 year old male that has creatinine equal to 92 micromol/L, AVA equal to 0.61 cm2, LV ejection fraction equal to 61 % and he is symptomatic at baseline. Finally, his BNP values are 381, ptide; AVA = aortic valve area; LV = left ventricular; HR = hazard ratio. * Trasnformed LV ejection fraction and Creatinine in the models Results As illustrated in Table 2 in the mixed-effects model de- scribing the evolution of BNP over time, all covariates have a strong association with the levels of BNP, except baseline gender. Specifically, a longer follow-up, lower AVA at baseline, older patient baseline age, symptomatic Fig. 1 Effect plot of AVA described from the joint model for Mr. Jones and another patient with the same age, with impaired LV ejection fraction of 61, creatinine level equal to 92 and both patients with no symptoms at baseline Fig. 1 Effect plot of AVA described from the joint model for Mr. Jones and another patient with the same age, with impaired LV ejection fraction of 61, creatinine level equal to 92 and both patients with no symptoms at baseline Andrinopoulou et al. BMC Cardiovascular Disorders (2015) 15:28 Page 4 of 8 Page 4 of 8 Table 3 Coefficients, standard error of coefficients and p-values for the joint model predicting death and aortic valve intervention Coef Exp(coef):HR Se(coef) p-value Death BNP at specific time point (pg/ml) 0.5 1.65 0.3 0.0962 AVA (cm2) −2.61 0.07 1.5 0.0815 Age (years) 0.02 1.02 0.04 0.5674 Male gender 1.12 3.06 0.6 0.0623 Symptoms 1.87 6.49 1.05 0.0753 *LV ejection fraction (%) 0.01 1.01 0.25 0.9539 *Creatinine (micromol/L) 0.18 1.2 0.15 0.2162 Aortic valve intervention BNP at specific time point (pg/ml) 0.18 1.2 0.25 0.4787 AVA (cm2) −1.12 0.33 1.04 0.2804 Age (years) −0.04 0.96 0.02 0.0077 Male gender 0.39 1.48 0.49 0.4287 Symptoms 1.08 2.94 0.46 0.0183 *LV ejection fraction (%) 0.24 1.27 0.21 0.2388 *Creatinine (micromol/L) −1.43 0.24 1.31 0.2761 BNP = brain natriuretic peptide; AVA = aortic valve area; LV = left ventricular; HR = hazard ratio. * Trasnformed LV ejection fraction and Creatinine in the models Table 3 Coefficients, standard error of coefficients and p-values for the joint model predicting death and aortic valve intervention Coef Exp(coef):HR Se(coef) p-value Fig. 2 Dynamic prediction for the survival probability for Mr. Jones for the survival probability for Mr Jones Fig. 2 Dynamic prediction for the survival probability for Mr. Jones Andrinopoulou et al. BMC Cardiovascular Disorders (2015) 15:28 Page 5 of 8 same can be seen in Fig. 3, where the intervention-free probabilities are presented. This can be explained by the fact that Mr. Jones’ BNP measurements are relatively low and stable. In contrast, Mr. Results Smith has more steep curves for both expected survival and aortic valve intervention- free probabilities indicating that the patient should be monitored frequently. Specifically, one year after his first follow-up visit Mr. Smith has a survival probability of 70 %, while one year after his last visit his survival probabi- lity is less than 50 %. The reason could be that Mr. Smith has a high BNP value at baseline and his progression is fas- ter within the 2 year period compared to Mr. Jones. Thus, Mr. Smith has a much lower survival probability one year after his last follow-up. patient at baseline, lower baseline LV ejection fraction and a higher baseline serum creatinine are highly associ- ated with an increased BNP. Moreover, Fig. 1 shows the evolutions of BNP over time of two hypothetical patients, Mr. Jones and another patient that has the same characteristics as Mr. Jones except for the AVA level which is 0.61. It is obvious in Fig. 1 that a smaller AVA is associated with a higher BNP at baseline. Furthermore, there is no difference in the progression of BNP between the two patients. From the joint model with the death as outcome, in Table 3, we observe that smaller AVA at base- line, male patient, symptoms at baseline and higher BNP at a specific time point (since we used all repeated mea- surements in the model for the specific covariate) tend to be associated with death. The joint model with the aortic valve intervention as outcome shows that a younger patient and symptoms at baseline are strongly associated with aortic valve intervention probabilities. Finally, from the bootstrapping we observe the area under the ROC curve for death and aortic valve inter- vention to be 0.88 and 0.59, respectively. This indicates a good discriminative capability of the BNP for death, and little added value for the prediction of aortic valve intervention. Figures 2, 3, 4 and 5 represent the dynamic prediction of survival and aortic valve intervention-free respectively for Mr. Jones and Mr. Smith, employing the joint mode- ling framework. It can be seen in Fig. 2 that as more BNP measurements accumulated over time for Mr. Jones, the survival curve does not show big changes. Moreover, the Discussion In this paper we illustrated the use of joint models of longitudinal and time-to-event data for individualized In this paper we illustrated the use of joint models of longitudinal and time-to-event data for individualized Fig. 3 Dynamic prediction for the aortic valve intervention-free probability for Mr. Jones ig. 3 Dynamic prediction for the aortic valve intervention-free probability for Mr. Jones Andrinopoulou et al. BMC Cardiovascular Disorders (2015) 15:28 Page 6 of 8 Fig. 4 Dynamic prediction for the survival probability for Mr. Smith Fig. 4 Dynamic prediction for the survival probability for Mr. Smith Fig. 4 Dynamic prediction for the survival probability for Mr. Smith dynamic event prediction using serial BNP measurements in patients with severe AS. Patient prognostication may be improved by the use of such models that take into account all available medical information that accumulates over time. In the case of Mr. Jones and Mr. Smith, their prob- abilities of survival and aortic valve intervention-free were calculated accounting for all BNP values that accumulated over time and were updated when new BNP mea- surements became available. This approach provides the cardiologist with a useful evidence-based tool to assess the impact of BNP on patient prognosis. Importantly, the calculated probabilities for survival and aortic valve intervention-free can be used as an early warning system, allowing the necessary time for the physicians to plan an intervention. Given the impaired quality of life (QOL) of symptomatic patients with AS [10] and the considerable improvement in QOL after the aortic valve replacement, dynamic event predictions may be of great value in opti- mizing the timing of the intervention [11]. realistic for biomarkers due to the fact that such cardio data as BNP values cannot be assumed to be constant between visits. Of course, the proposed methodology has several (potential) limitations, both from a clinical and a statis- tical point of view. From a clinical point of view, every patient is unique, and analysis based on group data may not account for the special characteristics of an indi- vidual patient. Moreover, there are factors that are not included in the statistical models that may play an im- portant role and thus influence the decision making. In this respect we acknowledge that the proposed metho- dology may be supportive in clinical decision making, but can never replace clinical expertise. Discussion Also, for clini- cians with limited understanding of advanced statistical models, the proposed methodology may be difficult to comprehend, and tutorials aimed at clinicians are needed to further educate clinical professionals [6]. From the stat- istical point of view, the analysis of more than one longitu- dinal outcomes such as BNP, AVA and symptoms over time together with survival outcomes requires advanced computational work and standard statistical packages do not yet provide these options. Moreover, there is not yet a package performing dynamic event prediction accounting for the competing risk problem: specifically, patients could The joint model of longitudinal and survival data [5] represents a powerful statistical tool capable of capturing the association between longitudinal and survival data. An alternative approach is to utilize the time-dependent Cox model. However, this model assumes a step func- tion between the repeated measurements, which is not Andrinopoulou et al. BMC Cardiovascular Disorders (2015) 15:28 Page 7 of 8 Fig. 5 Dynamic prediction for the aortic valve intervention-free probability for Mr. Smith ig. 5 Dynamic prediction for the aortic valve intervention-free probability for Mr. Smith to produce. Particularly, an easy web application could give the opportunity to every physician to derive up- dated predictions for new patients when more longitu- dinal outcomes are available. die or require an intervention, in this case is aortic valve intervention. The analysis, then, becomes more compli- cated by the fact that the two censored outcomes are not completely independent, thus it is clear that analyzing the two outcomes separately is not appropriate and may lead to bias. However, in order to keep the analysis simple and thus to use only available packages, in the paper we did not accounted for the competing risk problem. Further- more, a topic that was not addressed in this paper, con- cerns the validation of the derived predictions in terms of calibration. Within the joint modeling frame, some work has been done by Rizopoulos (2011) [7] and Proust-Lima and Taylor (2009) [8]. Specifically, they focus on predictive accuracy measures that compare the actual value of pre- dictions with the observed data using simulated data. Finally, a dataset consisting of more patients that are followed for a longer time period may provide better pre- dictions for future patients. From the analysis we obtained a non-significant asso- ciation between aortic valve intervention and the evolu- tion of BNP (Table 3). References 1. Katz M, Tarasoutchi F, Pesaro AE, Lopes RD, Spina GS, Vieira ML, et al. Natriuretic peptides and long-term mortality in patients with severe aortic stenosis. J Heart Valve Dis. 2012;21:331–6. 2. Lancellotti P, Moonen M, Magne J, O’Connor K, Cosyns B, Attena E, et al. Prognostic effect of long-axis left ventricular dysfunction and B-type natriuretic peptide levels in asymptomatic aortic stenosis. Am J Cardiol. 2010;105:383–8. 3. Otto CM, Burwash IG, Legget ME, Munt BI, Fujioka M, Healy NL, et al. Prospective study of asymptomatic valvular aortic stenosis. Clinical, echocardiographic, and exercise predictors of outcome. Circulation. 1997;95:2262–70. Authors’ contributions h f d f d Authors contributions Writing the first draft and data analysis: ERA. Data interpretation, discussion and preparation of the final manuscript: ERA, DR, MLG, EL, AJJCB, JJMT. All authors read and approved the final manuscript. Authors contributions Writing the first draft and data analysis: ERA. Data interpretation, discussion and preparation of the final manuscript: ERA, DR, MLG, EL, AJJCB, JJMT. All authors read and approved the final manuscript. Conclusions In conclusion, this paper has shown that temporal ad- justment of risk prediction models for patients with se- vere AS, as more measurements of BNP become available over time, provide the physician with an evi- dence based understanding of the prognostic implication of changes in the patient’s disease condition. With the cardiovascular medical practice increasingly moving to- wards personalized medicine [12], joint models may pro- vide an attractive tool for subject-specific predictions. The proposed joint model that was built and used to predict prognosis of patients suffering from severe AS, can be easily extended to other chronic disease entities that employ both longitudinal and survival data to dy- namically assess patient prognosis. Competing interests Competing interests The authors declare that they have no competing interests. Author details 1 f 1Department of Biostatistics, Erasmus MC, Rotterdam, The Netherlands. 2Department of Cardiology, Erasmus MC, Rotterdam, The Netherlands. 3KU Leuven, L-Biostat, Leuven, Belgium. 4Department of Cardiothoracic Surgery, Erasmus MC, Rotterdam, The Netherlands. Received: 26 May 2014 Accepted: 21 April 2015 Received: 26 May 2014 Accepted: 21 April 2015 Introduction to joint models * 4. Heuvelman HJ, van Geldorp MW, Kappetein AP, Geleijnse ML, Galema TW, Bogers AJ, et al. Clinical course of patients diagnosed with severe aortic stenosis in the Rotterdam area: insights from the AVARIJN study. Neth Heart J. 2012;20:487–93. Let Ti * denote the true failure time for the i-th individual (i = 1,…, n), and Ci the censoring time, then Ti = min(Ti *, Ci) represents the observed failure time for the i-th patient. Moreover, δi = 0,1 is the event indicator where 0 indicates censoring. For the longitudinal part, we let yi consist of longitudinal responses that may be obtained at different time points tij and have length ni. To describe the subject- specific evolutions over time of the longitudinal outcome we utilize a linear mixed-effects model. Specifically, it takes the form, 5. Rizopoulos D. Joint models for longitudinal and time-to-event data with applications in R. Boca Raton: Chapman and Hall/CRC Biostatistics Series; 2012. 6. Andrinopoulou ER, Rizopoulos D, Jin R, Bogers AJ, Lesaffre E, Takkenberg JJ. An introduction to mixed models and joint modeling: analysis of valve function over time. Ann Thorac Surg. 2012;93:1765–72. 6. Andrinopoulou ER, Rizopoulos D, Jin R, Bogers AJ, Lesaffre E, Takkenberg JJ. An introduction to mixed models and joint modeling: analysis of valve function over time. Ann Thorac Surg. 2012;93:1765–72. 7. Rizopoulos D. Dynamic predictions and prospective accuracy in joint models for longitudinal and time-to-event data. Biometrics. 2011;67:819–29. 8. Proust-Lima C, Taylor JM. Development and validation of a dynamic prognostic tool for prostate cancer recurrence using repeated measures of posttreatment PSA: a joint modeling approach. Biostatistics. 2009;10:535–49. 9. Rizopoulos D. JM: A package for the joint modelling of the longitudinal and time-to-event data. J Stat Software. 2010;35:1–33. g 7. Rizopoulos D. Dynamic predictions and prospective accuracy in joint models for longitudinal and time-to-event data. Biometrics. 2011;67:819–29. 8. Proust-Lima C, Taylor JM. Development and validation of a dynamic prognostic tool for prostate cancer recurrence using repeated measures of posttreatment PSA: a joint modeling approach. Biostatistics. 2009;10:535–49. 8. Proust-Lima C, Taylor JM. Development and validation of a dynamic prognostic tool for prostate cancer recurrence using repeated measures of posttreatment PSA: a joint modeling approach. Biostatistics. 2009;10:535–49. yi tð Þ ¼ f i tð Þ þ ∈i ¼ xT i tð Þβ þ zT i tð Þbi þ ∈i; p j g pp ; 9. Rizopoulos D. Discussion Hence, the validation showed that for the target group of patients the BNP as a marker for intervention does not exhibit great discrimination power. BNP levels have been previously found to be pre- dictors of reoperation. Therefore, although BNP profile is not a good predictor of intervention in our case, it is reliable in predicting mortality and thus can be very helpful in planning an intervention to prevent mortality due to AS disease progression. This non-significant re- sult could be explained by the fact that additional car- diovascular risk factors were not taken into account because either there were not available or the patients were not enough to include more factors in the model. Although all analysis was performed using standard statistical packages, a level of expertise in programming may be required. Thus, interactive web applications with friendly controls that easily incorporate plots and sum- maries are essential for adequate implementation of the proposed models in clinical practice may be interesting In this paper we assumed linear trajectories for the BNP biomarker since we did not have a big range of values per patient. However, in a different setting where more information would be available per patient it may be of interest to investigate for non-linear profiles. Andrinopoulou et al. BMC Cardiovascular Disorders (2015) 15:28 Page 8 of 8 corresponding vector of regression coefficients γ, and α denotes the strength of association between the longitu- dinal and survival outcome. Moreover, a Weibull base- line hazard h0(t) = ψtψ–1 was assumed. Specifically, patients could have highly non-linear evolu- tion that could not be described by a simple structure, such as linear one. Even though the interpretation then becomes more complex, it is evident that misspecifica- tion of the evolution of the biomarkers could lead to bias. In order to obtain valid results, it is important to postulate a mixed-effects model that is capable of appro- priately capturing such non-linear evolutions. Introduction to joint models * JM: A package for the joint modelling of the longitudinal and time-to-event data. J Stat Software. 2010;35:1–33. 10. van Geldorp MWA, Heuvelman HJ, Kappetein AP, Busschbach JJV, Takkenberg JJM, Bogers AJJC. The effect of aortic valve replacement on quality of life in symptomatic patients with severe aortic stenosis. Neth Heart J. 2013;21:28–35. where xi(t) denotes the design vector for the fixed effects regression coefficients β and zi(t) the design vector for the random effects bi. 11. van Geldorp MWA, Heuvelman HJ, Kappetein AP, Busschbach JJ, Cohen DJ, Takkenberg JJ, et al. Quality of life among patients with severe aortic stenosis. Neth Heart J. 2013;21:21–7. Finally, we assume that a normal distribution for the random effects describes the evolution of the longitu- dinal outcomes, i.e., 12. Joint Task Force on the Management of Valvular Heart Disease of the European Society of Cardiology (ESC); European Association for Cardio- Thoracic Surgery (EACTS), Vahanian A, Alfieri O, Andreotti F, Antunes MJ, Barón-Esquivias G, et al. Guidelines on the management of valvular heart disease (version 2012). Eur Heart J. 2012;33:2451–96. bi∼N 0; σb ð Þ; Where σb is the variance of the random intercept. For the survival process we have Where σb is the variance of the random intercept. F h i l h For the survival process we have hi t; θs ð Þ ¼ h0 tð Þe γTωiþaf i tð Þ f g; where θs is the parameter vector for the survival out- comes, ωi is a vector of baseline covariates with a where θs is the parameter vector for the survival out- comes, ωi is a vector of baseline covariates with a
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Zur Bewertung verbaler und physischer Gewalt im Amateurfußball. Eine computergestützte qualitative Inhaltsanalyse am Beispiel von Sportgerichtsurteilen des Bayerischen Fußballverbandes
Zeitschrift für Fußball und Gesellschaft
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cc-by
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Zur Bewertung verbaler und physischer Gewalt im Amateurfußball. Eine computergestützte qualitative Inhaltsanalyse am Beispiel von Sportgerichtsurteilen des Bayerischen Fußballverbandes Florian Koch, Clemens Bernd To cite this version: Florian Koch, Clemens Bernd. Zur Bewertung verbaler und physischer Gewalt im Amateurfußball. Eine computergestützte qualitative Inhaltsanalyse am Beispiel von Sportgerichtsurteilen des Bayerischen Fußballverbandes. FuG – Zeitschrift für Fußball und Gesellschaft, 2023, 4 (1), pp.27-42. �10.3224/fug.v4i1.03�. �hal-04108585� HAL Id: hal-04108585 https://u-bourgogne.hal.science/hal-04108585 Submitted on 27 May 2023 HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. 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. Distributed under a Creative Commons Attribution 4.0 International License Zur Bewertung verbaler und physischer Gewalt im Amateurfußball. Eine computergestützte qualitative Inhaltsanalyse am Beispiel von Sportgerichtsurteilen des Bayerischen Fußballverbandes. The evaluation of verbal and physical aggressions in amateur soccer. A computer-assisted qualitative content analysis using the example of a sports court ruling of the Bavarian Football Association. Florian Koch1 und Clemens Bernd Florian Koch (ORCID: 0000-0002-4897-7174) studierte Politikwissenschaften an der Universität Leipzig und den IEPs in Lille und Straßburg. Anschließend absolvierte er das binationale Promotionsprogramm Cotutelle de thèse zwischen der Universität Leipzig und der Université de Paris. Zurzeit lehrt er als Maître de conférences an der Université de Bourgogne (uB) sowie am IEP de Paris (Sciences Po) und forscht am Forschungslabor Centre Interlangues - Texte, Image, Langage (TIL) zum alltäglichen Sprachverhalten rund um den Sport/Fußball. Eine aktuelle Veröffentlichung: Koch, Florian (2019): Die Abwertung des Anderen mittels Sprache im Amateurfußball. Ein soziolinguistischer Vergleich in Deutschland und Frankreich. Berlin: Peter Lang (Sprache - Kultur - Gesellschaft). Clemens Bernd studierte Politikwissenschaften mit dem Schwerpunkt Internationale Beziehungen an der Johannes Gutenberg-Universität Mainz. Zurzeit arbeitet er als Projektmanager in der Hauptabteilung Innovationsmanagement und Digitale Transformation beim Südwestrundfunk (SWR). Er forscht über die alltägliche Diskriminierung im Sport/Fußball. 1 Korrespondierender Autor, florian.koch@u-bourgogne.fr Florian Koch und Clemens Bernd Zur Bewertung verbaler und physischer Gewalt im Amateurfußball. Eine computergestützte qualitative Inhaltsanalyse am Beispiel von Sportgerichtsurteilen des Bayerischen Fußballverbandes. Zusammenfassung Bisherige Studien über verbale und/oder physische Gewalt im Amateurfußball und deren Bewertung durch Sportrichter*innen legten Ihren Fokus auf rassistisch/fremdenfeindlich motivierte Konflikte. Nicht untersucht wurden Einflussfaktoren sowohl bei der Ausübung als auch bei der Bewertung verbaler und/oder physischer Gewalt aus – weiter gefassten – menschenfeindlichen Motiven. Ziel dieses Artikels ist es daher einen Beitrag zu leisten diese Forschungslücke zu schließen. Dafür werden mittels einer qualitativen Inhaltsanalyse 25 ausgewählte saisonübergreifende Urteile aus den Jahren 2016 bis 2020 des Sportgerichts des Bayerischen Fußball-Verband (BFV) softwaregestützt explorativ untersucht. Die Auswertung zeigt, dass Täter*innen und Opfer überwiegend männlich sind und sich Vorfälle verbaler und/oder physischer Gewalt zumeist bei Spielen der aktiven Herrenmannschaften ereigneten. Täter*innen waren dabei zumeist Trainer*innen/Betreuer*innen, gefolgt von Spieler*innen und Fans. Zudem hierarchisierten Sportrichter*innen verbale und/oder physische Gewalt. Während behindertenfeindlich motivierte verbale und/oder physische Gewalt milde bestraft wurde und bei sexistisch motivierter Gewalt nahezu gleichverteilt leichte/mittelschwere bzw. schwere/schwerste Strafen verhängt wurden, sprachen Sportgerichte des BFVs bei rassistisch/fremdenfeindlich motivierter verbaler und/oder physischer Gewalt mehrheitlich schwere/schwerste Strafen aus. Zukünftig sollte der BFV daher darauf achten, dass neben den bereits durchgeführten Präventionskampagnen und Anti-Gewalt-Kursen, die hauptsächlich auf Spieler*innen und Trainer*innen/Betreuer*innen abzielen, auch Kurse/Seminare angeboten werden, die gezielt für Amateurschiedsrichter*innen und/oder Sportrichter*innen konzipiert werden, um eine unterschiedliche Bewertung verbaler und/oder physischer Gewalt, die auf menschenfeindliche Einstellungen schließen lassen, zu vermeiden. Schlüsselwörter: verbale und/oder physische Gewalt; menschenfeindliche Einstellungen; Amateurfußball; Sportgerichtsbarkeit; 1 Abstract Previous studies on verbal and/or physical aggression in amateur football and its evaluation by sports judges have focused on conflicts motivated by racism and/or xenophobia. However, other factors that influence both the practice and evaluation of verbal and/or physical aggression from misanthropic motives have not yet been examined. The aim of this article is therefore to contribute to the closure of this gap in current research. To this end, 25 selected cross-seasonal rulings from 2016 to 2020 of the Bavarian Sports Court are explored utilising computer-assisted qualitative data analysis software (CAQDAS) by means of a qualitative content analysis. The analysis shows that perpetrators and victims are predominantly male, and that incidents of verbal and/or physical aggression mostly occur during matches involving teams of men. The perpetrators were found to be mostly coaches/staff, followed by players and spectators. In addition, the study shows that sports referees did hierarchically structure verbal and/or physical aggression. While verbal and/or physical aggression motivated by hostility towards persons with disabilities was punished leniently, and slight/medium, or severe/most severe penalties were imposed almost equally for aggression motivated by sexism, the majority of penalties imposed by the Bavarian Sports Court for verbal and/or physical aggression motivated by racism/xenophobia were severe/most severe. In future, the Bavarian Football Association (BFV) should therefore ensure that, in addition to their prevention campaigns and anti-violence courses that they already carry out, which are mainly aimed at players and coaches/staff, courses are also offered that are specifically designed for amateur referees and/or sports judges in order to avoid a hierarchical structure in the assessment of verbal and/or physical aggression that suggests group-focused enmity. Keywords: verbal and/or physical aggression; group-focused enmity; amateur football; sports court ruling; 2 Aktualität und Relevanz Der über sieben Millionen Mitglieder zählende Deutsche Fußball-Bund (DFB) zeichnete in seinem 6. Lagebericht des Amateurfußballs ein ambivalentes Bild (DFB 2020). 2 Danach kam es in der Saison 2019/2020 in 0,45% aller 60.000 erfassten Fußballspiele zu Gewalt- oder Diskriminierungsvorfällen. Das war – allerdings vor dem Hintergrund der Corona-Pandemie, die zahlreiche Absagen von saisonentscheidenden Spielen verursachte – ein Rückgang um ca. 0,05% an gemeldeten Gewalt- oder Diskriminierungsvorfällen im Vergleich zu den beiden vorherigen Saisons. Ähnlich verhält es sich mit Spielabbrüchen. Mussten in der Saison 2018/2019 noch 0,05% der Spiele abgebrochen werden, waren es in der Folgesaison lediglich 0,03%. Dieser Rückgang kann durchaus auch als (kurzfristiger) Erfolg der Strategie gewertet werden, die darauf abzielt, Druck auf verbandliche und politische Entscheidungsträger*innen durch verstärkte mediale Berichterstattung auszuüben. So wurden Brandbriefe von Amateurschiedsrichter*innen veröffentlicht, Schiedsrichterstreiks organisiert und somit Spielabsagen verursacht (Schulz/Teevs/Heier 2016). Zudem berieten sich die Innenminister*innen der Länder 2019, um verbale aber auch physische Gewalt v.a. gegen Amateurschiedsrichter*innen wirksam einzudämmen (BMI 2019). Der DFB veröffentlichte daraufhin einen offenen Brief, in dem allen aktiven Schiedsrichter*innen – auch aufgrund eines Verlustes um fast 22.000 in den letzten zehn Jahren (DFB 2018) – umfassende Unterstützung zugesichert wird (Curtius/Keller/Koch/Zimmermann 2019). Schließlich verweist der DFB (2021) auf sein Gewaltpräventionskonzept „Fair ist mehr“. Es bündelt bereits bestehende Angebote und Maßnahmen zur Gewaltprävention und Intervention bei Gewaltvorfällen. Ein wesentlicher Baustein befasst sich mit der effizienten Bearbeitung von Vorfällen und alternativen Sanktionsmaßnahmen der Sportgerichtsbarkeit, auch „im Sinne des Statuierens eines Exempels“ mit dem Ziel die Täter*innen zu einer Einstellungs- und Verhaltensänderung zu bewegen. Dennoch beklagt der 6. Lagebericht weiterhin das Auftreten gravierender Einzelfälle (DFB 2020). Ein besonders eindrückliches Beispiel, das darüber hinaus aufzeigt, dass aktuelle politische Konflikte mitunter auch auf dem Amateurfußballplatz ausgetragen werden, ereignete sich Mitte Oktober 2019 – drei Tage nach dem Einmarsch türkischer Truppen in Syrien – beim Kreisliga C Spiel in Essen (Poppe 2019). Die mehrheitlich aus syrischen Flüchtlingen bestehende zweite Mannschaft des BV Altenessen spielte gegen den türkischen Migrantenverein Barisspor. Aufgeheizt durch die politischen Ereignisse hetzten am Spielende Spieler des BV Altenessen den 2 Die Autoren bedanken sich herzlich bei den beiden anonymen Gutachter*innen für ihre zahlreichen kritischen Kommentare und konstruktiven Hinweise. 3 Schiedsrichter über den Platz und verletzten ihn. Der alteingesessene Verein mit mehr als 300 Mitgliedern aus 30 Nationen regierte prompt und meldete die Mannschaft umgehend ab, auch weil das zuständige Sportgericht ungewöhnlich hart durchgriff und fast alle Spieler mit einer Sperre von mindestens einem Jahr belegt hatte. Konkretes Ziel dieses Artikels ist es daher alltägliche verbale und/oder physische Gewalt im Amateurfußball anhand von 25 ausgewählten saisonübergreifenden Sportgerichtsurteilen des Bayrischen Fußball-Verbands (BFV) softwaregestützt explorativ zu untersuchen. Dabei konzentriert sich die vorliegende Analyse sowohl auf Einflussfaktoren bei der Ausübung konkreter alltäglicher verbaler und/oder physischer Gewalt im Amateurfußball als auch auf deren Bewertung durch Sportrichter*innen des BFVs. Ein besonderes Augenmerk wird dabei auf menschfeindliche Einstellungen gelegt. Dafür wird zunächst der theoretische Rahmen, der Forschungsstand sowie das methodische Vorgehen vorgestellt, um daran anschließend Einflussfaktoren bei der Ausübung verbaler und/oder physischer Gewalt und deren Bewertung durch Sportrichter*innen des BFVs herauszuarbeiten. Zuletzt werden die Ergebnisse diskutiert und konkrete Handlungsempfehlungen gegeben. Theoretischer Rahmen und thematische Hinführung Laut Delhom (2007: 229) lässt sich physische Gewalt nicht trennscharf von verbaler Gewalt unterscheiden. Vielmehr ist Gewalt nie „stumm“ und „wird meistens durch Sprache begleitet: Sie wird geplant und beschlossen, erzählt und kommentiert, gerechtfertigt oder legitimiert. Und darüber hinaus wird sie durch die Sprache und in der Sprache vollzogen.“ 3 Ähnlich argumentiert Krämer (2007: 31ff.). Sie definiert Gewalt ebenso als ein Phänomen, das sowohl auf den physischen Gewaltakt abzielt, aber auch den sprachlichen Gewaltakt einschließt mit dem Ziel die betroffene Person physisch oder symbolisch schmerzhaft zu verletzen.4 Verbale Gewalt ist ein Phänomen, das in allen Gesellschaften in vielfältiger Form auftritt (Krämer 2007: 32ff.). Klinker, Scharloth und Szczek (2018: 1ff.) verstehen darunter alltägliche sprachliche Normüberschreitungen wie Beleidigungen, Herabwürdigungen und zeichenhafte Aggressionen. Anders als physische Gewalt kann jedoch sprachlich-symbolische Gewalt dem Betroffenen nicht aufgezwungen werden und benötigt sowohl die Mitwirkung der Betroffenen als auch die 3 Lobenstein-Reichmann (2012: 215ff.) beschreibt detailliert die historische Entwicklung der einstigen Antipoden Gewalt und Sprache. Wesentlich ist dabei, dass in Anlehnung an Norbert Elias verbale Gewalt weitgehend physische Gewalt ersetzt und dieser stets vorausgeht. 4 Herrmann, Krämer und Kuch (2007: 7ff.) geben einen einführenden Überblick über das Konzept „Verletzende Worte“. 4 legitimierende Kraft Dritter (Lobenstein-Reichmann 2012: 219). Der Amateurfußballplatz ist nun ein spezifischer sozialer Raum, auf dem Normabweichungen eine weitaus höhere Toleranz erfahren und mögliche Sanktionen weniger konsequent durchgesetzt werden. Aggressives Verhalten wird vielmehr gefordert und gefördert, um in eingeübten unhinterfragten Routinen zu gerinnen (Dunning 1999: 30). Mennell (2006: 515) unterstreicht, dass übermäßige Gewaltausübung vor allem in Mannschaftssportarten vorkommt, die körperliche Robustheit mit einem komplexen Spiel kombinieren. Zahlreiche Autor*innen weisen zudem darauf hin, dass diese eingeübten Routinen zunehmend in den unteren Ligen in verbale und/oder physische Gewalt umschlagen (v.a. Ribler 2010: 13; Winands 2016: 7). Das sozialpsychologische Konzept der Gruppenbezogenen Menschenfeindlichkeit (GMF) untersucht nun den Zusammenhang zwischen der Ausübung alltäglicher Diskriminierung und menschenfeindlichen Einstellungen (v.a. Zick 2010: 19ff.). Kern des Konzepts ist die auf Vorurteilen beruhende Ideologie der Ungleichwertigkeit. Diese drückt sich durch menschenfeindliche Einstellungen gegenüber als fremd wahrgenommenen Gruppen aus. Die Zuschreibung zu den Gruppen erfolgt dabei aufgrund von invariablen Eigenschaften – wie der Hautfarbe, der (vermeintlichen) ethnischen Herkunft und/oder religiösen Zugehörigkeit, dem Geschlecht, der sexuellen Orientierung sowie der körperlichen Verfasstheit – entlang derer sich einzelne Elemente der GMF – insbesondere Rassismus und Fremdenfeindlichkeit, Islamophobie und Antisemitismus, Sexismus, Homophobie sowie Behindertenfeindlichkeit – konstituieren (Zick/Hövermann/Küpper 2011: 27ff.). Da die Abwertung einer als fremd wahrgenommenen Gruppe mit der Abwertung weiterer Gruppen einhergeht, bezeichnet Heitmeyer (2012: 15), Begründer und Vordenker des Ansatzes, die GMF als ein Syndrom. Zick, Scherer und Winands (2010: 133ff.) argumentieren, dass diese menschenfeindlichen Einstellungen nun durch eine Vielzahl von Akteur*innen – wie Spieler*innen, Fans, Funktionär*innen – in die Fußballarena hineingetragen werden. Dieses sei vor allem bei unparteiisch urteilenden verbandlichen Akteur*innen wie Schiedsrichter*innen und Sportrichter*innen höchst problematisch und hoch relevant, da dadurch menschenfeindliche Einstellungsmuster legitimiert und zementiert werden können.5 Koch (2019: 93) verbindet das reale alltägliche Sprachverhalten mit dem auf Vorurteilen basierenden Konzept der GMF. Er schlägt vor nunmehr von verbaler Abwertung zu sprechen, wenn die tatsächlich ausgeübte verbale Gewalt auf gruppenbezogene menschenfeindliche 5 Aufgrund der institutionellen Benachteiligung vor Gericht von Sportrichter*innen mit Migrationshintergrund, bezeichnen Zick, Scherer und Winands (2010: 133ff.) das Sportgericht gar pointiert als „ethnische Kampfarena“. 5 Einstellungen schließen lässt. Sportrichter*innen im Amateurfußball urteilen basierend auf der verbandlichen Rechts- und Verfahrensordnung (RVO) (BFV 2022a). Um als Sportrichter*in an ein unabhängiges bayrisches Sportgericht berufen zu werden, reicht es demnach aus älter als 30 Jahre alt zu sein, über ausreichend Erfahrung im Sport zu verfügen und sich im sportlichen Alltag bewährt zu haben. Die Befähigung zum Richteramt benötigen zwingend lediglich Vorsitzende des Sportgerichts sowie des Verbands-Sportgerichts (§ 9 RVO). Sanktioniert werden alle Formen unsportlichen Verhaltens (§ 47a RVO) in der Regel in nicht öffentlichen Sitzungen (§ 11 RVO). Dazu zählt insbesondere politisch extremistisches, obszön anstößiges oder provokativ beleidigendes Verhalten. § 47a RVO nimmt den Kern der Ideologie der Ungleichwertigkeit auf und definiert besonders schwere Fälle von Unsportlichkeit als solche, die durch herabwürdigende Äußerungen die Menschenwürde einer Person oder einer Gruppe von Personen verletzen. Das ist insbesondere dann der Fall, wenn Menschen aufgrund ihrer Herkunft, Hautfarbe, Sprache, Religion, Behinderung, Alter, geschlechtlichen oder sexuellen Identität diskriminiert werden. Aufgrund des verbandspolitischen Interesses leitet bei schweren Fällen unsportlichen Verhaltens in aller Regel der Verbandsanwalt das Verfahren (§ 35 RVO). Zudem erhöht sich die vorgesehene Mindeststrafe bei Fällen von festgestellten schweren Unsportlichkeiten auf eine Sperrstrafe von nicht unter fünf Wochen oder fünf Spielen bis hin zum Ausschluss oder einer Mindestgeldstrafe von 300 € (§ 47a RVO). Forschungsstand Obwohl verbale und/oder physische Gewalt im Amateurfußball vor allem gegen Amateurschiedsrichter*innen (Vester 2013; Rullang/Emrich/Pierdzioch 2015a, 2015b), auch als transnationales Phänomen (Koch 2019, 2021), wissenschaftlich untersucht und ebenso Einflussfaktoren bei der Ausübung von menschenfeindlicher Gewalt und deren Bewertung durch Amateurschiedsrichter*innen herausgearbeitet wurden (Koch 2020), ist der Sportgerichtsbarkeit in den letzten Jahren nur wenig Aufmerksamkeit geschenkt worden. Eine der wenigen Ausnahmen ist die Untersuchung von Pilz (2002). Anhand von ca. 4000 Sportgerichtsurteilen des Niedersächsischen Fußballverbandes (NFV) aus der Saison 1998/1999 analysiert dieser Konflikte im Jugendfußball, die auf rassistische/fremdenfeindliche Einstellungen schließen lassen. Er stellt fest, dass bei vergleichbaren Taten Spieler*innen nicht deutscher Herkunft härter bestraft wurden. So erhielten Spieler*innen nicht deutscher Herkunft bei „Tätlichkeiten ohne Verletzungen“ in rund zwei Drittel der Vorfälle eine Sperre von bis zu vier Wochen, 25,7% bis zu sechs Wochen und 9,2% über sechs Wochen, wohingegen deutsche 6 Spieler*innen in 78,9% der Vorfälle mit einer Sperre bis zu vier Wochen, 14,2% bis zu sechs und 5,8% über sechs Wochen belegt wurden. Noch deutlicher ist die unterschiedliche Strafmaßbemessung bei „Tätlichkeiten mit Verletzungen“. Während 54% der deutschen Spieler*innen für bis zu vier Wochen gesperrt wurden, waren es bei den nicht deutschen Spieler*innen nur 25%. Demgegenüber wurden 50% der nicht deutschen Spieler*innen und lediglich 20% der deutschen Spieler*innen mit einer Sperre von über sechs Wochen belegt. Bei den Ursachen dieser messbaren Diskriminierung von deutschen und nicht-deutschen Spieler*innen unterscheidet Pilz (2002: 11ff.) zwischen oberflächigen und tieferliegenden Ursachen. Zu ersteren zählt Pilz u.a. Erklärungen von Vertretern „deutscher Vereine und Verbände“, die auf das südländische Temperament und der damit einhergehenden Disziplinlosigkeit der Migrant*innen vor allem bei Entscheidungen von Schiedsrichter*innen abzielen, aber auch Klagen von Mitgliedern „ethnischer Vereine“, die sich (gezielt eingesetzter) verbaler Gewalt durch ihre deutschen Gegenspieler*innen und institutionelle Benachteiligung aufgrund vermeintlich (latent) rassistischer/fremdenfeindlicher Einstellungen durch Schiedsrichter*innen sowie Sportrichter*innen ausgesetzt sehen. Der Autor verweist ferner darauf, dass der sportliche Wettstreit auf dem Fußballplatz zwischen Jugendlichen deutscher und nichtdeutscher Herkunft nunmehr eine Stellvertreterfunktion einnimmt. Im Kern geht es um soziale Anerkennung und das Recht auf soziale Mobilität von Minderheiten in einer sich herausgeforderten Mehrheitsgesellschaft, die sich gegen eine Neuverteilung der Ressourcen und Privilegien wehrt. Die hohe Sensibilität der Migrant*innen auf dem Fußballplatz, die diese auch für Provokationen Teilhabemöglichkeiten anfällig macht, ist sowie herrschender Ausdruck ungleicher Diskriminierung gesellschaftlicher aufgrund von (latent) rassistischen/fremdenfeindlichen Einstellungen in der Mehrheitsgesellschaft. Scherer und Winands (2010: 47ff.) untersuchen ebenso Konflikte im Amateurfußball, die auf gruppenbezogene menschenfeindliche Einstellungen schließen lassen. Anhand von 1205 Sportgerichtsurteilen des Hessischen Fußball-Verbandes (HFV) aus der Saison 2007/2008 stellen sie fest, dass Täter*innen zumeist Spieler*innen aus dem Seniorenbereich und Opfer zumeist Schiedsrichter*innen waren. Zudem bestätigen sie, dass Spieler*innen anderer Herkunft vor dem Sportgericht überrepräsentiert waren und sowohl überproportional häufiger als auch öfter für härtere Vergehen verurteilt wurden. Darüber hinaus erhielten sie für vergleichbare Vergehen härtere Strafen. Obwohl die Autor*innen einräumen, dass ihre Ergebnisse keinen eindeutigen Zusammenhang zwischen den analysierten Sportgerichtsurteilen und GMF belegen, nehmen sie an, dass auch Sportrichter*innen mit einem selektiven Blick urteilen. 7 Laerum (2010: 55ff.) arbeitete mittels einer qualitativen Befragung von Sportrichtern heraus, dass diese, auch wenn kein direkter Zusammenhang zwischen Einstellung und Strafbeimessung nachgewiesen wurde, durchaus latent rassistische/fremdenfeindliche und islamophobe Stereotype und Vorurteile in die Sportgerichte hintragen. Das äußert sich auch dadurch, dass männliche Jugendliche mit islamischer Prägung, sowie die Rolle von Migrantenvereinen als besonders problematisch beschrieben wurden. Die Autorin sieht eine wesentliche Ursache dafür in der homogenen Zusammensetzung der Sportgerichte. So waren die Befragten ausschließlich männlich, deutscher Herkunft und über 60 Jahre alt.6 Die drei vorgestellten Studien legten ihren Fokus insbesondere auf Konflikte im Amateurfußball, die im Kern auf rassistische/fremdenfeindliche und islamophobe Einstellungen schließen lassen. Nicht untersucht wurden weitere Einflussfaktoren sowohl bei der Ausübung als auch bei der Bewertung verbaler und/oder physischer Gewalt von Sportrichter*innen im Amateurfußball sowie aus – weiter zu fassenden – menschenfeindlichen Motiven. Die vorliegende Untersuchung hat somit zum Ziel einen Beitrag zu leisten diese Forschungslücke zu schließen. Methodisches Vorgehen Aufgrund des explorativen Charakters und vorliegenden Datenmaterials nutzen wir für unsere Untersuchung die qualitative Inhaltsanalyse. Im Kern zielt diese spezifische Auswertungstechnik darauf ab, Textdokumente – wie die vorliegenden Sportgerichtsurteile – systematisch zu analysieren und zu interpretieren, indem aus dem vorhandenen Datenmaterial Zusammenhänge in Form eines Kategoriensystems induktiv herausgearbeitet werden (Mayring/Fenzl 2014: 543ff.; Mayring 2016: 114ff.).7 Die konkrete Zuordnung (Codieren) einzelner Textsegmente (Codings) – zumeist handelt es sich dabei um (unbestimmte) Rechtsbegriffe aus dem Sport, dem Strafmaß und wenigen kontextualisierenden Informationen (Ort, Datum, Liga, Spielphase, Opfer usw.) – zu den Kategorien (Codes) erfolgte ganz überwiegend durch Clemens Bernd. Um die Zuverlässigkeit des Codierens zu gewährleisten, wurden die sich entwickelnden Haupt- und Subkategorien (Subcodes) gemeinsam definiert, zur besseren Orientierung mit Ankerbeispielen versehen und fortlaufend 6 Zick, Hövermann und Küpper (2011: 14) belegen, dass gruppenbezogene menschenfeindliche Einstellungen mit zunehmendem Alter stärker ausgeprägt sind und mit dem Bildungsniveau und dem Einkommen korrelieren. Allerdings weisen die Autor*innen auch daraufhin, dass das Geschlecht keinen bedeutenden Effekt auf die GMF hat. 7 Mayring und Fenzl (2014: 544) unterscheiden zwischen induktiver und deduktiver Textanalyse. Während für erstere Variante ein Kategoriensystem genutzt wird, welches anhand des Datenmaterials entwickelt wird, nutzt die zweite Variante ein vorab entwickeltes theoriegeleitetes Kategoriensystem. 8 überprüft. Zudem erfolgte schließlich von beiden Autoren eine kritische Überprüfung des codierten Datenmaterials. Um das Codieren zu vereinfachen aber auch das spätere Korrelieren von Variablen sowie deren Präsentation zu unterstützen (Kuckartz 2014: 99ff.), verwendeten wir für die erste Analyse die QDA-Software MAXQDA Plus Version 2020, für die Überarbeitung des Datensatzes die Version 2022. Mayring unterstreicht ferner (2016: 140ff.), dass durch die Nutzung von QDASoftware ebenso wichtige Gütekriterien einer qualitative Inhaltsanalyse erfüllt werden, da diese einfacher intersubjektiv nachvollzogen, reproduziert und überprüft werden können.8 Als Datengrundlage nutzen wir öffentlich zugängliche, saisonübergreifende Sportgerichtsurteile des BFVs aus den Jahren 2016 bis 2020.9 Dieser mit ca. 1,6 Millionen Mitglieder größte der insgesamt 21 Landesverbände des DFBs verbindet ländliche und urbane Regionen und bietet mit ca. 13.000 Spielen an einem Spieltag die größtmögliche Grundgesamtheit. Zudem veröffentlicht dieser Verband – aus Datenschutzgründen lediglich eine begrenzte Auswahl – Sportgerichtsurteile auf seiner Homepage (BFV 2022b).10 Der Großteil dieser ca. 300 veröffentlichten Urteile sind für unsere Untersuchung irrelevante Vorfälle wie Freisprüche aber auch Spielsperren nach regulären roten Karten, Fragen zur Spielberechtigung einzelner Spieler*innen oder Anfragen zur Änderung von Spielterminierungen. Vor diesem Hintergrund haben wir folgende Kriterien entwickelt, die sicherstellen, dass die Urteile für unsere Untersuchung verwertbare Daten enthalten: (1) Die geahndeten Vergehen müssen losgelöst vom unmittelbaren Spielgeschehen sein. Dazu zählen u.a. Beleidigungen jeglicher Art sowie tätliche Angriffe auf Schiedsrichter*innen. Nicht dazu zählen somit grobe Fouls, selbst wenn diese den Spielausschluss und somit ein Sportgerichtsurteil zur Folge hatten. (2) Außerdem müssen aus dem Urteil die beteiligten individuellen Akteur*innen und das verhängte Strafmaß eindeutig hervorgehen. Nicht berücksichtigt werden daher u.a. Urteile gegen Vereine aufgrund von mangelnden Sicherheitsvorkehrungen. Vor diesem Hintergrund berücksichtigen wir für unsere nachfolgende Analyse 25 der ca. 300 gesichteten Urteile. Ergebnispräsentation der qualitativen Inhaltsanalyse von Urteilen des BFVs Die Ergebnispräsentation der qualitativen Inhaltsanalyse erfolgt in drei Schritten. Zuerst werden Einflussfaktoren – (1) Akteur*innenbezogene Faktoren (75 Codings), (2) Spielbezogene Faktoren (50 Codings), (3) Art der Vergehen (25 Codings) und (4) Verhängte Strafen (25 Codings) 8 So kann auf Anfrage der vorliegende Datensatz eingesehen und für weitere Untersuchungen genutzt werden. 9 Pandemiebedingt lagen in der Saison 2019/2020 lediglich zwei Urteile vor. 10 Unbegrenzter Zugang zu den Sportgerichtsurteilen ist lediglich Vereinsmitgliedern möglich, die über ein VereinsLogin verfügen. 9 zusammen mit zehn Subkategorien – bei der Ausübung verbaler und/oder physischer Gewalt und deren Bewertung durch Sportgerichte des BFVs isoliert vorgestellt, um daran anschließend in einem zweiten Schritt bedeutsame Einflussfaktoren miteinander zu kombinieren. Eine kritische Einordnung dieser Ergebnisse wird in der abschließenden Diskussion vorgenommen. Akteur*innenbezogene Faktoren Die erste Kategorie umfasst die Subkategorie Beteiligte Akteur*innen. Diese beschreibt, von wem das Vergehen ausging (Täter*in) und wer ihr Adressat war (Opfer). Zudem umfasst diese Subkategorie soziodemographische Faktoren wie das Geschlecht und Alter der Beteiligten Akteur*innen. Tabelle 1: Täter*innen- und Opfergruppen Täter*innengruppe Relative Häufigkeit Opfergruppe Relative Häufigkeit Trainer*innen/ Betreuer*innen Spieler*innen 40% Schiedsrichter*innen 68% 28% Spieler*innen 32% Fans 28% Schiedsrichter*innen 4% Gesamt 100% 100% Tabelle 1 zeigt, dass die Täter*innengruppe vier beteiligte Akteur*innen umfasst, wohingegen zur Opfergruppe lediglich zwei beteiligte Akteur*innen zählen. Des Weiteren springen drei prägnante Auffälligkeiten hervor: Erstens sind Schiedsrichter*innen in über zwei Drittel der Vorfälle Opfer von verbaler und/oder physischer Gewalt. Zweitens sind Spieler*innen in rund ein Viertel Täter*innen (28%) und in fast ein Drittel Opfer (32%). Drittens sind Trainer*innen/Betreuer*innen am häufigsten in der Täter*innenrolle zu finden (40%), gefolgt von den Spieler*innen (28%) und den Fans (28%). Analysiert man darüber hinaus die direkte Täter*innen-Opfer Konstellation, dann fällt auf, dass Trainer*innen/Betreuer*innen (28%), Spieler*innen (24%) und Fans (16%) in mehr als zwei Drittel der Vorfälle jeweils den Schiedsrichter*innen verbal und/oder physisch attackiert haben. Eigentlich erwartbare Vorfälle zwischen Spieler*innen sind dagegen äußerst selten (4%). Die Analyse zeigt zudem, dass – allerdings vor dem Hintergrund einer extrem ungleichen Verteilung der insgesamt ca. 300 gesichteten Sportgerichtsurteile – sowohl die Täter*innen (96%) als auch die Opfer (84%) ganz überwiegend männlich sind und sich die übergroße Mehrheit der Vorfälle nicht bei Spielen von Junior*innen ereignete (8%), sondern bei Spielen der aktiven 10 Herren- und/oder Damenmannschaften (92%). Spielbezogene Faktoren Diese Kategorie unterteilt sich in die Subkategorien Spielphase und Spielklasse. Erstere bezieht sich auf den Zeitpunkt – vor, während und/oder nach dem Spiel – der Ausübung verbaler und/oder physischer Gewalt. 65% der zuordenbaren Vorfälle fanden in der zweiten Halbzeit, mehr als 35% in der letzten Viertelstunde und rund ein Viertel (24%) nach dem Spiel, statt. Vor dem Spiel kam es dagegen nie und in der ersten Halbzeit eher selten zu verbaler und/oder physischer Gewaltanwendung (18%). Knapp die Hälfte der Vorfälle (48%) ereignete sich in der untersten Spielklasse, der Kreisliga, ein Fünftel (20%) in der ebenso unterklassigen Bezirksliga. In den höheren Ligen, wie Landesliga, Oberliga und der höchsten Spielklasse, der Regionalliga, wurde lediglich ein Vorfall vor dem Sportgericht abgeurteilt. Art der Vergehen Die Kategorie Art der Vergehen gliedert sich in die zwei Subkategorien Art der Attacke und Art der verbalen Abwertungen. Erstere beschreibt die Häufigkeitsverteilung der Vorfälle von verbaler und/oder physischer Gewalt mit dem Ziel deren Verknüpfung aufzuzeigen. In der Tat zeigt die Verteilung, dass in fast der Hälfte der Vorfälle (44%) verbale Gewalt Vorläufer für physische Gewalt war. In 56% der analysierten Urteile wurde ausschließlich verbale Gewalt angewandt. Bei keinem einzigen verhandelten Vorfall fand eine physische Attacke isoliert statt. Die Analyse zeigt zudem, dass sexistisch motivierte Gewalt am häufigsten vorkam (40%). Die meisten verbalen Abwertungen richten sich dabei gegen die Mütter der betroffenen Personen („Mutterficker“, „Hurensohn“). Fast ein Viertel der Beleidigungen (24%) haben eine behindertenfeindliche Motivation, wobei allerdings keine Worthäufung auszumachen ist. Lediglich die Beleidigung der Opfer als „blind“ kam mehrmals vor. Die aufgrund der thematischen Nähe zu einer Subkategorie zusammengefassten rassistischen/fremdenfeindlichen Beleidigungen traten selten auf (12%). Konkret wurden die Formulierungen „Ihr braucht euch nicht zu wundern, wenn ihr mit solch einem Pack verliert“ in Verbindung mit „Ich muss nicht mit Ausländern reden“,11 „Raus aus Deutschland“ und „Scheiß Neger“ verurteilt. Nicht in unserem Datensatz enthalten sind eigentlich aufgrund medialer Berichterstattung anzunehmende homophobe, 11 Im konkreten Fall wurde ein Schiedsrichter letztlich wegen provokativen Beleidigungen und einem Verhalten, das auf rassistische/fremdenfeindliche Einstellungen schließen lässt, gegenüber mehreren Spielern zu einer Geldstrafe in Höhe von 1000€ verurteilt. 11 antisemitische oder islamfeindliche Abwertungen. Verhängte Strafen Diese Kategorie bildet die Schwere der ausgesprochenen Strafe und deren Häufigkeit ab. Unterteilt ist diese in vier Subkategorien: (1) leichte Strafe, (2) mittelschwere Strafe, (3) schwere Strafe und (4) schwerste Strafe. In 28% der Vorfälle wurde eine leichte Strafe verhängt, d.h. es wurde eine Geldstrafe von bis zu 150€ oder ein Platzverweis mit maximal zwei Spielen Sperre ausgesprochen. In 24% der Vorfälle wurden die Täter*innen zu einer mittelschweren Strafe verurteilt, d.h. die verhängte Geldstrafe lag zwischen 150€ bis 500€ und Spieler*innen wurden für zwei Spieltage gesperrt. Eine schwere Strafe wurde in 36% der Vorfälle ausgesprochen. Diese lag vor, wenn eine Spielsperre von mehr als zwei Spielen oder eine Geldstrafe von bis zu 1.000€ verhängt wurde. In 12% der vorliegenden Vorfälle wurde die schwerste Strafe, d.h. der Ausschluss aus dem Verband ausgesprochen. Deskriptive bivariate Analyse Da die Präsentation einzelner Variablen und deren Häufigkeitsverteilung hinsichtlich unserer Forschungsfragen lediglich von begrenzter Aussagekraft ist, werden im Folgenden einzelne prägnante Variablen miteinander kombiniert, um deren Zusammenhänge deskriptiv herauszuarbeiten. Konkret wird zuerst gezeigt, wie sich das verhängte Strafmaß nach Art der Attacke verteilt. Tabelle 2: Das Strafmaß nach Art der Attacke Leichte & mittelschwere Strafe Verbale Gewalt 8 Schwere & schwerste Strafe 5 Gesamt 13 Verbale & physische Gewalt Absolute Häufigkeit 5 7 12 13 12 25 Relative Häufigkeit 52% 48% 100% Tabelle 2 zeigt, dass verbale Gewalt im Verhältnis milder bestraft wurde als verbale Gewalt, die in physische Gewaltanwendung mündete. Während ausschließlich verbale Gewalt in fast zwei Drittel der Vorfälle mit leichten/mittelschweren Strafen sanktioniert wurde (62%), verhängten Sportgerichte des BFVs bei Vorfällen verbaler Gewalt, die in physischer Gewalt überging, in fast 12 60% der Vorfälle schwere/schwerste Strafen. Schließlich wurde die schwerste Strafe, der Ausschluss aus dem Verband, nur bei Anwendung verbaler Gewalt in Verbindung mit physischer Gewalt ausgesprochen. Allerdings variiert das verhängte Strafmaß nach beteiligten Akteur*innen teils beträchtlich. Während Trainer*innen/Betreuer*innen, die verbale und/oder physische Gewalt gegen den Schiedsrichter*innen anwandten, ausschließlich zu leichten bis mittelschweren Strafen verurteilt wurden, verhängte das Sportgericht ausschließlich schwere bis schwerste Strafen gegen Spieler*innen, die verbale und/oder physische Gewalt gegen Schiedsrichter*innen ausübten. Trainer*innen/Betreuer*innen, die Spieler*innen verbal und/oder physisch attackierten, wurden stets mit schwersten Strafen belegt, während Zuschauer, die Spieler*innen attackierten mit mittelschweren und schweren Strafen sanktioniert wurden. Bei allen Verbandsausschlüssen lag ein tätlicher Übergriff von Spieler*innen auf Schiedsrichter*innen vor. Tabelle 3: Das Strafmaß nach Art der verbalen Abwertung Leichte & Schwere & mittelschwere Strafe schwerste Strafe Art der verbalen Abwertung Sexistisch 6 4 Gesamt 10 Behindertenfeindlich 5 1 6 Rassistisch/ fremdenfeindlich Ohne Zuordnung 1 2 3 1 5 6 Absolute Häufigkeit 13 12 25 Relative Häufigkeit 52% 48% 100% Die Tabelle 3 stellt den Zusammenhang zwischen dem Strafmaß und der Art der verbalen Abwertung dar. Fasst man wiederum die Kategorien leichte und mittelschwere Strafe sowie jene der schweren und schwersten Strafe zusammen, fällt auf, dass das Strafmaß über alle Arten der verbalen und/oder physischen Gewalt hinweg nahezu gleich verteilt ist. Das gilt insbesondere für sexistisch motivierte Gewalt. Allerdings ist ebenso festzustellen, dass während behindertenfeindlich motivierte Gewalt nahezu ausschließlich mit leichten und mittelschweren Strafen sanktioniert wurde, Sportgerichte des BFVs – allerdings bei einer geringen Fallzahl – mehrheitlich schwere und schwerste Strafen bei rassistisch/fremdenfeindlich motivierter Gewalt verhängten. Auffällig ist zudem, dass – wenngleich stark kontextspezifisch – das Strafmaß bei vergleichbaren 13 verbalen Abwertungen durchaus variiert. So wurde die behindertenfeindlich motivierte Abwertung „blind“ sowohl mit einer leichten Strafe als auch mit einer mittelschweren Strafe sanktioniert. Ähnlich verhält es sich bei der sexistisch motivierten Abwertung „Hurensohn“, die sowohl mit einer leichten, mittelschweren als auch schweren Strafe sanktioniert wurde. Erstaunlich ist zudem, dass – zumindest nach Meinung der Autoren – heftige verbale Abwertungen wie „du Mongo“ weniger hart bestraft wurden als vergleichbar harmlose Abwertung wie „du bist doch behindert“. Diskussionswürdig ist schließlich, dass die sexistisch und rassistisch/fremdenfeindlich motivierte Abwertung „Halt die Fresse du kubanische Hure“ lediglich mit einer mittelschweren Strafe verhältnismäßig milde sanktioniert wurde. Diskussion und praktische Handlungsempfehlung Ziel dieses Artikels war es Einflussfaktoren bei der Ausübung konkreter verbaler und/oder physischer Gewalt und deren Bewertung durch Sportrichter*innen des BFVs anhand von 25 ausgewählten saisonübergreifenden Urteilen aus den Jahren 2016 bis 2020 explorativ zu untersuchen. Da bisherige Studien Ihren Fokus auf rassistisch/fremdenfeindlich motivierte Konflikte im Amateurfußball und deren Bewertung durch Sportrichter*innen legten, konzentriert sich die vorliegende Untersuchung auf das Herausarbeiten von – weiter zu fassenden – menschfeindlichen Einstellungen und leistet somit einen ersten Beitrag diese Forschungslücke zu schließen. Die softwaregestützte qualitative Inhaltsanalyse zeigt die herausragende Bedeutung der Beteiligten Akteur*innen bei der Ausübung verbaler und/oder physischer Gewalt. Opfer wurden zumeist Schiedsrichter*innen, Täter*innen waren mehrheitlich Trainer*innen/Betreuer*innen, Spieler*innen und Fans. Beide Befunde bestätigen vorherige Studien, die die prekäre Situation der Schiedsrichter*innen belegen (siehe vor allem Vester 2013: 35ff.)12 und den konfliktverschärfenden Einfluss der Trainer*innen/Betreuer*innen herausarbeiten (Koch 2019: 161ff.). Bestätigt werden kann ebenso, dass verbale und/physische Gewalt zuvörderst ein Phänomen ist, das bei Männermannschaften auftritt und nicht bei Spielen von Junior*innen (Scherer und Winands 2010: 47ff.). Bedeutsam bei der Ausübung verbaler und/oder physischer Gewalt ist zudem die Spielphase. Dass fast zwei Drittel (65%) der Vorfälle in der zweiten Halbzeit und mehr als 35% der Vorfälle in der 12 Vester (2013: 35ff.) untersucht detailliert in einer Vollerhebung die Opferwerdung von Amateurschiedsrichter*innen im Württembergischen Fußball-Verband (wfv). Sie stellt fest, dass Schiedsrichter*innen geradezu „Zielscheibe“ verbaler und/oder physischer Gewalt sind. 14 letzten Viertelstunde stattfanden, deutet darauf hin, dass die Zunahme verbaler und/oder physischer Gewalt mit zunehmender körperlicher Erschöpfung und/oder sich aufstauender negativer Emotionen, wie zunehmende Resignation und Frustration über die sich abzeichnende Niederlage, einhergeht. Bestätigt werden kann zudem, dass sich verbale und/oder physische Gewalt tatsächlich in die unteren Ligen verlagert, sicherlich auch deshalb, weil diese weitaus weniger im Fokus der Öffentlichkeit stehen und medial begleitet werden als die höheren (Profi-)Ligen (Ribler 2010: 13; Winands 2016: 7). Das häufige Auftreten sexistisch motivierter Abwertungen (40%) bestätigt wiederum, dass im Amateurfußball, in dem nach wie vor mehrheitlich männliche Akteure agieren, sexistische Einstellungen eine höhere Akzeptanz erfahren als in anderen Bereichen der Gesellschaft (Degele/Janz 2011: 10ff.). Bestätigt werden kann zudem, dass verbale Gewalt weitgehend physische Gewalt ersetzt und dieser stets vorausgeht. Daraus kann geschlussfolgert werden, dass verbale Gewalt keineswegs nur harmlose Provokation ist, sondern als Vorbote das Potential einer Eskalation hin zur Ausübung physischer Gewalt in sich trägt (Lobenstein-Reichmann 2012: 215ff.). Ratsam ist daher ein frühzeitiges Eingreifen auch bei vermeintlich harmloser verbaler Gewalt, um ein sich gegenseitiges Hochschaukeln zu vermeiden. Die vorgenommene Einteilung nach der Schwere der Strafe zeigt auf, dass Sportrichter*innen – wie Amateurschiedsrichter*innen – einzelne Vergehen stark kontextspezifisch bewerten (Koch 2021: 205ff.). Erwartungsgemäß wurde verbale Gewalt milder bestraft als verbale Gewalt, die in physische Gewaltanwendung mündete. Die schwerste Strafe, der Ausschluss aus dem Verband, wurde nur bei Anwendung verbaler Gewalt in Verbindung mit physischer Gewalt ausgesprochen. Allerdings variiert das verhängte Strafmaß nach beteiligten Akteur*innen beträchtlich. Während verbale und/oder physische Gewalt von Trainer*innen/Betreuer*innen gegenüber den Schiedsrichter*innen ausschließlich mit leichten bis mittelschweren Strafen sanktioniert wurde, belegte das Sportgericht Spieler*innen, die verbale und/oder physische Gewalt gegen den Schiedsrichter*innen ausübten, mit schweren bis schwersten Strafen. Bei der schwersten Strafe, dem Verbandsausschluss, lag stets ein tätlicher Angriff von Spieler*innen auf Schiedsrichter*innen vor. Beide Befunde lassen darauf schließen, dass der BFV darauf bedacht ist, die Sicherheit seiner Spielleiter*innen zu gewährleisten, auch durch die Verhängung schwerster Strafen, insbesondere bei physischen Übergriffen von Spieler*innen gegen Schiedsrichter*innen. Das ist konsequent, da der 6. Lagebericht des DFBs zeigt, dass Amateurschiedsrichter*innen, wenn auch äußerst selten, durchaus das Ziel von körperlichen Angriffen mit teilweise schweren gesundheitlichen Folgen sind. Andererseits belegen die vergleichsweise milden Strafen für Trainer*innen/Betreuer*innen und Fans, dass das konfliktverschärfende Eingreifen ins 15 Spielgeschehen durch Dritte als weniger gravierend bewertet wird, obwohl Amateurschiedsrichter*innen selbst über die bedeutende Rolle der Trainer*innen als Schlichter einerseits oder andererseits als Konfliktauslöser und/oder Verstärker berichten (Koch 2019: 181). Schließlich zeigt die Analyse, dass das Strafmaß und die Art der verbalen Gewalt über alle Arten verbaler und/oder physischer Gewalt hinweg nahezu gleich verteilt sind. Das trifft insbesondere auf sexistisch motivierte Gewalt zu. Demgegenüber steht der Befund, dass behindertenfeindlich motivierte Gewalt fast ausschließlich mit leichten und mittelschweren Strafen sanktioniert wurde, wohingegen Sportgerichte des BFVs – allerdings vor dem Hintergrund einer geringen Fallzahl – mehrheitlich schwere und schwerste Strafen bei rassistisch/fremdenfeindlich motivierter Gewalt verhängte. Daraus kann geschlussfolgert werden, dass nicht nur Amateurschiedsrichter*innen (Koch 2019: 172ff.), sondern eben auch Sportrichter*innen durchaus eine unterschiedliche Bewertung von verbalen Abwertungen abhängig von der ihnen zugrunde liegenden Motivation vornehmen. Darüber hinaus kann festgehalten werden, dass – wenn auch stark kontextspezifisch – das Strafmaß bei vergleichbaren verbalen Abwertungen variiert. So wurde die behindertenfeindlich motivierte Abwertung „blind“ und die sexistisch motivierte Abwertung „Hurensohn“ unterschiedlich bestraft. Schließlich konnte aufgezeigt werden, dass einige heftige verbale Abwertungen teils milder bestraft wurden als vergleichbar harmlose verbale Abwertungen. Ratsam ist daher – in Anlehnung an Hilpert (2009: 128) – die Erarbeitung einer Orientierung gebenden Kasuistik, nun auch von verbalen Abwertungen, also von verbaler Gewalt, die menschenfeindliche Einstellungen begründen. Darüber hinaus sollte der BFV zukünftig darauf bedacht sein, dass die verschiedenen Präventionsund Konfliktmanagementstrategien, wie das Einsetzen von Konfliktmanager*innen, aber auch das Anbieten von Anti-Gewalt-Kursen und das Durchführen von Anti-Rassismus-Kampagnen (BVF 2022), um Kurse erweitert werden, die gezielt für Amateurschiedsrichter*innen und/oder Sportrichter*innen konzipiert werden, um eine unterschiedliche Bewertung von verbaler und/oder physischer Gewalt, die auf menschenfeindliche Einstellungen schließen lassen, insbesondere gegenüber Menschen mit Behinderung, zu vermeiden. Die vorliegende Untersuchung ist lediglich ein erster thematisch begrenzter Beitrag Einflussfaktoren sowohl bei der Ausübung konkreter verbaler und/oder physischer Gewalt aus menschenfeindlichen Motiven als auch deren Bewertung durch neutrale Dritte herauszuarbeiten. Weiterer Forschungsbedarf besteht insbesondere aufgrund des geringen verwertbaren Datenmaterials. Ebenso kann in Analogie zu vergleichbaren Studien ein hohes Dunkelfeld angenommen werden, da oftmals verbale Abwertungen als Banalität, dem (Fußball-)Sport 16 innewohnend und/oder dem alltäglichen Stress geschuldet abgetan und somit erst gar nicht dokumentiert werden kontextualisierende (Koch im Informationen, Erscheinen: die zur 16). Zudem fehlen Herausarbeitung oftmals von wichtige vermeintlich menschenfeindlichen Einstellungen allerdings wesentlich sind. Wünschenswert ist es daher, dass grundlegende Annahmen und Ergebnisse der vorliegenden Untersuchung aufgenommen, mit Erkenntnissen weiterer Studien angereichert, auf vergleichbare Forschungsfelder übertragen und um quantitative Folgestudien erweitert werden. 17 Literatur BfV. 2022. “Das ist der Bayerische Fußball-Verband e.V.” (https://www.bfv.de/der-bfv/wer-wirsind/wer-wir-sind-und-standpunkte/bfv---wer-wir-sind-und-wofur-wir-stehen). BMI. 2019. 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Frankfurt am Main: Brandes & Apsel. 20
https://openalex.org/W3174951177
https://www.revistas.usp.br/rlae/article/download/188063/173644
Spanish; Castilian
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Validation of a questionnaire on the use of Interactive Response System in Higher Education
Revista latino-americana de enfermagem
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Validación del cuestionario sobre uso de Mandos Interactivos de Respuesta en la Educación Superior Ángel Custodio Mingorance-Estrada1 https://orcid.org/0000-0003-4478-3011 Juan Granda-Vera2 https://orcid.org/0000-0001-6888-7785 Gloria Rojas-Ruiz1 https://orcid.org/0000-0001-8541-383X Inmaculada Alemany-Arrebola3 https://orcid.org/0000-0002-4127-3502 Objetivo: el presente estudio tiene como objetivo diseñar y validar un cuestionario que permita medir la percepción de los estudiantes universitarios del uso de los MIR como recurso tecnopedagógico en el aula. Método: se aplicó el cuestionario Mandos Interactivos de Respuesta para la Mejora del Proceso de Enseñanza-Aprendizaje, creado ad hoc para esta investigación, que está formado por 24 ítems, aplicado a 142 estudiantes universitarios. Resultados: tanto el análisis factorial exploratorio como el confirmatorio arrojan tres dimensiones: ambiente de clase, los procesos de enseñanza-aprendizaje y la evaluación. Los resultados obtenidos tanto en la fiabilidad (alfa de Cronbach 0.955) como en el análisis factorial confirmatorio (χ2/gl=1.944, CFI=0.97; GFI=0.78; RMR=0.077; RMSEA=0.08) arrojan índices altamente satisfactorios. Conclusión: los análisis estadísticos muestran que este instrumento es una herramienta válida, fiable y de fácil aplicación para el docente que le permitirá evaluar la experiencia del alumnado sobre el aprendizaje centrado en el estudiante. Descriptores: Educación; Educación Superior; Aprendizaje; Estudios de Validación; Encuestas y Cuestionarios; Tecnología Educacional. Cómo citar este artículo Mingorance-Estrada AC, Granda-Vera J, Rojas-Ruiz G, Alemany-Arrebola I. Validation of a questionnaire on the use of Interactive Response System in Higher Education. Rev. Latino-Am. Enfermagem. 2021;29:e3418. [Access día mes año ]; Available in:  URL . DOI: http://dx.doi.org/10.1590/1518-8345.3374.3418. 1 University of Granada, Department of Didactics and School Organization, Melilla, ES, España. 2 University of Granada, Department of Didactics of Corporal Expression, Melilla, ES, España. 3 University of Granada, Department of Developmental and Educational Psychology, Melilla, ES, España. Ángel Custodio Mingorance-Estrada1 https://orcid.org/0000-0003-4478-3011 Juan Granda-Vera2 https://orcid.org/0000-0001-6888-7785 Gloria Rojas-Ruiz1 https://orcid.org/0000-0001-8541-383X Inmaculada Alemany-Arrebola3 https://orcid.org/0000-0002-4127-3502 Rev. Latino-Am. Enfermagem 2021;29:e3418 DOI: 10.1590/1518-8345.3374.3418 www.eerp.usp.br/rlae Artículo Original Introducción Los estudios consultados muestran cómo la utilización de los MIR en las aulas universitarias permiten mejorar tres grandes ámbitos: el ambiente de clase, los procesos de enseñanza-aprendizaje y la evaluación de los alumnos(15-20), siendo cada vez más importante definir las posibilidades y limitaciones de esta herramienta para la mejora de la calidad de la Educación Superior(6). La convergencia de la Educación Superior debe establecer un espacio de intercambio de experiencias prácticas que permitan avanzar en el conocimiento e investigación desarrollados en el marco común del Espacio Europeo e Iberoamericano de Educación Superior(1-2). La puesta en marcha del aprendizaje centrado en el estudiante conlleva el interés por establecer un modelo que integre eficazmente la tecnología con los conocimientos de mediación didáctica, evolucionando hacia el Modelo Tecnopedagógico (TPACK), que combina los conocimientos disciplinar, pedagógico y tecnológico, pero siempre teniendo presente el contexto en el que se interviene(3-4), con mayor interacción entre profesores y estudiantes desde un análisis dialógico-crítico(2). Centrándonos en el factor ambiente del aula, los MIR aumentan la asistencia(21) y la participación de los estudiantes(22-25), ya que se produce una mayor participación en clase que con la metodología más tradicional(23,26-27). Dentro del factor aprendizaje, algunos estudios establecen que la interacción frecuente y positiva dinamizan las clases cuando se usan los MIR(13,28- 29) promoviendo el aprendizaje activo(30-32). Además, se fomenta la atención(33-34), la concentración(35) y la memoria(36-37) en el estudio. Existe una amplia investigación que sugiere que la mejora en el rendimiento es fruto del uso de los MIR tal como indican algunos estudios(31-32,38-41), otros trabajos no encuentran dicho efecto(42). En relación con el factor evaluación, los hallazgos de la literatura apoyan la capacidad de los MIR como herramienta para la evaluación y la retroalimentación(43-44). Se considera que tanto estudiantes como docentes consiguen beneficios a través de la retroalimentación que reciben con el uso de esta tecnopedagogía(20,27). Todo ello, conlleva a un proceso de aprendizaje clave para la interacción del saber y saber hacer(45). Algunos autores muestran la necesidad de establecer el conocimiento didáctico mediante la relación entre los distintos tipos de conocimiento (de la disciplina, pedagógico general y del alumnado) y la biografía del profesor(5). Cómo citar este artículo Mingorance-Estrada AC, Granda-Vera J, Rojas-Ruiz G, Alemany-Arrebola I. Validation of a questionnaire on the use of Interactive Response System in Higher Education. Rev. Latino-Am. Enfermagem. 2021;29:e3418. [Access día mes año ]; Available in:  URL . DOI: http://dx.doi.org/10.1590/1518-8345.3374.3418. 2 Rev. Latino-Am. Enfermagem 2021;29:e3418. Introducción Así, se pone en evidencia que en la formación inicial de los enfermeros tanto los aspectos pedagógicos, usando la monitorización mediante los Mandos Interactivos de Respuesta (en adelante MIR), como la importancia del aprendizaje con docentes expertos, puede conllevar el desarrollo de una enseñanza de calidad, entre otras cuestiones(6). En este sentido, es importante estimular a los docentes a integrar la tecnología en las aulas, tal y como se pone de manifiesto en el Informe Horizon(7), logrando un impacto importante sobre la educación en los próximos años. Para ello, el profesorado universitario debe utilizar las herramientas tecnológicas que más conocen y utilizan, así como acceder a los nuevos recursos tecnológicos para la mejora de los procesos docentes(8). El presente estudio tiene como objetivo diseñar y validar un cuestionario que permita medir la percepción de los estudiantes universitarios del uso de los MIR como recurso tecnopedagógico en el aula. www.eerp.usp.br/rlae www.eerp.usp.br/rlae Resultados En primer lugar, se analizó la confiabilidad del cuestionario MIR-MPEA con 35 elementos a través del coeficiente de consistencia interna α de Cronbach, siendo 0.965. Aunque este índice era elevado se procedió a eliminar aquellos ítems cuya correlación ítem-total fue menor a 0.20. De tal forma, el cuestionario final quedó formado por 24 ítems con un α=0.955, oscilando los índices de homogeneidad entre 0.42 a 0.85. La experiencia tuvo lugar en diferentes Grados de la Universidad de Granada (España), en materias concernientes a la formación básica del alumnado. Para ello, se pide la colaboración del profesorado de forma voluntaria para participar en dicho estudio y que utilice los MIR en sus clases. Posteriormente se midió las medias, desviaciones típicas, asimetría y las correlaciones ítem-total de cada uno de los ítems que componen el cuestionario. Tal como se observa en la Tabla 1, la asimetría es negativa en todos los ítems, lo que muestra mayor concentración de respuestas que denotan puntuaciones altas en esos ítems. El uso de los MIR en estas materias básicas se llevó a cabo a lo largo de todo el semestre del curso académico 2016-17, antes, durante y al finalizar las clases. A la finalización del semestre, se aplicó el cuestionario MIR- MPEA para conocer la percepción de la experiencia llevada Tabla 1 - Valores descriptivos de los ítems del cuestionario MIR-MPEA. Granada, España, 2017 Nº Ítems M* DT† Asimetría Correl. ítem-total 1 Con el uso de los MIR estoy más concentrado en las clases. 3.40 1.15 -0.477 0.689 2 Con el uso de los MIR mido si estoy siguiendo correctamente los contenidos dados de la asignatura durante las clases. 3.81 1.01 -0.658 0.619 5 Durante mi experiencia con los MIR lo paso bien aprendiendo. 3.52 1.18 -0.559 0.566 9 Los MIR se utilizan para conocer los conocimientos iniciales de los alumnos. 3.54 1.258 -0.595 0.488 10 El uso de los MIR se lleva a cabo por docentes experimentados para proporcionar una buena retroalimentación (feedback). 3.97 0.891 -0.616 0.610 14 La utilización de los MIR me ayuda a desarrollar mi razonamiento sobre los contenidos trabajados. 3.59 1.162 -0.666 0.639 15 El uso de los MIR hace que las clases sean amenas y dinámicas. 3.82 1.119 -0.877 0.656 18 El uso de los MIR permite aumentar mi rendimiento en el aprendizaje. 3.76 1.129 -0.653 0.762 19 El uso continuado de los MIR mejora mi asistencia a las clases. Método Del mismo modo se manifiesta que los cambios tecnológicos en los docentes universitarios se orientan a la variación dentro de la estabilidad, introduciendo las que son coherentes con sus prácticas docentes en las actividades de aprendizaje que normalmente desarrollan(9). El tipo de diseño fue transeccional descriptivo, ya que se recolectaron los datos en un tiempo único con el fin de poder describir el fenómeno y analizar su incidencia en un momento determinado. La investigación se desarrolla en la Universidad de Granada (España) en su Campus de Melilla, situado en el Norte de África, cuyo alumnado pertenece a las Facultades de Ciencias de la Salud, Ciencias Sociales y Jurídicas, y Ciencias de la Educación y del Deporte. Para ello, se ha utilizado un muestreo no probabilístico de tipo intencional. Los criterios de selección han sido: en primer lugar, docentes que utilicen tecnometodología en sus aulas, los Mandos Interactivos de Respuesta en sus clases y en segundo lugar, la voluntariedad del alumnado en participar en este estudio. Por todo ello, la muestra está formada por 142 estudiantes, 110 son mujeres (77.5%) y 32 son hombres (22.5%). En relación con el curso académico, Para afrontar estos retos tecnológicos es importante invertir el aprendizaje, proporcionando materiales a los alumnos, en diversos formatos, para que desarrollen un trabajo previo antes de llegar al aula, incorporando el uso de los MIR para constatar las mejoras del proceso de aprendizaje centrado en el estudiante(10-12). A su vez, se expone que los MIR son parte de la experiencia que se está llevando a cabo en las aulas universitarias hoy día(13). Así, la literatura actual en la Educación Superior sobre el uso de esta tecnología en los últimos años se centra en los ámbitos de Ciencia, Tecnología, Ingeniería y Matemáticas, Sociología, Humanidades, Salud (Medicina y Enfermería), Administración de Empresas y Negocios e Inglés(13-14). 3 Mingorance-Estrada AC, Granda-Vera J, Rojas-Ruiz G, Alemany-Arrebola I. 17 alumnos son de primero (12%), 95 de segundo curso (66.9%) y 30 de tercero (21.1%). a cabo. Dicho cuestionario tuvo lugar la última semana del semestre, con una duración de unos 15 minutos, aproximadamente. Se pide conformidad a los alumnos para participar voluntariamente y de forma anónima en esta experiencia, siguiendo las normas del Committee on Publication Ethics (COPE). Método Para la realización de esta investigación se ha elaborado un cuestionario ad hoc para esta investigación, “Mandos Interactivos de Respuestas para la Mejora del Proceso de Enseñanza-Aprendizaje (MIR-MPEA)”. En relación con los ítems del MIR-MPEA, fueron redactados después de una amplia revisión bibliográfica sobre los 3 factores destacados anteriormente(21,46-47), que permitió establecer la validez de contenido, ya que no se disponía de expertos en esta materia. Se partió de un banco inicial de 65 ítems agrupados por categorías(48), después del análisis de los ítems, se obtuvieron 35 ítems que fueron agrupados en las tres dimensiones: ambiente, proceso y evaluación. En cuanto al formato de respuesta del cuestionario se utilizó una escala tipo Likert, con 5 alternativas de respuesta, que abarca desde 1 Totalmente en Descuerdo a 5 Totalmente de Acuerdo. Para el tratamiento estadístico de los datos se ha utilizado el programa informático SPSS en su versión 20.0. Para conocer la fiabilidad de cada bloque se utilizó el alfa de Cronbach y para la validez del cuestionario se realizó análisis factorial exploratorio. Así como el programa LISREL 8.8 para el análisis factorial confirmatorio. (continúa en la página siguiente...) Resultados Además, las comunalidades de los ítems están por encima de h2=0.40, oscilando entre 0.421 “El uso continuado de los MIR mejoram mi asistencia a las clases” a 0.791 “El uso de los MIR le permitiría conocer y comparar sus respuestas con las respuestas de los compañeros”. El AFE muestra la existencia de 3 factores que explican el 61.61% de la varianza total, siendo esta proporción aceptable. Además, las comunalidades de los ítems están por encima de h2=0.40, oscilando entre 0.421 “El uso continuado de los MIR mejoram mi asistencia a las clases” a 0.791 “El uso de los MIR le permitiría conocer y comparar sus respuestas con las respuestas de los compañeros”. Debido a que previamente no se había realizado ninguna publicación acerca del análisis factorial del cuestionario MIR-MPEA, antes de realizar un Análisis Factorial Confirmatorio era conveniente llevar a cabo un Análisis Factorial Exploratorio (AFE) con el objetivo de conocer cómo se agrupan los ítems en factores. Para garantizar que los datos se ajustan a un modelo de análisis factorial se sometieron los datos a la prueba de Kaiser, Meyer y Olkin (KMO = 0.941) y a la prueba de esfericidad de Barlett (c2 = 2446.206; gl = 300; p<0.001), encontrando valores que permiten la utilización del análisis factorial como técnica idónea para interpretar la información contenida en esta matriz. En la Tabla 2 se muestran los factores, los ítems, los pesos factoriales y la fiabilidad de cada dimensión, así como la interpretación de dichos factores. Para determinar las dimensiones se ha seguido el criterio de las cargas factoriales, siendo este valor de 0.30(49). Tabla 2 - Factores, ítems, pesos obtenidos en el Análisis Factorial Exploratorio del MIR-MPEA. Granada, España, 2017 Alfa, Peso factorial Ítems, factores y varianza explicada 1 2 3 h2* α† FACTOR 1: Ambiente F1 51.07% 15.El uso de los MIR hace que las clases sean amenas y dinámicas. 0.759 0.625 0.926 30.El uso de los MIR permite debatir activamente las ideas erróneas para construir el conocimiento. 0.748 0.303 0.736 48.El uso de los MIR mejora mi participación en las clases desde el anonimato. 0.731 0.650 42.El uso de los MIR permite tener más confianza para hacer preguntas durante las clases. 0.685 0.403 0.673 53.Las respuestas proporcionadas a través de los MIR aumentan mi confianza en las clases al ver que contesta de manera acertada. Resultados 3.70 1.266 -0.793 0.579 20 El uso de los MIR permite conocer y comparar mis respuestas con las respuestas de los compañeros. 3.42 1.234 -0.646 0.441 21 El uso de los MIR permite corregir un error o falta de comprensión sobre los contenidos del tema durante las clases. 3.71 1.121 -1.032 0.558 22 Podrías estar más interesado/a en las clases cuando se utilizan los MIR. 3.65 1.066 -0.698 0.768 24 El uso de los MIR me gusta como control de la asistencia. 3.65 1.005 -0.605 0.699 29 El uso de los MIR mejora la motivación durante las clases. 3.83 1.111 -0.825 0.753 (continúa en la página siguiente...) www.eerp.usp.br/rlae 4 Rev. Latino-Am. Enfermagem 2021;29:e3418. Nº Ítems M* DT† Asimetría Correl. ítem-total 30 El uso de los MIR permite debatir activamente las ideas erróneas para construir el conocimiento. 3.80 1.168 -0.771 0.792 35 El uso de los MIR evalúa mi conocimiento comprensivo de los contenidos en cada uno de los temas tratados durante las clases. 3.99 0.971 -1.211 0.788 36 El uso de los MIR promueve el estudio regular de la asignatura para ir mejor. preparados a las clases. 3.64 1.094 -0.762 0.715 42 El uso de los MIR permite tener más confianza para hacer preguntas durante las clases. 3.76 1.254 -0.739 0.787 46 La utilización de los MIR se hace al finalizar las clases para repasar los contenidos dados cada día. 3.80 1.100 -0.736 0.772 47 La utilización de los MIR hace que las clases tengan un ambiente más agradables e interactivo en comparación con las clases tradicionales. 3.86 1.082 -0.804 0.769 48 El uso de los MIR mejoran mi participación en las clases desde el anonimato. 3.87 1.104 -0.773 0.712 53 Las respuestas proporcionadas a través de los MIR aumentan mi confianza en las clases al ver que contesta de manera acertada. 3.44 1.164 -0.420 0.700 59 Los MIR proporcionan una información valiosa para mejorar mi proceso de aprendizaje. 4.09 1.017 -1.007 0.687 65 El uso de los MIR mejora la comprensión de los contenidos explicados en clase. 3.87 1.160 -0.911 0.727 *M = Media; †DT = Desviación típica (continuación...) *M = Media; †DT = Desviación típica *M = Media; †DT = Desviación típica El AFE muestra la existencia de 3 factores que explican el 61.61% de la varianza total, siendo esta proporción aceptable. *M = Media; †DT = Desviación típica Resultados 0.640 0.537 5.Durante mi experiencia con los MIR lo paso bien aprendiendo. 0.602 0.347 0.484 22.Estoy más interesado/a en las clases cuando se utilizan los MIR. 0.577 0.497 0.641 47.La utilización de los MIR hace que las clases tengan un ambiente más agradables e interactivo en comparación con las clases tradicionales. 0.562 0.491 0.644 24.El uso de los MIR me gusta como control de la asistencia 0.538 0.480 0.590 1.Con el uso de los MIR estoy más concentrado en las clases. 0.483 0.560 0.564 (continúa en la página siguiente ) s, ítems, pesos obtenidos en el Análisis Factorial Exploratorio del MIR-MPEA. Granada, España, 2017 5 Mingorance-Estrada AC, Granda-Vera J, Rojas-Ruiz G, Alemany-Arrebola I. Alfa, Peso factorial Ítems, factores y varianza explicada 1 2 3 h2* α† FACTOR 2: Proceso Enseñanza-aprendizaje F2 5.47% 18.El uso de los MIR permite aumentar mi rendimiento en aprendizaje. 0.404 0.726 0.740 0.869 9.Los MIR se utiliza en la evaluación inicial de los contenidos que se van a empezar a explicar para saber los conocimientos iniciales de los alumnos. 0.725 0.550 2.El uso de los MIR mide si estoy siguiendo correctamente los contenidos dados de la asignatura durante las clases. 0.702 0.599 59. Los MIR proporcionan una información valiosa para mejorar su proceso de aprendizaje. 0.628 0.512 10.El uso de los MIR se lleva a cabo por docentes experimentados para proporcionar una buena retroalimentación (feedback). 0.324 0.576 0.483 46.La utilización de los MIR se hace al finalizar las clases para repasar los contenidos dados cada día. 0.402 0.687 19.El uso continuado de los MIR mejora mi asistencia a las clases. 0.516 0.422 FACTOR 3: Evaluación F3 5.07% 20.El uso de los MIR permite conocer y comparar sus respuestas con las respuestas de los compañeros. 0.864 0.755 0.871 36.El uso de los MIR promueve el estudio regular de la asignatura para ir mejor preparados a las clases. 0.403 0.678 0.686 21.El uso de los MIR permite corregir un error o falta de comprensión sobre los contenidos del tema durante las clases. 0.361 0.641 0.576 35.El uso de los MIR evalúa mi conocimiento comprensivo de los contenidos en cada uno de los temas tratados durante las clases. 0.604 0.523 0.715 29.El uso de los MIR mejora la motivación durante las clases. 0.391 0.491 0.680 65.El uso de los MIR mejora la comprensión de los contenidos explicados en clase. *h2 = Comunalidad; †α = Alfa de Cronbach Resultados 0.332 0.486 0.602 (continuación...) (continuación...) (RMSEA) de 0.080, ambos índices se consideran aceptables ya que se encuentran entre 0.5 y .08(49). Otros indicadores fueron el índice de bondad de ajuste (GFI) y el índice de ajuste comparativo (CFI) (con valores de 0.78 y 0.97, respectivamente), se encuentran dentro de los límites de la tolerancia. Estos resultados confirman que el modelo de los 3 factores se ajusta a los datos, por esto, consideramos que el modelo puede mantenerse como una explicación plausible a la estructura dimensional que se propone. Posteriormente, se realizó el Análisis Factorial Confirmatorio y se sometieron a prueba el modelo de 3 factores. Se empleó el método de estimación de máxima verosimilitud para analizar la matriz de correlaciones. La bondad de ajuste del modelo propuesto se evaluó mediante diversos indicadores, se utilizó la χ2/gl (484.13/249) siendo de 1.944, este índice se encuentra dentro de los estándares establecidos. El Residuo Cuadrático Medio (RMR) es de 0.077 y el Error de Aproximación Cuadrático Medio www.eerp.usp.br/rlae www.eerp.usp.br/rlae 6 Rev. Latino-Am. Enfermagem 2021;29:e3418. 6 Figura 1 - Análisis factorial confirmatorio del cuestionario “Mandos Interactivos de Respuestas para la Mejora del Proceso de Enseñanza-Aprendizaje” Figura 1 - Análisis factorial confirmatorio del cuestionario “Mandos Interactivos de Respuestas para la Mejora del Proceso de Enseñanza-Aprendizaje” Tabla 3 - Fiabilidad Compuesta y Varianza Media Extraída de los factores del cuestionario MIR-MPEA. Granada, España, 2017 Factores FC* VME† F1: Ambiente de Aprendizaje 0.955 0.544 F2: Proceso de enseñanza-aprendizaje 0.930 0.508 F3: Evaluación del aprendizaje 0.863 0.540 *FC = Fiabilidad compuesta; †VME = Varianza media extraída En cuanto a la fiabilidad del instrumento se realiza el alfa de Cronbach, tanto total con un α= 0.955 como de las dimensiones que lo componen, obteniendo valores que oscilan entre α=0,922 para el factor 1, “Ambiente”, hasta α= 0,869 en el factor 2, “Proceso de enseñanza- aprendizaje”. Estos datos demuestran que la fiabilidad del cuestionario es buena en todos los factores, siendo más bajo en el Factor 3, “Evaluación”. Aunque este estadístico ha sido ampliamente utilizado en la investigación social se debe complementar con otro tipo de análisis como los índices de Fiabilidad Compuesta (FC) y la Varianza Media Extractada (VME). Los resultados obtenidos se muestran en la Tabla 3, siendo ambos en todos los casos muy adecuados. www.eerp.usp.br/rlae Discusión En relación con la calidad de los ítems, se midió a través de la correlación ítem-total, los datos indican índices altos, que oscilan de 0.488 a 0.787, lo que muestra una alta consistencia interna apoyando la idea de que los ítems están correlacionados y la escala es precisa. Además, en el análisis descriptivo realizado de los ítems que componen el cuestionario, los datos muestran que existe asimetría negativa, lo que revela que los universitarios tienen tendencia a responder de acuerdo o muy de acuerdo. En cuanto a la primera dimensión, el factor “Ambiente de aprendizaje”, el alfa de fiabilidad medido a través del alfa de Cronbach es de 0.926, por lo que muestra alta consistencia interna y el AFE explica el 51.7% de la varianza total, siendo el factor con mayor peso en este cuestionario. Entre los ítems que componen esta dimensión se encuentran: ítem 15: el uso de los MIR hace que las clases sean amenas y dinámicas, ítem 48: el uso de los MIR mejora mi participación en las clases desde el anonimato, ítem 47: la utilización de los MIR hace que las clases tengan un ambiente más agradable e interactivo en comparación con las clases tradicionales, entre otros. Este Factor es determinante ya que el alumnado universitario con un ambiente adecuado de clase aumentará la asistencia a esta asignatura(22) y mejora su participación(13,17,44). Además, incrementa la interacción entre el profesorado y el alumnado(13), influye de forma positiva en la atención(33-34) y en su concentración(35), tal como avalan los estudios analizados. En cuanto a la fiabilidad de la escala, los datos arrojan un alfa de Cronbach de 0.965, lo que indica una alta fiabilidad. Estos datos están en consonancia con los índices de fiabilidad compuesta de los factores que componen MIR- MPEA, que alcanzan unos niveles óptimos en los 3 factores (Ambiente, Proceso de enseñanza-aprendizaje y Evaluación) que oscilan entre 0.955, 0.930 y 0.863, respectivamente, siendo el valor mínimo recomendado de 0.70. La segunda dimensión, que corresponde al factor “Proceso de enseñanza-aprendizaje”, muestra un índice de consistencia interno de 0.869 y explica el 5.4% de la varianza total, bajando de forma significativa el peso del factor. Discusión El objetivo del presente estudio es la construcción de un cuestionario válido y fiable para medir el uso de los MIR en el aprendizaje centrado en el estudiante 7 Mingorance-Estrada AC, Granda-Vera J, Rojas-Ruiz G, Alemany-Arrebola I. 61.61% de la varianza total. Posteriormente, se procedió con el AFC para confirmar el modelo de los tres factores. Los índices de bondad utilizados han sido χ2/gl siendo de 1.944, este índice se encuentra dentro de los estándares establecidos, el RMR es 0.077 y el RMSEA es de 0.080, índices que se consideran aceptables ya que se encuentran entre 0.5 y 0.08(49). Además, el GFI que representa el ajuste conjunto es de 0.78 y CFI con un valor de 0.97, se encuentran dentro de los límites de la tolerancia. Estos resultados confirman que el modelo de los 3 factores se ajusta a los datos, considerando que el modelo puede mantenerse como una explicación plausible a la estructura dimensional que se propone. universitario. Los resultados muestran que este trabajo constituye un aporte empírico a la validez de este modelo de intervención en el uso de los MIR, permitiendo a los profesores universitarios en general, y de manera particular, a los docentes de Ciencias de la Salud transformar el proceso de enseñanza-aprendizaje para incentivar e involucrar a los estudiantes a través de un enfoque más activo para profesores del siglo XXI, que puede medir la percepción del alumnado en el uso de los MIR, habiendo avanzado a pasos agigantados, además de ser un recurso tecnopedagógico, se ha convirtiendo en una forma de juego para los estudiantes, adaptándolo al contexto que no son juego, incorporándose como una metodología emergente denominada gamificación, que proporciona la oportunidad de transformar drásticamente las aulas tradicionales de manera que puedan mejorar el ambiente de clase, el proceso de aprendizaje y su rendimiento académico de manera lúdica y divertida. Por todo lo anterior, estos resultados permiten confiar en la fiabilidad y validez de este instrumento, por lo que con la construcción y validación de este cuestionario se hace posible medir la percepción del estudiante en el uso de los MIR en el proceso de aprendizaje. El cuestionario MIR-MPEA está formado por 24 ítems que se agrupan en 3 factores: Ambiente, Proceso de enseñanza-aprendizaje y Evaluación. www.eerp.usp.br/rlae Discusión Esta dimensión hace referencia a aquellos elementos que son básicos para acceder al conocimiento, tales como los debates, la interacción entre el profesorado- alumnado, que inciden positivamente en su proceso de aprendizaje, ya que ayuda al repaso y a la comprensión de los contenidos. Entre los ítems que lo componen se encuentran, ítem 47: la utilización de los MIR hace que las clases tengan un ambiente más agradable e interactivo en comparación con las clases tradicionales, ítem 59: los MIR proporcionan una información valiosa para mejorar mi proceso de aprendizaje, ítem 46: la utilización de los MIR se hace al finalizar las clases para repasar los contenidos Pero, además, se realizó la validez del constructo, tanto a través del Análisis Factorial Exploratorio (AFE) como del Confirmatorio (AFC). En primer lugar, se procedió al AFE, ya que no había instrumentos similares validados que midieran la percepción del alumnado universitario hacia el aprendizaje y solo conocíamos las dimensiones que conforman el constructo objeto de estudio fruto de la bibliografía consultada. Para ello, en primer lugar, los datos se sometieron a prueba de de Kaiser, Meyer y Olkin, obteniendo un valor significativo (KMO = 0.941) y a la prueba de esfericidad de Barlett (c2 = 2446.206; gl = 300; p < 0.001), estos valores permiten la utilización del análisis factorial como técnica idónea para interpretar la información contenida en esta matriz. Este análisis arrojó tres factores claramente delimitados que explican el Rev. Latino-Am. Enfermagem 2021;29:e3418. 8 del Campus de Melilla, de la Universidad de Granada, que utilizan recursos tecnopedagógicos como los MIR en sus aulas, adaptándolos a procesos de metodología activa gamificada, por lo que se hace necesario impulsar su uso e investigarlo. Por esta razón, es fundamental concienciar y formar al profesorado universitario en el modelo TPACK (Technological Pedagogical Content Knowledge) como un marco conceptual que puede orientar al profesorado para la integración de la tecnología en los procesos de aprendizaje de los estudiantes, lo que promoverá las metodologías emergentes, tales como la gamificación. En segundo lugar, se debe seguir mejorando el instrumento para que pueda ser utilizado por el profesorado, de cualquier ámbito de conocimiento y en cualquier circunstancia que acontezca, tal y como se ha producido en el confinamiento del COVID-19, pudiéndose trabajar, en épocas venideras, según los planteamientos gubernamentales y universitarios, en entornos presenciales, B-Learning y E-Learning con su alumnado. Discusión Por lo que se tendrá que mejorar el instrumento, incluyendo nuevos ítems que valoren este nuevo modelo de enseñanza virtual. Además, debemos profundizar la influencia de este instrumento en función de las distintas disciplinas de las Ciencias de la Salud, hecho que pretendemos llevar a cabo en el futuro. La aplicación de los avances tecnológicos en el ámbito universitario mejora el proceso aprendizaje activo de los estudiantes, siendo necesario conocer la opinión de los mismos para lograr un avance y mejora en la docencia. En tercer lugar, es necesario realizar más investigación cuasi- experimental en el ámbito de la docencia universitaria, en sus diversas ramas de conocimiento, para analizar la influencia de la metodología activa gamificada a través de estos recursos tecnopedagógicos y la metodología tradicional, teniendo muy presente su alcance o relación con el rendimiento académico. dados cada día. Estos elementos coinciden con los estudios que consideran que esta metodología de trabajo mejora el rendimiento(31-32,38-41), con el cambio de paradigma que establece el aprendizaje activo de los estudiantes a través de la conexión entre el saber y el saber hacer(45), fruto de la interacción frecuente y positiva que puede dinamizar las clases cuando se usan los MIR(13,28). Todo esto permite un razonamiento sobre el material, adquiriendo un conocimiento más profundo, ayudando al repaso y a la comprensión de los contenidos y mejora de la retención a largo plazo(36-37), con la consiguiente optimización del proceso de aprendizaje. En relación con la tercera dimensión, “Evaluación del aprendizaje” presenta un alfa de Cronbach de 0.871, y el AFE explica el 5.07% de la varianza total, siendo un porcentaje similar al factor anterior. Este dimensión se relaciona con la retroalimentación y evaluación formativa, que ayudan a corregir errores o falta de comprensión sobre los contenidos del tema trabajados, tal como indican las investigaciones consultadas(4,20,40). Está formado por el ítem 20: el uso de los MIR permite conocer y comparar sus respuestas con las respuestas de los compañeros, el ítem 36: el uso de los MIR promueve el estudio regular de la asignatura para ir mejor preparados a las clases, el ítem 35: el uso de los MIR evalúa mi conocimiento comprensivo de los contenidos en cada uno de los temas tratados durante las clases, entre otros. Discusión Esta retroalimentación inmediata incide de forma positiva tanto en el alumnado en su proceso de aprendizaje como en el docente en su proceso de enseñanza, lo que permite la evaluación formativa de los alumnos. De este modo, el mundo global en el que vivimos, sin fronteras académicas, los Espacios de Educación Superior Europeo e Iberoamericano cumplen la premisa en la que se pone de manifiesto que la calidad del sistema universitario debe estar en continuo proceso de transformación con el cambio de paradigma hacia el aprendizaje activo de los alumnos y al aprendizaje permanente a lo largo de toda la vida. Además, es necesario establecer procesos de evaluación y acreditación del trabajo que se realiza en las universidades, con la consiguiente transferencia del conocimiento de las investigaciones realizadas entre las distintas investigaciones en las diversas ramas del saber, estableciendo un entendimiento interdisciplinar que resalte la realidad y cuestione el pensamiento tradicional de las distintas ramas del saber como compartimentos aislados y separados de las fronteras disciplinares. www.eerp.usp.br/rlae Conclusión La contribución de este estudio al conocimiento es el diseño y desarrollo de un instrumento y su transferencia, que permita medir y valorar la percepción de los estudiantes en el uso de los MIR con respecto al desarrollo de la docencia universitaria para el dominio de la materia de cualquier rama del conocimiento, ya sean Ciencias de la Salud, Ciencias Sociales y Jurídicas, Artes y Humanidades, e Ingeniería y Arquitectura, mediante un recurso tecnopedagógico, en tres factores fundamentales: ambiente, proceso de enseñanza-aprendizaje y evaluación. Este cuestionario puede ser aplicado en clase durante el tiempo que dure la formación, lo que ayudará a mejorar los procesos de enseñanza-aprendizaje. En este sentido, los profesores de Ciencias de la Salud deben establecer Entre las limitaciones del presente estudio enumeramos las siguientes: en primer lugar, es necesario seguir aumentando el número de participantes, ya que la muestra no es excesivamente amplia, pero permite tener un punto de partida para transferirlo a otras ramas de conocimiento. La razón es que son escasos los docentes 9 9 Mingorance-Estrada AC, Granda-Vera J, Rojas-Ruiz G, Alemany-Arrebola I. Literature Review and Case Studies, EUR 29882. Luxembourg: European Commission; 2019. doi: https://doi.org/10.2760/552774 y explorar nuevas formas innovadoras de involucrar a los estudiantes y estimular el aprendizaje activo, siendo importante la incorporación de metodologías activas a través del uso de los mandos interactivos de respuestas en disciplinas como Enfermería, Medicina, Farmacia, Educación Paramédica, Psicología, Odontología, Fisioterapia, Logopedia, Biotecnología, Epidemiología, Genética, Bioquímica, Terapia Ocupacional, Nutrición Humana y Dietética, entre otras. Este instrumento proporciona un enfoque pedagógico positivo en el proceso de enseñanza-aprendizaje para docentes y estudiantes de Ciencias de la Salud, lo que conlleva una mejora del rendimiento con el propósito de adquirir un conocimiento profundo de las materias. 9. Marcelo C, Yot C, Mayor C. Enseñar con tecnologías digitales en la Universidad. Comunicar. 2015;45:117-24. doi: https://doi.org/10.3916/C45-2015-12 10. 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[Internet]. 18. Castro MJ, López M, Cao MJ, Fernández-Castro M, García S, Frutos M, et al. Impact of educational games on academic outcomes of students in the Degree in Nursing. PLoS One. 2019;14(7):e0220388. doi: https:// doi.org/10.1371/journal.pone.0220388 8. www.eerp.usp.br/rlae Referencias Enhancing student engagement and immediate feedback with clickers and response cards. Int J Innov Learn. 2018;24(1):81-97. doi: https://doi. org/10.1504/IJIL.2018.092924 28. Banks D. Reflections on the use of ARS with small groups. In: ______. Audience Response Systems in Higher Education: Applications and Cases. Pennsylvania: IGI Global; 2006. p. 373-86. 29. Wood A. Nurse Perceptions of Interactivity during Their Onboarding Orientation: Effect of an Audience Response System [thesis]. Boiling Springs: Hunt School of Nursing; 2017 [cited Dec 12, 2019]. Available from: https:// digitalcommons.gardner-webb.edu/cgi/viewcontent.cgi ?article=1268&context=nursing_etd 30. Ismaile S, Alhosban F, Hawamdeh S. Making learning fun to increase nursing students’ success: Formative feedback in communication learning. Australas Med J. 2017;10(12):1014-21. doi: https://doi.org/10.21767/ AMJ.2017.3228 27. Mohan R. Enhancing student engagement and immediate feedback with clickers and response cards. Int J Innov Learn. 2018;24(1):81-97. doi: https://doi. org/10.1504/IJIL.2018.092924 38. Rana NP, Dwivedi YK. Can clicking promote learning? measuring student learning performance using clickers in the undergraduate information systems class. J Int Educ Bus. 2017;10(2):201-15. doi: http://doi.org/10.1108/ JIEB-06-2016-0010 38. Rana NP, Dwivedi YK. Can clicking promote learning? measuring student learning performance using clickers in the undergraduate information systems class. J Int Educ Bus. 2017;10(2):201-15. doi: http://doi.org/10.1108/ JIEB-06-2016-0010 org/10.1504/IJIL.2018.092924 28. Banks D. Reflections on the use of ARS with small groups. In: ______. Audience Response Systems in Higher Education: Applications and Cases. Pennsylvania: IGI Global; 2006. p. 373-86. 28. Banks D. Reflections on the use of ARS with small groups. In: ______. Audience Response Systems in Higher Education: Applications and Cases. Pennsylvania: IGI Global; 2006. p. 373-86. Education: Applications and Cases. Pennsylvania: IGI Global; 2006. p. 373-86. 39. Iwamoto D, Hargis J, Taitano E, Vuong K. Analyzing the efficacy of the testing effect using kahoottm on student performance. Turkish Online J Distance Educ. [Internet]. 2017;18(2):93-80. doi: https://doi.org/10.17718/ tojde.306561 29. Wood A. Nurse Perceptions of Interactivity during Their Onboarding Orientation: Effect of an Audience Response System [thesis]. Boiling Springs: Hunt School of Nursing; 2017 [cited Dec 12, 2019]. Available from: https:// digitalcommons.gardner-webb.edu/cgi/viewcontent.cgi ?article=1268&context=nursing_etd 29. Wood A. Nurse Perceptions of Interactivity during Their Onboarding Orientation: Effect of an Audience Response System [thesis]. Boiling Springs: Hunt School of Nursing; 2017 [cited Dec 12, 2019]. Available from: https:// digitalcommons.gardner-webb.edu/cgi/viewcontent.cgi ?article=1268&context=nursing_etd 40. George C, Gallegos C, Tesar AJ, Connor K, Martz K. The use of a game-based learning platform to engage nurs- ing students: A descriptive, qualitative study. Nurse Educ Pract. 2017;27:101-6. doi: https://doi.org/10.1016/j. nepr.2017.08.019. 40. Referencias Kapsalis G, Ferrari A, Punie Y, Conrads J, Collado A, Hotulainen R, et al. Evidence of Innovative Assessment: 8. Kapsalis G, Ferrari A, Punie Y, Conrads J, Collado A, Hotulainen R, et al. Evidence of Innovative Assessment: www.eerp.usp.br/rlae 10 Rev. Latino-Am. Enfermagem 2021;29:e3418. 31. Castro MJ, López M, Cao MJ, Fernández-Castro M, García S, Frutos M, et al. Impact of educational games on academic outcomes of students in the Degree in Nursing. PLoS One 2019;14(7):e0220388. doi: https:// doi.org/10.1371/journal.pone.0220388 19. Nishimura A. Effects of different methods of reflection on nurses’ gaze and judgement in a task using a touch panel. J Clin Nurs. 2018;27:569-77. doi: https://doi. org/10.1111/jocn.14096 19. Nishimura A. Effects of different methods of reflection on nurses’ gaze and judgement in a task using a touch panel. J Clin Nurs. 2018;27:569-77. doi: https://doi. org/10.1111/jocn.14096 20. Toothaker R. Millennial’s perspective of clicker technology in a nursing classroom: A mixed methods h t d N Ed T d 2018 62 80 4 d i 20. Toothaker R. Millennial’s perspective of clicker technology in a nursing classroom: A mixed methods research study. Nurse Educ Today. 2018;62:80-4. doi: https://doi.org/10.1016/j.nedt.2017.12.027 32. Corell A, Regueras LM, Verdú E, Verdú MJ, Castro JP. Effects of competitive learning tools on medi- cal students: A case study. PLoS One. 2018;13(3):e0194096. doi: https://doi.org/10.1371/journal.pone.0194158 32. Corell A, Regueras LM, Verdú E, Verdú MJ, Castro JP. Effects of competitive learning tools on medi- cal students: A case study. PLoS One. 2018;13(3):e0194096. doi: https://doi.org/10.1371/journal.pone.0194158 https://doi.org/10.1016/j.nedt.2017.12.027 21. Chen TL, Lan YL. Using a personal response system as an in-class assessment tool in the teaching of basic college 33. Oswald KM, Rhoten SE. Improving classroom clicker practices: Effects of incentives and feedback on retention. N Am J Psychol. [Internet]. 2014 [cited Dec 6, 2017];16(1):79-88. Available from: https://www. researchgate.net/publication/285958781_Improving_ classroom_clicker_practices_Effects_of_incentives_and_ feedback_on_retention chemistry. Australas J Educ Technol. 2013;29(1):32-40. doi: https://doi org/10 14742/ajet 95 chemistry. Australas J Educ Technol. 2013;29(1):32-40. chemistry. Australas J Educ Technol. 2013;29(1):32-40. doi: https://doi.org/10.14742/ajet.95 doi: https://doi.org/10.14742/ajet.95 22. Aktekin NÇ, Çelebi H, Aktekin M. Let’s kahoot! Anatomy. Int. J. Morphol. 2018;36(2):716-21. doi: http:// dx.doi.org/10.4067/S0717-95022018000200716 23. Barr ML. Encouraging college student active engagement in learning: The influence of response methods. Innov High Educ. 2013;39(4):307-19. doi: https://doi.org/10.1111/jcal.12205 34. Marshall LL, Varnon AW. An Empirical Investigation of Clicker Technology in Financial Accounting Principles. J Learn High Educ. [Internet]. 2012 [cited Jun 3, 2018];8(1):7-18. Available from: https://files.eric.ed.gov/ fulltext/EJ1144930.pdf 34. Marshall LL, Varnon AW. Referencias An Empirical Investigation of Clicker Technology in Financial Accounting Principles. 34. Marshall LL, Varnon AW. An Empirical Investigation of Clicker Technology in Financial Accounting Principles. 34. Marshall LL, Varnon AW. An Empirical Investigation of Clicker Technology in Financial Accounting Principles. 34. Marshall LL, Varnon AW. An Empirical Investigation of Clicker Technology in Financial Accounting Principles. J Learn High Educ. [Internet]. 2012 [cited Jun 3, 2018];8(1):7-18. Available from: https://files.eric.ed.gov/ fulltext/EJ1144930.pdf J Learn High Educ. [Internet]. 2012 [cited Jun 3, 2018];8(1):7-18. Available from: https://files.eric.ed.gov/ fulltext/EJ1144930.pdf J Learn High Educ. [Internet]. 2012 [cited Jun 3, 2018];8(1):7-18. Available from: https://files.eric.ed.gov/ fulltext/EJ1144930.pdf 24. Iskander M. Systematic review of the implementation 24. Iskander M. Systematic review of the implementation of audience response systems and their impact on participation and engagement in the education of healthcare professionals. BMJ Simulation & Technology Enhanced Learning. 2018;4(2):47-50. doi: http://dx.doi. org/10.1136/bmjstel-2017-000245 of audience response systems and their impact on participation and engagement in the education of 35. Bojinova ED, Oigara JN. Teaching and learning with Clickers: Are Clickers good for students? Interdiscip J E-Learning Learn Objects. 2011;7:169-83. doi: https:// doi.org/10.28945/1506 healthcare professionals. BMJ Simulation & Technology org/10.1136/bmjstel-2017-000245 25. Licorish SA, Owen HE, Daniel, B, George, JL. Students’ perception of Kahoot!’s in uence on teaching and learning. RPTEL. 2018;13(9):1-23. doi: https://doi.org/10.1186/ s41039-018-0078-8 36. Benson JD, Szucs KA, DeIuliis E, Leri A. Impact of Student Response Systems on Initial Learning and Retention 36. Benson JD, Szucs KA, DeIuliis E, Leri A. Impact of Student Response Systems on Initial Learning and Retention of Course Content in Health Sciences Students. J Allied Health. [Internet]. 2017 [cited Feb 12, 2018];46(3):158- 63. Available from: https://www.ingentaconnect.com/ content/asahp/jah/2017/00000046/00000003/art00008 37. Njie-Carr VP, Ludeman E, Lee MC, Dordunoo D, Trocky NM, Jenkins LS. An Integrative Review of Flipped Classroom Teaching Models in Nursing Education. J Prof Nurs. 2017;33(2):133-44. doi: 10.1016/j. profnurs.2016.07.001 RPTEL. 2018;13(9):1-23. doi: https://doi.org/10.1186/ s41039-018-0078-8 of Course Content in Health Sciences Students. J Allied Health. [Internet]. 2017 [cited Feb 12, 2018];46(3):158- 63. Available from: https://www.ingentaconnect.com/ content/asahp/jah/2017/00000046/00000003/art00008 26. Oliveira C, Tirapelli C, Rodrigues CT, Domaneschi C, Caldeira SA. Interactive audience response systems in oral and maxillofacial radiology undergraduate lectures. Eur J Dent Educ. [Internet]. 2017;22:63-9. doi: https:// doi.org/10.1111/eje.12258 26. Oliveira C, Tirapelli C, Rodrigues CT, Domaneschi C, Caldeira SA. Interactive audience response systems in oral and maxillofacial radiology undergraduate lectures. Eur J Dent Educ. [Internet]. 2017;22:63-9. doi: https:// doi.org/10.1111/eje.12258 27. Mohan R. Referencias George C, Gallegos C, Tesar AJ, Connor K, Martz K. The use of a game-based learning platform to engage nurs- ing students: A descriptive, qualitative study. Nurse Educ Pract. 2017;27:101-6. doi: https://doi.org/10.1016/j. nepr.2017.08.019. 30. Ismaile S, Alhosban F, Hawamdeh S. Making learning fun to increase nursing students’ success: Formative feedback in communication learning. Australas Med J. 2017;10(12):1014-21. doi: https://doi.org/10.21767/ AMJ.2017.3228 41. Kalaian SA, Kasim RM. Effectiveness of various innovative learning methods in health science class- www.eerp.usp.br/rlae www.eerp.usp.br/rlae 11 Mingorance-Estrada AC, Granda-Vera J, Rojas-Ruiz G, Alemany-Arrebola I. Copyright © 2021 Revista Latino-Americana de Enfermagem Este es un artículo de acceso abierto distribuido bajo los términos de la Licencia Creative Commons CC BY. Esta licencia permite a otros distribuir, mezclar, ajustar y construir a partir de su obra, incluso con fines comerciales, siempre que le sea reconocida la autoría de la creación original. Esta es la licencia más servicial de las ofrecidas. Recomendada para una máxima difusión y utilización de los materiales sujetos a la licencia. Contribución de los Autores: rooms: a meta-analysis. Adv Health Sci Educ Theory Pract. [Internet]. 2017;22(5):1151-67. doi: https://doi. org/10.1007/s10459-017-9753-6 Concepción y dibujo de la pesquisa: Ángel Custodio Mingorance-Estrada. Obtención de datos: Ángel Custodio Mingorance-Estrada, Gloria Rojas-Ruiz. Análisis e interpretación de los datos: Ángel Custodio Mingorance-Estrada, Juan Granda-Vera, Inmaculada Alemany-Arrebola. Análisis estadístico: Juan Granda- Vera, Inmaculada Alemany-Arrebola. Redacción del manuscrito: Ángel Custodio Mingorance-Estrada, Gloria Rojas-Ruiz. Revisión crítica del manuscrito en cuanto al contenido intelectual importante: Ángel Custodio Mingorance-Estrada. 42. Maloney LM, Dilger JP, Werfel PA, Cimino LM. Are Audience Response Systems Worth the Cost ? Comparing Question-Driven Teaching Strategies for Emergency Medical Technician Education. Internet J Allied Health Sci Pract. [Internet]. 2017 [cited May 7, 2018];16(1):1- 8. Available from: https://nsuworks.nova.edu/cgi/ viewcontent.cgi?article=1704&context=ijahsp Medical Technician Education. Internet J Allied Health 43. Ismail MA, Mohammad JA. Kahoot: A promising tool for formative assessment in medical education. Educ Med J. 2017;9(2):19-26 doi: https://doi.org/10.21315/ eimj2017.9.2.2 Todos los autores aprobaron la versión final del texto. 44. Nikou SA, Economides AA. Mobile-based assessment: A literature review of publications in major referred journals from 2009 to 2018. Comput Educ. 2018;125:101- 19. doi: https://doi.org/10.1016/j.compedu.2018.06.006 45. Montejano-Lozoya R, Gea-Caballero V, Miguel-Montoya I, Juárez-Vela R, Sanjuán-Quiles A, Ferrer-Ferrandiz E. Validation of a questionnaire designed to measure nursing student satisfaction with practical training. Rev. Latino- Am. Enfermagem. 2019;27:e3206. doi: https://doi. org/10.1590/1518-8345.3102.3206 44. Nikou SA, Economides AA. Mobile-based assessment: A literature review of publications in major referred journals from 2009 to 2018. Comput Educ. 2018;125:101- 19. doi: https://doi.org/10.1016/j.compedu.2018.06.006 45. Montejano-Lozoya R, Gea-Caballero V, Miguel-Montoya I, Juárez-Vela R, Sanjuán-Quiles A, Ferrer-Ferrandiz E. Validation of a questionnaire designed to measure nursing student satisfaction with practical training. Rev. Latino- Am. Enfermagem. 2019;27:e3206. doi: https://doi. org/10.1590/1518-8345.3102.3206 Conflicto de intereses: los autores han declarado que no existe ningún conflicto de intereses. Conflicto de intereses: los autores han declarado que no existe ningún conflicto de intereses. 46. Miles NG, Soares TP. Acceptance of clickers in a large multimodal biochemistry class as determined by student evaluations of teaching: Are they just an annoying distraction for distance students? Biochem Mol Biol Educ. 2016; Oct;44(1):99-108. doi: https://doi.org/10.1002/ bmb.20917 47. Yeh CR, Tao YH. How benefits and challenges of personal response system impact students’ continuance intention? A Taiwanese context. Educ Technol Soc. [Internet]. 2013 [cited Jan 10, 2018];16(2):257-70. Available from: https://pdfs.semanticscholar.org/283d/ f5d6cddabfc9abb8d2806efbf809975818cb.pdf 48. Lin YC, Liu TC, Chu CC. Implementing clickers to assist learning in science lectures: The clicker-assisted conceptual change model. Australas J Educ Technol. 2011;27(6):979-96. Contribución de los Autores: doi: https://doi.org/10.14742/ ajet.924 47. Yeh CR, Tao YH. How benefits and challenges of personal response system impact students’ continuance intention? A Taiwanese context. Educ Technol Soc. [Internet]. 2013 [cited Jan 10, 2018];16(2):257-70. Available from: https://pdfs.semanticscholar.org/283d/ f5d6cddabfc9abb8d2806efbf809975818cb.pdf 48. Lin YC, Liu TC, Chu CC. Implementing clickers to assist learning in science lectures: The clicker-assisted conceptual change model. Australas J Educ Technol. 2011;27(6):979-96. doi: https://doi.org/10.14742/ ajet.924 49. Kline R. Principles and Practice of Structural Equation Modeling. New York: The Guilford Press; 2011. 49. Kline R. Principles and Practice of Structural Equation Modeling. New York: The Guilford Press; 2011. Recibido: 12.12.2019 Aceptado: 13.08.2020 Editora Asociada: Regina Aparecida Garcia de Lima Copyright © 2021 Revista Latino-Americana de Enfermagem Este es un artículo de acceso abierto distribuido bajo los términos de la Licencia Creative Commons CC BY. Esta licencia permite a otros distribuir, mezclar, ajustar y construir a partir de su obra, incluso con fines comerciales, siempre que le sea reconocida la autoría de la creación original. Esta es la licencia más servicial de las ofrecidas. Recomendada para una máxima difusión y utilización de los materiales sujetos a la licencia. Autor de correspondencia: Ángel Custodio Mingorance Estrada E-mail: amingoe@ugr.es https://orcid.org/0000-0003-4478-3011 www.eerp.usp.br/rlae
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Data-driven polarimetric imaging: a review
Opto-electronic science
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Data-driven polarimetric imaging: a review Citation: Yang K, Liu F, Liang SY, et al. Data-driven polarimetric imaging: a review. Opto-Electron Sci 3, 230042 (2024). https://doi.org/10.29026/oes.2024.230042 Received: 14 November 2023; Accepted: 19 January 2024; Published online: 29 February 2024 Received: 14 November 2023; Accepted: 19 January 2024; Published online: 29 February 2024 1School of Optoelectronic Engineering, Xidian University, Xi’an 710071, China; 2School of Physics, Xidian University, Xi’an 710071, China; 3Graduate School of Information, Production and Systems, Waseda University, Kitakyushu 808-0135, Japan; 4Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; 5Hangzhou Institute of Technology, Xidian University, Hangzhou 311200, China. Data-driven polarimetric imaging: a review Kui Yang1†, Fei Liu1†*, Shiyang Liang3†, Meng Xiang1, Pingli Han1, Jinpeng Liu1, Xue Dong1, Yi Wei4, Bingjian Wang2, Koichi Shimizu3* and Xiaopeng Shao1,5* This study reviews the recent advances in data-driven polarimetric imaging technologies based on a wide range of prac- tical applications. The widespread international research and activity in polarimetric imaging techniques demonstrate their broad applications and interest. Polarization information is increasingly incorporated into convolutional neural net- works (CNN) as a supplemental feature of objects to improve performance in computer vision task applications. Polarimetric imaging and deep learning can extract abundant information to address various challenges. Therefore, this article briefly reviews recent developments in data-driven polarimetric imaging, including polarimetric descattering, 3D imaging, reflection removal, target detection, and biomedical imaging. Furthermore, we synthetically analyze the input, datasets, and loss functions and list the existing datasets and loss functions with an evaluation of their advantages and disadvantages. We also highlight the significance of data-driven polarimetric imaging in future research and develop- ment. Keywords: deep learning; polarimetric imaging; image processing Yang K, Liu F, Liang SY et al. Data-driven polarimetric imaging: a review. Opto-Electron Sci 3, 230042 (2024). Yang K, Liu F, Liang SY et al. Data-driven polarimetric imaging: a review. Opto-Electron Sci 3, 230042 (2024) 1School of Optoelectronic Engineering, Xidian University, Xi’an 710071, China; 2School of Physics, Xidian University, Xi’an 710071, China; 3Graduate School of Information, Production and Systems, Waseda University, Kitakyushu 808-0135, Japan; 4Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; 5Hangzhou Institute of Technology, Xidian University, Hangzhou 311200, China. †These authors contributed equally to this work. *Correspondence: F Liu, E-mail: feiliu@xidian.edu.cn; K Shimizu, E-mail: shimizu@xidian.edu.cn; XP Shao, E-mail: xpshao@xidian.edu.cn Received: 14 November 2023; Accepted: 19 January 2024; Published online: 29 February 2024 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2024. Published by Institute of Optics and Electronics, Chinese Academy of Sciences. 230042-1 †These authors contributed equally to this work. *Correspondence: F Liu, E-mail: feiliu@xidian.edu.cn; K Shimizu, E-mail: shimizu@xidian.edu.cn; XP Shao, E-mail: xpshao@xidian.edu.cn Received: 14 November 2023; Accepted: 19 January 2024; Published online: 29 February 2024 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2024. Published by Institute of Optics and Electronics, Chinese Academy of Sciences. 230042-1 Crosstalk-free achromatic full Stokes imaging polarimetry metasurface enabled by polarization-dependent phase optimization Crosstalk-free achromatic full Stokes imaging polarimetry metasurface enabled by polarization-dependent phase optimization Yaxin Zhang, Mingbo Pu, Jinjin Jin, Xinjian Lu, Yinghui Guo, Jixiang Cai, Fei Zhang, Yingli Ha, Qiong He, Mingfeng Xu, Xiong Li, Xiaoliang Ma, Xiangang Luo http://www.oejournal.org/oes Review February 2024, Vol. 3, No. 2 Review Short history Polarimetric imaging, first observed by Sir Isaac Newton and Christiaan Huygens in the 17th century, is a tech- nique used to capture and analyze the polarization prop- erties of light. Moreover, they noticed that light could be separated into two polarized lights when interacting with calcite crystals. In the 19th century, scientists such as Malus and Fresnel made significant contributions to the understanding of polarization. Since its inception, polari- metric imaging has progressed and been applied in three essential fields in the 20th century: polarization micro- scopy, which allows scientists to study the birefringence properties of materials under polarized light, remote sensing and polarimetry, enhancing the detection and discrimination of objects based on their polarization characteristics; medical imaging; and tissue optical prop- erties. In recent years, digital cameras and other innovat- ive imaging techniques have become capable of captur- ing valuable information on surfaces, materials, and bio- logical samples. Recent developments in computer vis- ion have enabled the incorporation of polarization in- formation into various tasks, offering potential advant- ages over traditional RGB imaging. Deep learning, a subfield of machine learning, is in- spired by the human brain. Artificial neural networks emerged during the 1940s and 1950s. Research on neur- al networks continued during the 1960s and 1980s, and perceptron and backpropagation algorithms were de- veloped. However, neural networks face limitations ow- ing to computational constraints and insufficient data. In the early 2000s, traditional machine learning techniques, such as support vector machines and decision trees, out- performed deep learning in many tasks, leading to a de- cline in interest in deep learning. After 2010, the propos- al of a new network structure and improvements in com- putational power propelled the advancement of deep learning. Key developments during this decade include the use of CNN for computer vision and recurrent neur- al networks for natural language processing. With ongo- ing research, deep learning continues to evolve into more efficient training algorithms, model architectures, and applications across various domains, making it a central part of the AI field of artificial intelligence. The remainder of this study is organized as follows: First, we review the recent trends in data-driven polari- metric imaging from four aspects. Second, seven exist- ing research fields are categorized and analyzed with their corresponding algorithms. Third, we discuss vari- ous algorithmic approaches used in each field. Yang K et al. Opto-Electron Sci 3, 230042 (2024) Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 theory and practice37−41. To overcome the lack of a physical theoretical model, the use of deep learning technology to optimize the in- formation processing procedure has been proposed. Sev- eral experiments have proven its advantages in extract- ing potential features from images and determining the relationship between information transmission systems based on massive datasets. However, intensity informa- tion is commonly used in existing data-driven methods, which lose other three-dimensional information. In ad- dition, the unicity of information would make the ima- ging performance suffer from various challenges; for ex- ample, in target detection tasks, spoofing targets and camouflaged targets can reduce the accuracy rate; in se- mantic segmentation, water hazards and metallic sur- faces are the key challenges in the segmentation of road scenes; in pathological diagnosis, the only color informa- tion in medical images would increase the risk of misdia- gnosis; and for transparent objects, parts of imaging pro- cesses would be challenging to implement, such as 3D re- construction and segmentation in the intensity field. Therefore, the introduction of additional light-field physical information, such as polarization, is gaining in- creasing attention as a supplemental feature of objects for improved performance in higher-level visual tasks, expanding beyond intensity-only coverage. Therefore, polarimetric imaging and deep learning will contribute to future research and development. This review sum- marizes the existing methods of combining polarimetric imaging and deep learning and demonstrates the cur- rent gains visually and comprehensively. Introduction gnosis24−29, remote sensing30−33, and semantic segmenta- tion34−36. Furthermore, harnessing the features of polariz- ation opens new possibilities for research and techno- logy development. Polarization is a fundamental physical property of light that expresses the characteristics of vector shear wave1,2. When light interacts with objects or media, it exhibits various characteristics and representations of polariza- tion that correspond to the intrinsic characteristics of the material. This unique quality is an additional dimension of information that has many applications in various fields, such as polarimetric descattering3−9, 3D shape re- construction10−13, reflection removal14,15, target detection16−20, biomedical imaging21−23, pathological dia- The conventional polarization method is limited in ac- curately capturing and utilizing information owing to complicated interactions in the transmission process. Moreover, convolutional neural networks (CNN) excel at nonlinear expression and information extraction based on large datasets, making them better suited for modeling and interpreting polarization information than traditional algorithms, which can bridge the gap between Short history Next, we discuss three critical aspects of their existing datasets and loss functions and their advantages and disadvantages. Next, we discuss the strengths and weaknesses of the practical applications of data-driven polarimetric ima- ging and possible opportunities to address these chal- lenges. Finally, we provide conclusions, highlighting the importance and potential of data-driven polarimetric imaging in various applications and research areas that could extend the current uses of data-driven polarimet- ric imaging and provide insights to advance its future development. Until the 21st century, the combination of deep learn- ing and polarimetric imaging has become more pro- nounced. In the mid-2010s, data-driven techniques were 230042-2 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 applied to polarimetric imaging. Machines and deep learning have been used for tasks, including object recog- nition in polarimetric images. Subsequently, researchers explored the potential of deep learning for processing and analyzing polarimetric data in more diverse do- mains because deep learning helps extract valuable in- formation from polarization data. Additionally, the syn- ergy between deep learning and polarimetric imaging continues to evolve with advancements in models, al- gorithms, and applications, forming the significant field called “data-driven polarimetric imaging”, which is the novel method that combines the learning method and polarimetric imaging. Data-driven polarimetric imaging can revolutionize various fields and represents a prom- ising interdisciplinary research area and technological innovation. A diagram of this short history is shown in Fig. 1. With the exploration of data-driven polarimetric ima- ging, the application fields and utilization of polariza- tion information have gradually increased. Regarding the input and use of polarization information, directly cap- tured images from detectors, such as division of focal plane (DoFP) images42−45 or 0°, 45°, 90°, and 135°46−52 po- larization images, are the most common inputs. It should be noted that, despite having four components, the last one is often neglected in practical applications, leading to the predominant use of only the initial three parameters. This is because, theoretically, the last parameter can be derived from the first three, thereby reducing the diffi- culty in image acquisition. In addition, polarimetric parameter features were calculated to represent the po- larization information more intuitively, such as the de- gree of polarization (DoP) and angle of polarization(AoP)52−57, [S0, S1, S2]52,55,58,59, Mueller matrix images60−63 and other combinations of these elements. Short history Features based on the corresponding physical model during different tasks were also computed to guide net- work training, such as zenith and azimuth angle maps derived from specular and diffuse reflection64,65 in 3D shape reconstruction tasks. With the exploration of data-driven polarimetric ima- ging, the application fields and utilization of polariza- tion information have gradually increased. Regarding the input and use of polarization information, directly cap- tured images from detectors, such as division of focal plane (DoFP) images42−45 or 0°, 45°, 90°, and 135°46−52 po- larization images, are the most common inputs. It should be noted that, despite having four components, the last one is often neglected in practical applications, leading to the predominant use of only the initial three parameters. Trends Data-driven polarimetric imaging is a novel approach aimed at compensating for the defects and difficulties of a single-information interpretation model. Recently, the advantages of combining polarimetric imaging and deep learning have been applied in several fields, and dozens of algorithms based on data-driven polarimetric ima- ging have been proposed, covering several application areas. This section describes more recent trends in data- driven polarimetric imaging from four perspectives. Schematics of the trends in existing data-driven polari- metric imaging are shown in Fig. 2. Furthermore, physical modes are crucial during net- work training, such as polarimetric descattering models, three-dimensional imaging models, Fresnel equations, Mueller matrix interpretation models, and other tradi- tional polarization models. The preliminary methods are often end-to-end architectures43,46,47,56,64,66−70, suggesting that the polarization information is input into the 17th century 19th century 20th century 21th century More valuable information Mid-2010s Birefringence of calcite crystals Malus law Fresnel laws Detector Primary phenomena Proposal of theory 1940s-1950s 1960s-1980s 2010s 2010s Neure Perceptron Backpropagation algorithm Advances of hardware Applications Data-driven polarization imaging Extraction of potential features Computer Fig. 1 | Schematic of short history about data-driven polarimetric imaging. 230042-3 21th century More valuable information 17th century Birefringence of calcite crystals Malus law Detector Fresnel laws Data-driven polarization imaging Fig. 1 | Schematic of short history about data-driven polarimetric imaging. 230042-3 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 From raw polarimetric images to preprocessing features DoFP image [S0, S1, S2] [S0, DoP, AoP] Polarimetric sub-images Mueller matrix images Mueller matrix parameters maps Physical priors Epoch Loss Epoch Loss Epoch Loss Epoch Loss Other features Network training From end-to-end architecture to physical-model-combined structure End-to-end network training Physical formulation Physical formulation Physical-model-guided network training Physical-model-integrated network training Network output Network input Epoch Loss From single to multi physical properties Polarimetric information Intensity information Pre-trained network maps Spectral information Phase information Speckle field information Network training From image processing to semantic tasks Image enhancement Semantic segmentation Target detection lmage reconstruction Biomedical imaging a d b c 2 | Schematics of the trends of existing data-driven polarimetric imaging. The solid black arrows refer to the data flow, the dotted bla ws refer to that the data may or not flow. The gradient blue arrows mean the trends of corresponding aspects. Trends (a) The utilization of polarim nformation has been gradually deepened from raw polarimetric images to preprocessing features. (b) The physical modes have been cruc ng the training of network from end-to-end architecture to physical-model-combined structure. (c) The various physical properties have be ually fused to network. (d) The tasks have been developed from image processing to semantic tasks. Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 From raw polarimetric images to preprocessing features DoFP image [S0, S1, S2] [S0, DoP, AoP] Polarimetric sub-images Mueller matrix images Mueller matrix parameters maps Physical priors Epoch Loss Other features Network training a a Epoch Loss Epoch Loss Epoch Loss From end-to-end architecture to physical-model-combined structure End-to-end network training Physical formulation Physical formulation Physical-model-guided network training Physical-model-integrated network training Network output Network input b From end-to-end architecture to physical-model-combined structure b Physical formulation Physical formulation Ep Physical-model-integrated network training Ep Physical-model-integrated network training Physical model integrated network training Epoch Loss From single to multi physical properties Polarimetric information Intensity information Pre-trained network maps Spectral information Phase information Speckle field information Network training From image processing to semantic tasks Image enhancement Semantic segmentation Target detection lmage reconstruction Biomedical imaging d c From single to multi physical properties c Phase information Intensity information Fig. 2 | Schematics of the trends of existing data-driven polarimetric imaging. The solid black arrows refer to the data flow, the dotted black arrows refer to that the data may or not flow. The gradient blue arrows mean the trends of corresponding aspects. (a) The utilization of polarimet- ric information has been gradually deepened from raw polarimetric images to preprocessing features. (b) The physical modes have been crucial during the training of network from end-to-end architecture to physical-model-combined structure. (c) The various physical properties have been gradually fused to network. (d) The tasks have been developed from image processing to semantic tasks. 230042-4 https://doi.org/10.29026/oes.2024.230042 Yang K et al. Opto-Electron Sci 3, 230042 (2024) network to generate the desired outputs directly; however, the physical models are gradually guided46,47,49,71 or integrated into network training48,72−74. Furthermore, the physical model and its inverse process can form a self-supervised closed loop to achieve better perform- ance. Furthermore, data-driven polarimetric imaging en- ables the physical interpretability of networks compared with conventional deep network methods. Polarimetric parameters were initially adopted to perform different tasks without exploring the hidden mechanisms. Trends In more in-depth research on data-driven polarimetric imaging, the estimation of specific physical parameters based on the nonlinear representation of deep networks36,58 and the physical interpretation of network layers63 have been gradually studied. This process contributes to future re- search and development. Based on the polarimetric in- formation fused into the network, other physical proper- ties of light have also been introduced in network train- ing, such as spectrum172 and phase71,75. Finally, the applic- ation fields tend to be semantic tasks based on convolu- tional enhancement or restoration imaging. Initially, im- age processing was the main field applied by data-driven polarimetric imaging, such as descattering imaging46−49,66,70,72,76, denoising45,77,78, demosaicing43,44,68, dynamic range enhancement79, reflection removal53,73,74, low-light imaging42,50, and even 3D reconstruction shape64,65,67,71,80,81. Next, semantic tasks appeared gradu- ally similar to semantic segmentation56,57,69,82,83, camou- flage object detection84, classification60,61, pathological diagnosis62,63,85,86,87. larization parameters of the nonlinear operators are sensitive to noise. Consequently, the effective restora- tion and enhancement of polarization information are crucial for subsequent applications. In this section, we analyze the limitations of polarimetry techniques and re- view the existing restoration and enhancement of accur- ate polarization information methods. Restoration and enhancement of accurate After capturing the images using the aforementioned polarimetric techniques, Stokes vectors were adopted to display the polarization characteristics. The relationship between them can be characterized by the follow equation: Applications of data-driven polarimetric imaging According to the application field, existing data-driven polarimetric imaging methods can be classified into sev- en categories: polarimetric descattering, 3D shape recon- struction, reflection removal, restoration and enhance- ment of polarization information, target detection, bio- medical imaging and pathological diagnosis, and se- mantic segmentation, as shown in Fig. 3. Polarimetry techniques Polarimetry techniques are crucial in obtaining polariza- tion information. Several sub-polarized direction images (0°, 45°, 90°, and 135° or 0°, 60°, and 120°) must be cap- tured to obtain the polarization characteristics based on the Stokes vector model. However, the obtaining and cal- culating process, which acquires polarization informa- tion indirectly, introduces extra imaging noise to sharply reduce the accuracy of the polarization information. To date, there are four typical methods for measuring polar- ization images: division of time/rotating elements, divi- sion of amplitude88−90, division of aperture91,92, and divi- sion of the focal plane93−95. The division of time/rotating elements is the most common method, depending on the time-sequential activity. The polarizers and retarders were rotated, and measurements were made at different positions of the polarimetry elements. Furthermore, the time gap between operations may cause misregistration of polariz- ation images in a dynamic scene or the motion of the camera case. The division of amplitude could capture multiple images simultaneously; however, the inherent drawback is that the intensity of images would decrease to less than a quarter of the intensity of the original sig- nal, which causes the image contrast to decrease sharply and amplify the image noise. Moreover, misregistration must be solved. The aperture captures images simultan- eously using four coaxial cameras with different polariza- tion directions. Distinctly, it is expensive and has a fixed misregistration. The division of the focal plane is the method used in real-time polarimetric imaging, even in dynamic scenes. However, instantaneous field-of-view errors (i.e., mosaicking and low-resolution problems) af- fect the calculation of polarization parameters. Table 1 presents a comparison of the polarimetric elements. polarization information Accurate polarization information is the foundation of imaging and its applications. In the real world, theoretic- al constraints and technological limitations lead to dis- tortion of polarization information. Additionally, the po- 230042-5 230042-5 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 Pathological diagnosis Species classification Spoofing target Camouflaged target Pseudo-color target Target detection Biomedical imaging and pathological diagnosis Data-driven polarimetric imaging Semantic segmentation Polarimetric descattering 3D shape reconstruction Reflection removal Polarization channels Hypers- pectral imaging Remote sensing Medicine Transparent object Scene in wild Underwater descattering Dehazing Remote sensing Indoor object Scene in wild Transparent object Human face MMPD Holographic amplitude & phase Stokes vectors Low-light imaging Restoration and enhancement of polarization information HDR reconstruction MMPD denoising Polarimetric parameters denoising Demosaicing lndoor & outdoor Real & synthetic Fig. 3 | Applications of data-driven polarimetric imaging. Holographic li d & Stokes vectors MMPD denoising Fig. 3 | Applications of data-driven polarimetric imaging. P = √ S2 1 + S2 2 + S2 3 S0 , PL = √ S2 1 + S2 2 S0 , PC = S3 S0 , ϕpol = 1 2arctan (S2 S1 ) , (3) Iφ = (S0 + S1cos2φ + S2sin2φ)/2 , (1) Iφ = (S0 + S1cos2φ + S2sin2φ)/2 , (1) (1) where Iφ is the image with the polarization direction φ. Then, the Stokes vectors can be calculated from the sub- polarization direction images96,97: (3) ϕpol where P, PL, PC are DoP, degree of linear polarization (DoLP), degree of circular polarization (DoCP), respect- ively. is the AoP. S0 = 1 2 (I0 + I45 + I90 + I135) = 2 3 (I0 + I60 + I120) , S1 = I0 −I90 = 4 3 ( I0 −1 2I60 −1 2I120 ) , S2 = I45 −I135 = 2 √ 3 3 (I0 −I120) , S3 = I45, π 2 −I135, π 2 = 2 √ 3 3 ( I0, π 2 −I120, π 2 ) , (2 Furthermore, the Mueller matrix is another common but comprehensive parameter that describes the modula- tion of light after interaction with a material or media98−100. The Mueller matrix contained 16 elements using a 4×4 matrix. https://doi.org/10.29026/oes.2024.230042 https://doi.org/10.29026/oes.2024.230042 Yang K et al. Opto-Electron Sci 3, 230042 (2024) Table 1 | Comparisons of various polarimetric elements. Advantages Disadvantages Division of time ● Robust ● Small system size and inexpensive ● Easiest to implement ● Not suitable for dynamic scenes ● Not simultaneously imaging ● Probable linear misregistration Division of amplitude ● Simultaneous imaging ● Largest system size ● Expensive and complex ● Nonlinear misregistration ● Loss of image intensity ● Highly mechanical flexibility required Division of aperture ● Simultaneous imaging ● Robust ● Expensive ● Loss of field of view ● Fixed misregistration Division of focal plane ● Simultaneous imaging ● No misregistration ● Small and portable ● Loss of spatial resolution ● Mosaicking problem ● Fabrication difficult ● Expensive Table 1 | Comparisons of various polarimetric elements. Advantages Disadvantages Division of time ● Robust ● Small system size and inexpensive ● Easiest to implement ● Not suitable for dynamic scenes ● Not simultaneously imaging ● Probable linear misregistration Division of amplitude ● Simultaneous imaging ● Largest system size ● Expensive and complex ● Nonlinear misregistration ● Loss of image intensity ● Highly mechanical flexibility required Division of aperture ● Simultaneous imaging ● Robust ● Expensive ● Loss of field of view ● Fixed misregistration Division of focal plane ● Simultaneous imaging ● No misregistration ● Small and portable ● Loss of spatial resolution ● Mosaicking problem ● Fabrication difficult ● Expensive the other 15 elements encode the vectorial properties of the object. Furthermore, Mueller matrix polar decom- position (MMPD)101, Mueller matrix transformation (MMT)102, Mueller matrix anisotropy coefficients (MMAC)103, and other decompositions of the Mueller matrix104−106 have been proposed to quantitatively charac- terize the properties of an object. tion parameters amplify errors and negatively affect sub- sequent imaging performance. tion parameters amplify errors and negatively affect sub- sequent imaging performance. In summary, the acquisition device for raw sub-polar- ization images, the use of nonlinear operators to calcu- late polarization characteristic parameters, and special imaging environments also decrease the inaccuracy of the polarization information. Therefore, polarization in- formation with high accuracy must be restored and en- hanced. In this section, we examine photostarvation, mo- saicking, and noising to demonstrate the restoration and enhancement of polarization information, as depicted in Fig. 4. The estimation of these parameters, which represent different polarizations, is always based on nonlinear op- erations, which are unavoidable when introducing er- rors and noise. https://doi.org/10.29026/oes.2024.230042 To achieve the desired performance, the calculated polarization parameters must be refined or re- stored to obtain highly accurate information. In addi- tion, obtaining precise polarization information can be challenging when operating in special imaging environ- ments, such as low light and noise, or with special ima- ging devices. In these scenarios, the disturbed polariza- polarization information The Stokes vector of the output light Sout can be expressed by the Mueller matrix after the in- cident light Sin propagates through the medium as follows: (2) where S0, S1, S2, S3 are the components of Stokes vectors. The parameters [S0, S1, S2] are the most common repres- entations of the linear polarization components. S3 shows the circular polarization component. Therefore, the polarization characteristic parameters can be ob- tained using Eq. (3):     Sout 0 Sout 1 Sout 2 Sout 3    =   m00 m01 m02 m03 m10 m11 m12 m13 m20 m21 m22 m23 m30 m31 m32 m33       Sin 0 Sin 1 Sin 2 Sin 3    , (4) where m00 represents the transformation of intensity and where m00 represents the transformation of intensity and 230042-6 information methods In photostarved environments, imaging always suffers Demosaicing Holographic amplitude & phase Stokes vectors Low-light imaging MMPD images Muller matrix denoising HDR reconstruction Polarimetric parameters denoising Polarization channels L1 S1 S2 S3 25 μm 0° 45° 135° 90° Fig. 4 | The considered tasks of restoration and enhancement of polarization information, including low-light imaging, Muller matrix de- noising, high dynamic range (HDR) reconstruction, polarimetric parameters denoising, demosaicing, transformation between holo- graphic amplitude & phase and polarization channels, transformation between Stokes vectors and MMPD images. 230042-7 D i i Holographic lit d & Fig. 4 | The considered tasks of restoration and enhancement of polarization information, including low-light imaging, Muller matrix de- noising, high dynamic range (HDR) reconstruction, polarimetric parameters denoising, demosaicing, transformation between holo- graphic amplitude & phase and polarization channels, transformation between Stokes vectors and MMPD images. 230042-7 https://doi.org/10.29026/oes.2024.230042 Yang K et al. Opto-Electron Sci 3, 230042 (2024) from a low signal-to-noise ratio, which affects the ima- ging quality, making low-light imaging challenging. In polarimetric imaging, the accuracy of polarization in- formation is degraded. Existing methods consider de- noising, correction of color bias, and exposure time in intensity imaging107,108. However, the difference between polarimetric and conventional intensity imaging has not been fully considered. Based on the power of being data driven, Hu et al. presented a one-to-three (intensity, DoLP, and AoP) hybrid network called IPLNet to simul- taneously enhance the image quality of intensity and po- larization information42, as shown in Fig. 5(a). The en- hanced RGB image generated by the chromatic RGB subnetwork is divided into three channels, and each channel is fed into the polarization subnetwork to pre- dict the polarization information. Enhanced results and corresponding comparisons with mainstream methods are shown in Fig. 5(b) with corresponding values of structural similarity index (SSIM) of each image. However, many parameters are generated in this net- work, which sharply reduces the operational efficiency. The image color is also inaccurate. Therefore, Xu et al. first performed initial denoising and color deviation cor- rection of four polarization orientation images by net- work named ColorPolarNet and used the polarization difference network to enhance intensity details, DoLP, and AoP maps50. The results demonstrate that the pro- posed methods have faster processing speed and better performance regarding signal fidelity, contrast enhance- ment, and color reproduction50,109. information methods Compared with IPL- Net, ColorPolarNet demonstrates slightly superior per- formance and achieves higher peak signal-to-noise ratio (PSNR), SSIM and patch-based contrast quality index (PCQI) in terms of S0, DoLP, and AoP. Additionally, ColorPolarNet achieves notably lower color difference (CD), indicating reduced distortion compared to IPLNet. In processing speed, ColorPolarNet (2.88 s) is more than twice as fast as the IPLNet model (6.10 s). Division of the focal plane polarimeter is one of the most common polarimetric imaging sensors that can in- stantaneously capture dynamic polarization information. Each individual pixel with different polarization orienta- tions is situated in a 2×2 superpixel, recording only one from the four essential intensity measurements; there- fore, demosaicing and reconstruction of full-resolution and accurate polarization information are indispensable. Zhang et al. proposed a convolutional demosaicing net- work called the PDCNN to learn end-to-end mapping between coarse interpolation results and full-resolution polarization images68, which is the first typical demosa- icing architecture, as shown in Fig. 6(a). The bicubic-in- terpolated results are used as the input, which intro- duces an interpolation bias, resulting in inaccurate re- constructed results. After comparison with several main- stream methods, the reconstruction results of DoLP and AoP outperformed those of other methods, as shown in Fig. 6(b) and 6(c) with corresponding values of PSNR of each image110−113. Zeng et al. proposed a four-layer end- to-end fully convolutional neural network that directly learns mapping from DoFP to three polarization proper- ties: intensity, DoLP, and AoP43. However, the noise in the AoP images remained significant. Wu et al. provided R G B Polar-Net RGB-Net S0 DoLP AoP S0 DoLP AoP Detail Residual dense network Residual dense block (RDB) Convolutional layer Concatenate layer 0 0 0.50 1 −π/2 π/2 SSIM=0.66 SSIM=0.24 SSIM=0.15 SSIM=0.86 SSIM=0.62 SSIM=0.40 SSIM=0.71 SSIM=0.61 SSIM=0.36 SSIM=0.91 SSIM=0.71 SSIM=0.44 SSIM=0.95 SSIM=0.76 SSIM=0.48 lPLNet CBM3D SIDNet ColorPolarNet lnput a b Fig. 5 | Enhanced method of polarization information. (a) Architecture of the Hu et al.’s method. (b) Enhanced results and corresponding comparisons with mainstream methods. Figure reproduced with permission from ref.42, Optical Society of America Publishing AG. 230042-8 S0 DoLP AoP Detail 0 0 0.50 1 −π/2 π/2 SSIM=0.66 SSIM=0.24 SSIM=0.15 SSIM=0.86 SSIM=0.62 SSIM=0.40 SSIM=0.71 SSIM=0.61 SSIM=0.36 SSIM=0.91 SSIM=0.71 SSIM=0.44 SSIM=0.95 SSIM=0.76 SSIM=0.48 th d (b) E h d lt d di a Fig. 5 | Enhanced method of polarization information. (a) Architecture of the Hu et al.’s method. information methods (b) Enhanced results and corresponding comparisons with mainstream methods. Figure reproduced with permission from ref.42, Optical Society of America Publishing AG. 230042-8 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 PSNR=37.23 dB PSNR=36.59 dB PSNR=36.95 dB PSNR=42.85 dB Ground truth PSNR=40.36 dB PSNR=25.34 dB PSNR=22.95 dB PSNR=25.27 dB PSNR=30.61 dB Ground truth PSNR=24.31 dB Block Block Block Block Block Block Conv Concatenation Input OutputN Output2 Output1 Output Skip connection S0 S1 S2 Stokes layer a b b1 b2 b3 b4 b5 b6 c1 c2 c3 c4 c5 c6 c Fig. 6 | The convolutional demosaicing network proposed by Zhang et al. and results68. (a) Architecture of the Zhang et al. method. (b) Re- constructed DoLP images of different methods on the test images. (b1) The results of method113. (b2) The results of method110. (b3) The results of method111. (b4) The results of method112. (b5) and (b6) are respectively PDCNN and ground truth. (c) Reconstructed AoP images of different methods on the test images. (c5) and (c6) are respectively PDCNN and ground truth. Figure reproduced with permission from ref.68, Optical Soci- ety of America Publishing AG. Fig. 6 | The convolutional demosaicing network proposed by Zhang et al. and results68. (a) Architecture of the Zhang et al. method. (b) Re- constructed DoLP images of different methods on the test images. (b1) The results of method113. (b2) The results of method110. (b3) The results of method111. (b4) The results of method112. (b5) and (b6) are respectively PDCNN and ground truth. (c) Reconstructed AoP images of different methods on the test images. (c5) and (c6) are respectively PDCNN and ground truth. Figure reproduced with permission from ref.68, Optical Soci- ety of America Publishing AG. quality45. However, all channel-wise features are treated equally, resulting in a lack of flexibility. Inspired by the attention mechanism, Liu et al. proposed an attention- based residual neural network to remove noise and re- store the polarization information of polarimetric im- ages77, as Fig. 7. Therefore, the proposed methods can suppress noise more effectively and restore polarization information more accurately, as shown in Fig. 7(b)109. Additionally, SSIM is used to compare the quality of im- ages obtained by different methods. Focusing on the de- noising of Mueller matrix images, Yang et al. information methods The intensity image describes the reflectivity and transmissivity of the object, whereas the polarization image describes the texture de- tails, material properties, shape, shading, and roughness. These two types of images provide complementary in- formation from different aspects to obtain images with rich physical features and improve the performance of practical tasks. Conventional fusion methods are challen- ging to handle various scenes because a manually de- signed fused factor is adopted. Based on the excellent performance of CNN, Zhang et al. proposed an unsuper- vised deep network called PFNet to fuse the intensity and DoLP images126. The feature extraction module trans- forms the images of S0 and DoLP into high-dimensional nonlinear feature maps using two Dense Blocks, and the concatenation operator is used to fuse the feature maps to reconstruct the fused image using the reconstruction module. The architecture of PFNet is shown in Fig. 8(a). Therefore, the method based on deep learning outper- forms other state-of-the-art methods, as shown in Fig. 8(b)122−125. SSIM is used to compare the quality of images obtained by different methods. Contrarily, they modi- fied the architecture to enhance performance. A Dense Block is used to encode the input images and the fusion subnetwork rather than a concatenation operator to fuse the feature maps127. New loss function strategies are ad- opted, such as the loss between fused and input images and the loss between fused and encoded features. The proposed architecture can also be used for infrared and visible image fusion, and multi-focus image fusion. the camera, a digital camera always captures only a lim- ited fraction of the range, resulting in low-dynamic- range images with over- or underexposed areas that can- not reflect real-world scenes in high-dynamic-range im- ages128,129. Ting et al. studied the relationship between po- larization parameters and the exposure time of a pixel in a polarization image and trained the reconstruction framework to recover a high-dynamic-range image us- ing polarization images79. Other applications of restoration and enhancement are based on data-driven polarimetric imaging. The po- larization parameters and other physical properties are interconvertible. Liu et al. used a deep neural network to transform holographic images reconstructed from a single state of polarization into images equivalent to those captured using a single-shot computational polar- ized light microscope75. Si et al. information methods built a deep residual U-Net that incorporated channel attention with many paired low- and high-SNR Mueller matrix images78. The ground truth is obtained based on the low equally weighted variance (EWV), which can be ex- pressed as: a more physically relevant loss function for the angle of linear polarization (AoLP) reconstruction, establishing a two-stage lightweight approach for reconstructing the intensity and polarization information in real time44. The improved version meets the demand for real-time infer- ence. Wen et al.114, Sargent et al.115, Sun et al.116 and Pis- tellato et al.117 also proposed the data-driven demosa- icing methods to ensure the fidelity of polarization signa- tures and enhance image resolution. Besides deep learn- ing-based methods, other approaches also yield favor- able results in this domain, such as the sparse tensor fac- torization-based model, which introduces a combina- tion of tensor factorization and sparse coding for the first time118. Because the polarimetric parameters are always de- rived from the measured intensities through nonlinear operators, which would amplify the noise for the AoP, removing noise to precisely restore the polarization in- formation is a significant task107,119,120. CNN have distinct advantages regarding extracting image features and hid- den structures; thus, they are suitable for image restora- tion and enhancement in noisy environments. Li et al. employed deep neural networks to significantly suppress the noise in polarimetric images and enhance the image EWV = 220 N ( σ2 + m00 4 ) , (5) (5) σ2 where is the variance of Gaussian noise and N denotes the number of states of polarization. The larger the value of N, the higher is the signal-to-noise ratio. The pro- posed method can effectively resolve the conflict between the measurement accuracy and acquisition time. The fused images contain more information than The fused images contain more information than 230042-9 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 Fd Fd ωFd ω δ σ ˆ ˆ ˆ ω CARDB d−1 Channel attention Channel attention Global residual learning 1 2 d d d−1 d−2 Local residual learning Concatenation (0°,45°,90°,135°) (0°,45°,90°,135°) DoLP DoLP AoP AoP 3D Conv (5×3×3) Reshape 2D Conv (3×3) Concatenation 2D Conv (1×1) Global pooling Dense SSIM=0.60 SSIM=0.12 SSIM=0.05 SSIM=0.89 SSIM=0.77 SSIM=0.16 SSIM=0.97 SSIM=0.81 SSIM=0.22 SSIM=0.92 SSIM=0.84 SSIM=0.25 Ours GT BM3D PDRDN lnput a b S0 Sample A: polyresin statue = Rescale Fig. 7 | Liu et al. method. (a) Architecture of the proposed method. information methods fed Stokes images to a well-designed deep learning network to generate Mueller matrix-based parameter images, such as linear retard- ance and diattenuation parameters58. information methods (b) Enhanced results and corresponding comparisons with mainstream methods. Figure reproduced with permission from ref.77, Optical Society of America Publishing AG. DoLP AoP 3) SSIM=0.60 SSIM=0.12 SSIM=0.05 SSIM=0.89 SSIM=0.77 SSIM=0.16 SSIM=0.97 SSIM=0.81 SSIM=0.22 SSIM=0.92 SSIM=0.84 SSIM=0.25 Ours GT BM3D PDRDN lnput b S0 Sample A: polyresin statue Global residual learning Fig. 7 | Liu et al. method. (a) Architecture of the proposed method. (b) Enhanced results and corresponding comparisons with mainstream methods. Figure reproduced with permission from ref.77, Optical Society of America Publishing AG. single physical properties because the mixed informa- tion describes various characteristics. The fusion of po- larization and intensity is the most common method used in practical applications121−125. The intensity image describes the reflectivity and transmissivity of the object, whereas the polarization image describes the texture de- tails, material properties, shape, shading, and roughness. These two types of images provide complementary in- formation from different aspects to obtain images with rich physical features and improve the performance of practical tasks. Conventional fusion methods are challen- ging to handle various scenes because a manually de- signed fused factor is adopted. Based on the excellent performance of CNN, Zhang et al. proposed an unsuper- vised deep network called PFNet to fuse the intensity and DoLP images126. The feature extraction module trans- forms the images of S0 and DoLP into high-dimensional nonlinear feature maps using two Dense Blocks, and the concatenation operator is used to fuse the feature maps to reconstruct the fused image using the reconstruction module. The architecture of PFNet is shown in Fig. 8(a). Therefore, the method based on deep learning outper- forms other state-of-the-art methods, as shown in Fig. 8(b)122−125. SSIM is used to compare the quality of images obtained by different methods. Contrarily, they modi- fied the architecture to enhance performance. A Dense Block is used to encode the input images and the fusion subnetwork rather than a concatenation operator to fuse the feature maps127. New loss function strategies are ad- opted, such as the loss between fused and input images and the loss between fused and encoded features. The proposed architecture can also be used for infrared and visible image fusion, and multi-focus image fusion. single physical properties because the mixed informa- tion describes various characteristics. The fusion of po- larization and intensity is the most common method used in practical applications121−125. Polarimetric descattering Clear vision in scattering media is critical for various ap- plications such as industrial and civil fields130, traffic sur- veillance systems131, automatic drives132, remote sensing133, rescue operations134, seabed mapping135, mon- itoring of marine species migration and coral reefs, and scene analysis136. However, when capturing images in a scattered environment, the visibility of objects is typic- ally sharply degraded, which is caused by the scattering of suspended particles such as clouds, water, haze, smoke, smog, fog, and mist in the air, soil particles, float- ing excrement of marine animals, algae, and mineral salt in underwater scenes. The backscattered light was mixed Because of the limitations of the response function of 230042-10 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 DoLP Input S0 Dense block Dense block Concatenation Fusion Feature map Feature extraction Conv+ReLU Conv Reconstruction Output Fused image DoLP GF GTF S0 SR MSVD PFNet CVT a b SSIM=0.672 SSIM=0.676 SSIM=0.701 SSIM=0.712 SSIM=0.661 SSIM=0.673 Fig. 8 | The unsupervised deep network PFNet proposed by Zhang et al.126 (a) The architecture of PFNet. (b) Fused results compared with conventional methods. Figure reproduced with permission from ref.126, Optical Society of America Publishing AG. Fig. 8 | The unsupervised deep network PFNet proposed by Zhang et al.126 (a) The architecture of PFNet. (b) Fused res conventional methods. Figure reproduced with permission from ref.126, Optical Society of America Publishing AG. ervised deep network PFNet proposed by Zhang et al.126 (a) The architecture of PFNet. (b) Fused results compared with ds. Figure reproduced with permission from ref.126, Optical Society of America Publishing AG. with the object signal during its propagation towards the camera. Because the physical properties of imaging in haze and underwater environments are similar, we ana- lyzed the dehazing and descattering processes based on deep learning and polarimetric imaging. where z is the distance between the object and the cam- era; β is the degraded coefficient, and Lobject is the origin- al object signal not attenuated by the scattering media along the line of sight. The transmitted process e–βz is the delay of exponential function, which also expressed as t. Second, backscattered light A is an undesired com- ponent that veils the object light to reduce the contrast of the image. The backscattered light can be expressed as: Polarimetric imaging model in scattering media Based on the atmospheric transport model137,138, image I captured by the camera after propagation in scattering media consists of two components: (1) direct transmis- sion signal D, which represents the light that an object reflection is scattered by the media during propagation towards the camera; and (2) backscattered light A, which denotes the light backscattered by the particles in the ob- ject light line of sight without an object signal139. The model can be expressed as: A = A∞ ( 1 −e−βz) , (8) (8) where A∞ is the saturated backscattering light as the dis- tance increases. We aimed to reconstruct the original ob- ject signal Lobject by combining Eq. (6) and (7). Then, Lob- ject can be expressed as: Lobject = I −A 1 −A/A∞ , (9) (9) I⊥ I∥ as a result, the estimation of two unknown components A and A∞ is key to reconstructed Lobject. Schechner et al. proposed a polarization descattering model based on an atmospheric scattering model. The proposed method ob- tains two orthogonally polarized images, and through two orthogonal polarization states of the polar- izer139. This method assumes that the air light is usually I⊥ I∥ as a result, the estimation of two unknown components A and A∞ is key to reconstructed Lobject. Schechner et al. proposed a polarization descattering model based on an atmospheric scattering model. The proposed method ob- tains two orthogonally polarized images, and through two orthogonal polarization states of the polar- izer139. This method assumes that the air light is usually I = D + A . (6) (6) First, as the light reflected from the object propagates towards the camera via the medium, the object radiance suffers from absorption and scattering, yielding a de- graded signal. This process is described as follows: D = Lobjecte−βz , (7) (7) https://doi.org/10.29026/oes.2024.230042 Yang K et al. Opto-Electron Sci 3, 230042 (2024) backscattered light by extrapolation fitting to overcome the first and third assumptions. However, selecting the fitting function when the light was irregular was challen- ging142. Wei et al. considered the difference between the AoP of backscattering light and the object signal, using independent component analysis (ICA) to estimate the object signal with nonuniform polarization characterist- ics to avoid the limitation of assumptions (3) and (4)143. Polarimetric imaging model in scattering media These methods are based on physical models that lack the robustness and effectiveness of a single method in complex scenes because accurate estimation of key para- meters may not be achieved. However, the latent factors influencing image quality have not been explored. partially polarized, whereas the object is not polarized. Thus, the captured images can be described by: Ii = D 2 + Ai, i ∈[⊥, ∥] . (10) (10) Because the object is not polarized, the DoP of the im- ages is equal to that of the backscattered light. Further- more, the backscattered light is estimated using the po- larization model: ˆA = IP PA , (11) (11) where PA and P are the DoPs of the backscattered light and captured images, respectively. The saturated backs- cattering light A∞ is the mean of the background region selected in the image where there is no object and the PA is calculated by the selected region which is the DoP of total backscattering light. Finally, the reconstructed Lobject can be expressed as: Deep learning methods based on CNN are adept at ex- tracting hidden features and fitting the nonlinear rela- tionships between backscattering light and object signals. Thus, it is a promising choice for descattering and dehaz- ing. Polarimetric data-driven descattering methods com- bine polarization information and deep learning. Moreover, the existing model can guide the training of the descattering network to combine its advantages. Three different pipelines were used in the data-driven polarization descattering method. First, the end-to-end architecture without the physical model, which always uses the polarization images to feed into the network. Second, the physical-model-guided network methods, which are guided by the existing or proposed but non- participation in network training; Third, the physical- model-integrated network methods, which integrate the physical model into the network to train the descattering network together. The following section provides a de- tailed explanation of these three perspectives. Lobject = I −A 1 −A/A∞ = I (1 −P) 1 −IP/A∞PA . (12) (12) A polarimetric imaging model is a physical, low-cost, and effective method for restoring clear images in a scat- tering environment. However, the method is based on the following assumptions: 1) The backscattered light at infinity is uniform, but in the real world, clouds, the solar radiation angle, and oth- er factors may influence the distribution. 2) The DoP of the object is not polarized, but it does not make sense. Polarimetric imaging model in scattering media 3) The DoP of the backscattered light is constant; however, it must be spatially variable. 4) The polarized direction of the image is equal to that of the backscattered light and the object signal, but there are possible differences. End-to-end descattering network 5) The degraded coefficients of backscattering light and object signal are similar; however, Akkaynak et al.’s study has proved that attenuation coefficient of object signal depends on the distance z, reflectance ρ, spectrum of ambient light E, spectral response of the camera Sc, and beam attenuation coefficients of the water body βb; however, the backscattering light is related to E, Sc, βb and scattering of the water body b84,140,141. The end-to-end architecture is a common structure that enhances image quality and relies on higher-order non- linear representations. The descattering process of a net- work is the fitting of the descattering transmission of a high-order function, which has been successfully applied to complete scattering removal using intensity informa- tion136,144−146. In recent studies, the introduction of polar- ization information has proven that the input of polariz- ation images into the network can improve the quality of qualitative and quantitative evaluations. This section dis- cusses the polarization end-to-end descattering network. Several improved methods focusing on this insuffi- ciency have been proposed. For example, Huang et al. es- timated the polarization difference image (PDI) of an ob- ject signal using feasible region fitting to overcome the limitations of the second assumption. Hu et al. estim- ated the spatial distributions of the DoP of an object and In 2020, Hu et al. first employed a deep learning tech- nique in polarimetric underwater imaging46, as shown in Fig. 9(a), which is a typical end-to-end descattering 12 230042-12 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 Zhang et al., which contained four pairs of networks consisting of polarized and gray versions. Furthermore, this method assumes that the intensity and polarization images are information streams, two types of informa- tion flows in their respective networks, and join together at the end of the model. Furthermore, the addition of po- larization information to gray information and feeding these two parts into fused networks, called DENSE-U- NET BLOCK, at the forefront of the network could achieve better results than the front or end of the net- work70. The experiments with different turbidity levels performed better than the other methods and demon- strated the excellent robustness of the proposed method. architecture. End-to-end descattering network The three dimensional inputs of different linear polarization orientations with 0°, 45°, 90° are fed into an end-to-end polarimetric dense network (PDN) rather than intensity images, which contains three main com- ponents: Shallow Feature Extraction (SFE) used to ex- tract shallow polarization features, Residual Dense Block (RDB) as the basic structure to connect with each other, and concatenated by Dense Feature Fusion (DFF). The third crucial component was utilized to fuse all the fea- tures and output the descattering results. A dataset con- taining abundant polarization image pairs was built using a commercial DoFP camera, and a water tank filled with milk was used to capture turbid and clear object signals. The same network structure, based only on intensity im- ages, was trained to verify the significance of the polariz- ation information. Compared with the intensity network and existing methods, the polarization network demon- strated higher values of image contrast (IC), measure of enhancement (EME), PSNR, and SSIM, which indicate higher image quality6,147, as shown in Fig. 9(b). In remote sensing, the scattering medium signific- antly impacts on the results of object reconstruction, even producing speckle patterns. Obtaining the corres- pondence between the original object and the imaging process is challenging and crucial. Li et al. combined an object’s polarization information with a modified U-net- based deep learning network (MU-DLN) to retrieve the original object’s information influenced by the scattering Another end-to-end architecture was proposed by b1 b2 b3 b4 b5 a b S0 90° 0° 45° Polarimetric-Net 3D input d−1th d−1th 1th dth dth nth Residual learning SFE RDB RDB DFF Recovered image (Output) Single input Intensity-Net Conv. layer ReLU Concat. layer Raw Polarimetric-Net He Liang Ground truth EME=9.10 SSIM=0.77 EME=8.07 SSIM=0.66 EME=4.86 SSIM=0.36 EME=5.17 SSIM=0.39 EME=5.63 SSIM=0.31 EME=6.81 SSIM=0.31 Fig. 9 | The flow chart and results of the Hu et al. method, which is a typical end-to-end descattering architecture46. (a) The architecture of proposed method. (b) Comparison of the enhanced images by different methods. Figure reproduced with permission from ref.46, Elsevier BV. 230042-13 d−1th dth Residual learning d−1th 1th dth nth g SFE RDB DFF Recovered image (Output) Recovered image Recovered image EME=5.17 SSIM=0.39 EME=5.17 SSIM=0.39 EME=6.81 SSIM=0.31 EME=8.07 SSIM=0.66 EME=8.07 SSIM=0.66 Fig. 9 | The flow chart and results of the Hu et al. method, which is a typical end-to-end descattering architecture46. (a) The architecture of proposed method. Physical-model-integrated descattering network The physical-model-integrated descattering network in- tegrates the descattering model into the network as the backbone to guide the descattering process, which intro- duces constraints compared to the physical-model- guided descattering network. Therefore, the main task of the network is to generate or refine specific parameters before they are utilized to generate improved results. Furthermore, the physical model and its inverse process can form a self-supervised closed loop to achieve im- proved performance. Guided by a physical imaging model, Ren et al. trained a lightweight dehazing CNN to rapidly process turbid images, comparing it with conventional dehazing meth- ods and introducing additional circular polarization in- formation148. Furthermore, this is the first time circular polarization information has been fed into a network. Two unknown parameters in the polarimetric descatter- ing model resulted in an underdetermined function by directly generating these parameters. Therefore, the pro- posed method combines these two parameters into a single formula to avoid an underdetermination problem and minimize the reconstruction error. The new para- meter K is expressed as:149 To further combine the physical formation model with deep learning methods, some researchers have em- bedded existing dehazing approaches into the proposed pipeline. Zhou et al. proposed a robust polarization- based dehazing architecture with a generalized physical formation model that requires no specific clues to estim- ate the required physical parameters or handcrafted pri- ors48. Figure 10(a1) and 10(a2) show the network archi- tecture and corresponding example with evaluation in- dex(PSNR and multi-scale SSIM). The transmitted light D (T in Fig. 10(a1)) and original scene radiance Lobject (R in Fig. 10(a2)) can be calculated using the following equations: K = (I −A∞)/t + A∞−1 I −1 . (13) (13) Lobject =KI −K + 1 The Eq. (12) can be rewritten as an , after which the descattering process is viable once the parameter K is obtained. Tests were conducted in differ- ent turbid environments to verify the feasibility of the proposed method, and the results indicated the effective- ness and high efficiency of the lightweight architecture. Subsequently, Ding et al. adopted a multi-polarization fusion adversarial generative network to enhance turbid images47. End-to-end descattering network (b) Comparison of the enhanced images by different methods. Figure reproduced with permission from ref.46, Elsevier BV. 230042-13 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 ˆA = I0 −I[1 −P]/2 PAcos2ϕA , (14) medium66. Data were acquired using a Monte Carlo sim- ulation system and deep learning technology to learn a physical model of the scattering process. The experi- mental results show that the object’s information for the Q-component can be reconstructed very well because of the suppression of scattering light and highlighting of ballistic light. Several fixed optical thickness environ- ments are tested to reflect the superiority of the trained MU-DLN. (14) ϕA where the is AoP of backscattering light computed by the selected background region. They built the first color polarization image datasets in the natural underwater en- vironment, which selected the visually better enhanced results among the results produced by several conven- tional methods150−154. Compared with the underwater res- ults by four supervised data-driven polarimetric meth- ods mentioned above, the experimental results in labor- atory simulated by the milk have huge improvement; however, the natural results are improved to a lesser ex- tent because the complicated environment would in- crease the diversities of known or hidden parameters and then result in the networks become more generalized with higher robustness but lower performance in a spe- cific example. ϕA where the is AoP of backscattering light computed by the selected background region. They built the first color polarization image datasets in the natural underwater en- vironment, which selected the visually better enhanced results among the results produced by several conven- tional methods150−154. Compared with the underwater res- ults by four supervised data-driven polarimetric meth- ods mentioned above, the experimental results in labor- atory simulated by the milk have huge improvement; however, the natural results are improved to a lesser ex- tent because the complicated environment would in- crease the diversities of known or hidden parameters and then result in the networks become more generalized with higher robustness but lower performance in a spe- cific example. Physical-model-guided descattering network The physical-model-guided descattering network is trained to remove the scattering effect guided by the physical model but does not participate in network train- ing. Based on the physical model, the theoretical feasibil- ity was proven before guiding the design of the network architecture. Therefore, the middle stage of combining the physical priors and models is instructive for further fusing the physical model into the design of the network pipeline. Physical-model-integrated descattering network Compared with the conventional model, the proposed method introduces an angle of polarization to calculate the backscattered light, expressed as: D = PI −IPA PT −PA and Lobject = DA∞ A∞−(I −D) , (15) (15) where PT and PA define the DoP of the transmitted light and backscattered light, respectively, which are estim- ated by the subnetworks. The symbols I and P are the 230042-14 230042-14 230042-14 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 g2 Estimated PA and PT b1 a1 g1 g3 g4 Refined T Refined R Calculated R Calculated T Estimated infinite airlight Precomputed haze image and DoP PSDNet-T2 PSDNet-T1 PSDNet-A PSDNet-L Estimated target radiation Orthogonal scene image Fused transmission map Estimated scene airlight Estimated transmission map I⊥ Composite haze image I|| I⊥ I|| b2 a2 Polarization sub-images Fig. 10 | The methods combining the physical formation model with deep learning methods and corresponding examples. (a1) Zhou et al. method and (a2) the corresponding example. (b1) Shi et al. method and (b2) the corresponding examples. g2 Estimated PA and PT a1 g1 g3 g4 Refined T Refined R Calculated R Calculated T Estimated infinite airlight Precomputed haze image and DoP a2 Polarization sub-images a2 Fig. 10 | The methods combining the physical formation model with deep learning methods and corresponding examples. (a1) Zhou et al. method and (a2) the corresponding example. (b1) Shi et al. method and (b2) the corresponding examples. corresponding results, with assessment criteria(IC and entropy-based no-reference image quality assessment (ENIQA)) which consist of three subnetworks to com- pute the object radiation, transmitted light, and backs- cattered light. The pipeline processed a self-supervised closed loop to optimize the network. The end-to-end descattering network is part of the total pipeline, which effectively reduces the scale of the network and enhances performance. Additionally, a secondary product with an accurate transmission map was produced, which may be helpful for other computer vision tasks, such as 3D re- construction. Several experiments demonstrated that the proposed architecture can effectively improve the visibil- ity of object details and is highly robust for the scene. haze image and its DoP, respectively, which can be calcu- lated from the polarized images. The DoPs of the object signal and scattered light can be generated by subnet- work g1 before estimating the transmitted light using the imaging model. The refined light transmitted by subnet- work g2 is utilized to calculate the original scene radi- ance, in which the saturated backscattered light is ob- tained by subnetwork g3. Finally, the refined subnet- work g4 was adopted to generate the refined results. The raw polarization direction images join each subnetwork. The generation of the synthetic dataset was instruct- ive. Clear images with depth and semantic segmentation maps must be provided for the generation process. 230042-14 Specificly, α is a com- pensation factor for the water absorption is set to 0.99 as the subject was placed in a comparably shallow position with pure water in the underwater environment, where the influence on the absorption of natural light could be ignored. η is a bias factor and ps° is degree of the scatter- ing light, where the range of η is from 1 to 1/ ps°. The measurement value of ps° is set to 0.8333 and η=1.13 to meet the range of 1 ≤ η ≤ ps°. S∞ is the scattered light ra- diation at an infinite distance estimated by the intensit- ies of the brightest region of the no object. Lobject = I −(A/ε) 1 −(A/εA∞) , (17) (17) where the ε is bias factor. Consequently, the object radi- ance was optimized and recovered based on the under- water image recovery process. The proposed method has a much lower cost for preparing the training datasets and demonstrates the capability of recovering underwater images under different nonuniform optical fields. The flow chart, proposed architecture of the Yang et al. meth- od76, corresponding results, and comparisons with exist- ing methods142,147,164,165 are shown in Fig. 12. The proposed methods not only overcome the acquisi- tion of polarization characteristics of the environment and object in the conventional process but also minim- ize the dependency on datasets even when training on only one image. Moreover, the mismatch between the model and a real scene lacking environmental priors is significantly reduced. Figure 11(a) and 11(b)49 show the proposed architecture and a visual comparison among the different descattering methods147,160−163 together with statistical index of image contrast. The method repres- ents a pioneering attempt in the realm of unsupervised descattering imaging. However, its capacity for enhan- cing imaging outcomes remains somewhat limited. Data-driven polarization descattering methods have been gradually introduced into physical models to guide network training, which resolves the limitations of tradi- tional methods. Data-driven methods learn more com- prehensive features and adjust them for more complex media. Furthermore, it improves the training perform- ance and provides another training process for a self-supervised closed loop to optimize the network. 230042-14 The Foggy Cityscapes-DBF dataset was eligible, and reason- able values of the corresponding parameters were set to generate the synthetic dataset155−157. Gaussian noise was introduced to make them spatially variant and conform to real-world scattering conditions to improve the ro- bustness of the network158,159. R(·) Because it is hard to capture the ground truth corres- ponding to the object in the underwater environment, unsupervised learning polarimetric underwater methods were proposed. Zhu et al. synergistically make use of an untrained network and polarimetric imaging formation model to recover images from scattering in underwater scenarios without requiring additional datasets49. There are two stages during the network training. First, the raw input images are input to the network to generate Contrarily, Shi et al. processed a polarization-based self-supervised dehazing network called PSDNet to elim- inate the influence of haze on images72. Figure 10(b1) and 10(b2) show the proposed network architecture and 230042-15 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 Yang et al. trained a network to inpaint backscattered light with different polarization orientations, which was used to calculate the DoP and AoP of the background light76. Furthermore, this is another unsupervised meth- od that does not require clear ground truth. The primary task of the proposed method is to calculate the complete backscattered light. After removing the region of the ob- ject in the captured polarized images using the GrabCut algorithm, the incomplete image, randomly erasing a re- gion, is input to the network to generate a mission re- gion before using a filtering method that compares the gray value of one pixel with those in other nearby pixels and replacing the singular point with the averaged value in a 7×7 square. Furthermore, a clear image is calculated using the following modified function: M (·) the optimized image. Furthermore, using the imaging formation model to recalculate the degraded im- age. After the circular process, the loss of raw image and calculated image is adopted to optimize the network: [R (·; w∗) , Θ∗] = argmin w,Θ ∥M{R(I(x, y); w), Θ} −I(x, y)∥2 2 , (16) [R (·; w∗) , Θ∗] = (16) where w is the weights, Θ is parameters of image forma- tion model including {α, η, ps°, S∞}. These parameters are estimated using a neural network, where the initial value is selected by traditional methods. Yang K et al. Opto-Electron Sci 3, 230042 (2024) Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 lmage acquisition and preprocessing Collected polarization images I (x, y) Recovery and optimization of L (x, y) Optimization and recovery of L (x, y) by Eq. (10) Calculate EME of L (x, y) Get incomplete A∞ (x, y) (Remove the target object in I (x, y)) Obtain B (x, y) from Eq. (6) Estimation of A∞ (x, y) and B (x, y) Use unsupervised learning to obtain the complete A∞ (x, y) Calculate stokes parameters of A∞ (x, y) and filter them Estimate DOP of B from DOP of A∞ (x, y) Estimate the intensity of the polarized part of B (x, y) from DoP of I (x, y) 128×128×1 128×128×1 4×4×512 4×4×512 4×4×512 4×4 Trans 4×4 Conv 4×4 Conv 1×1 Conv 1×1×4096 128×128×1 Conv ReLU ReLU ReLU ReLU Upsample block ×5 Upsample block: Feature extraction block×5 Feature extraction block: Feature extraction block×5 Generative network Discriminative network 3×3 same Conv Max-pooling ReLU 4×4 s=2 p=1 TransConv ReLU c1 c2 c3 c4 EME=0.4007 EME=0.3994 EME=0.3829 EME=1.3175 EME=1.2754 EME=1.3378 a b d real or fake Fig. 12 | Unsupervised underwater descattering methods and corresponding results. For the equations and variables, see the ref.76. (a) The flow chart. (b) Proposed architecture of Yang et al. method. (c) The results of generated backscattering light; (c1) the captured polarized im- age; (c2) the generated backscattering image of proposed trained network; (c3) the backscattering image after smooth filtering; (c4) the corres- ponding ground truth. (d) Final descattering results and comparisons with other methods. Figure reproduced with permission from ref.76, Optical Society of America Publishing AG. g p ( ) p g lmage acquisition and preprocessing Collected polarization images I (x, y) Recovery and optimization of L (x, y) Optimization and recovery of L (x, y) by Eq. (10) Calculate EME of L (x, y) Get incomplete A∞ (x, y) (Remove the target object in I (x, y)) Obtain B (x, y) from Eq. (6) Estimation of A∞ (x, y) and B (x, y) Use unsupervised learning to obtain the complete A∞ (x, y) Calculate stokes parameters of A∞ (x, y) and filter them Estimate DOP of B from DOP of A∞ (x, y) Estimate the intensity of the polarized part of B (x, y) from DoP of I (x, y) a c2 c1 c3 c4 c4 c3 Fig. Yang K et al. Opto-Electron Sci 3, 230042 (2024) 12 | Unsupervised underwater descattering methods and corresponding results. For the equations and variables, see the ref.76. (a) The flow chart. (b) Proposed architecture of Yang et al. method. (c) The results of generated backscattering light; (c1) the captured polarized im- age; (c2) the generated backscattering image of proposed trained network; (c3) the backscattering image after smooth filtering; (c4) the corres- ponding ground truth. (d) Final descattering results and comparisons with other methods. Figure reproduced with permission from ref.76, Optical Society of America Publishing AG. Additionally, the physical model in the network may generate extra parameters that are probably helpful for other tasks, such as depth maps, backgrounds, and inher- ent coefficients of media. The acquisition of dataset is key for network training in scattering media. The syn- thetic and generated methods may solve the problem despite remaining crucial and challenging in the future. Additionally, the physical model in the network may generate extra parameters that are probably helpful for other tasks, such as depth maps, backgrounds, and inher- ent coefficients of media. The acquisition of dataset is key for network training in scattering media. The syn- thetic and generated methods may solve the problem despite remaining crucial and challenging in the future. the DoP, AoP, and micro-surface zenith and azimuth. An intrinsic drawback of deriving a shape from polariza- tion is the ambiguous estimation of surface orientation. The suitable arctangent function in the model results in a multivalued azimuth, commonly known as azimuth am- biguity. Cues from various aspects, such as geometry168−170, spectrum171,172, photometry173−176. However, relying only on a physical-based imaging mod- el, recovering the shape with high accuracy remains chal- lenging under nonlaboratory conditions. The excellent nonlinear representation ability of deep neural networks can narrow the gap between ideal and real-world condi- tions. This section reviews the existing 3D shapes from polarization methods combined with deep learning (DL). 230042-14 90° 0° 45° Input (I0°, I45°,I90°) Generated measurement Loss θ* Underwater IFM M (·, Θ) Network (R (·; w)) w w, θ w* arg min||M {R (I (x, y); w),Θ)−I (x, y)||2 2 Input IFM Ours MIP HE RGHS DCP GC RoWS a b Contrast=0.257 Contrast=0.273 Contrast=0.354 Contrast=0.279 Contrast=0.286 Contrast=0.335 Contrast=0.263 Contrast=0.216 Contrast=0.252 Fig. 11 | Unsupervised underwater descattering method. (a) Architecture of Zhu et al. method49 and (b) visual comparison among different de-scattering methods. Figure reproduced with permission from ref.49, Optical Society of America Publishing AG. 230042-16 MIP Contrast=0.279 HE RoWS Contrast=0.286 Contrast=0.252 HE Contrast=0.286 Ours Contrast=0.354 IFM Contrast=0.273 Generated measurement HE Contrast=0.286 MIP Contrast=0.279 Ours Contrast=0.354 MIP GC C 0 9 Contrast=0.216 HE HE RoWS Contrast=0.252 RoWS Contrast=0.252 RoWS Fig. 11 | Unsupervised underwater descattering method. (a) Architecture of Zhu et al. method49 and (b) visual comparison among different de-scattering methods. Figure reproduced with permission from ref.49, Optical Society of America Publishing AG. 230042-16 Principles of polarization 3D shape reconstruction where n denotes the refractive index, θ refers to the zenith angle, assuming that ηi =1 because in most condi- tions light is incident from air. The refractive index of specular surface is denoted ηt = n. Because the azimuth angle is perpendicular to the phase of the specular polar- ization178, leading to the π/2 shift of azimuth angle. This is another ambiguity problem regarding shape owing to polarization. The surface shape changed the polarization states of the incident illumination, providing the possibility of recov- ering the shape from polarization. In polarization detec- tion, polarization information can be obtained using a camera and a rotated linear polarizer mounted in front of it or a camera with a pixelated polarizer. The captured image intensity varied sinusoidally. ˆn = n (1 + iκ) Material object reconstruction is more complicated than that of regular specular surfaces. The refraction in- dex of metal is a complex number defined as , where κ is the attenuation coefficient. And then Eq. (20) can be derived as: I ( ϕpol ) = Imax + Imin 2 + Imax −Imin 2 cos ( 2ϕpol −2ϕ ) , (18) where ϕpol denotes the angle of polarizer axis relative to a chosen reference orientation, ϕ denotes the azimuth angle of micro-surface of an object, Imax and Imin refer to the observed maximum and minimum intensity during ϕpol from 0 to π. A whole 2π period of sinusoidal func- tion results in a π ambiguity. For instance, if ϕ=ϕpol, the maximum intensity is obtained. However, the minimum intensity corresponds to two azimuth angles, i.e. ϕ±π/2. The π-ambiguity problem is the one of shape from polar- ization. Psm = 2ntanθsinθ tan2θsin2θ + |ˆn| 2 . (21) (21) Diffuse reflection originates from the light refracted by the shallow surface of an object, in which it is partially polarized owing to the irregular interactions between the light and the interior particles. Therefore, the DoP of dif- fuse reflection is determined by the Fresnel transmission coefficients. The relationship between the DoP and the Fresnel coefficients of diffuse reflection is defined as: The polarization state of the reflected light directly de- pends on the reflection type occurring over the surface, which is primarily specular or diffuse reflection, as shown in Fig. 13. Pd = T∥−T⊥ T∥+ T⊥ = R⊥−R∥ 2 −R⊥−R∥ . (22) (22) Under these conditions, light is refracted to the air in the object. Three-dimensional shape reconstruction By analyzing the interactions between light and surface geometry, we can reconstruct the 3D shapes of objects166,167, where polarization is crucial. Natural illu- mination becomes partially polarized after reflection from an object’s surface. Polarized reflection implies shape information because the Fresnel equations relate 230042-17 https://doi.org/10.29026/oes.2024.230042 Yang K et al. Opto-Electron Sci 3, 230042 (2024) Yang K et al. Opto-Electron Sci 3, 230042 (2024) Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 ing conditions using only polarization images. n (u) = [nx (u) , ny (u) , nz (u)] T In addition to the ambiguity problem, other limita- tions to the shape owing to polarization exist. For ex- ample, estimating the zenith angle requires an unknown prior for the refractive index, which limits the recon- struction of complex objects and natural scenes. Second, when the zenith angle is close to zero, the influence of noise increases because the DoP is small. Third, mixed reflections were common in real-world scenarios. Moreover, achieving satisfactory reconstruction results in complex scenarios by using the linear superposition of a single physical model is challenging. Finally, the dis- continuous depth is also a significant challenge for re- covering the shape from the derived surface normal by integration. Consequently, the introduction of other in- formation is essential to avoid the problems mentioned above and expand the application fields. = [sinϕ (u) sinθ (u) , cosϕ (u) sinθ (u) , cosθ (u)] T , (24) where nx(u), ny(u), nz(u) are the normal vectors of ele- ment surface u. The normal vector can be expressed us- ing the surface gradient, as shown in Eq. (25) n (u) = 1 √ p(u) 2 + q(u) 2 + 1 [ −p(u) 2 −q(u) 2 1 ]T , (25) p (u) = ∂zx (u) q (u) = ∂zy (u) ∇z (u) = [ p (u) q (u) ]T where , , i.e., . Finally, the surface shape z(u) was reconstructed. (25) The shape of the polarization differs according to the type of reflection. Compared to specular reflection, the DoP and zenith angles for diffuse reflection are one-to- one maps. However, diffuse reflection makes the recon- struction process challenging because of the lower signal- to-noise ratio and higher dependence of the DoP on the refractive index. For specular reflection, the mirror-like surface maintains a relatively uniform direction and phase, thus avoiding the influence of random noise. However, specular reflection leads to more ambiguous problems than diffuse reflection does. Moreover, it is challenging to find an object when the viewing direction exceeds the reflection direction. Table 2 compares the advantages and disadvantages of the shape from polariz- ation based on both specular and diffuse reflections. Yang K et al. Opto-Electron Sci 3, 230042 (2024) Typical methods include a combination of heuristic priors, such as the boundary and convexity of objects180, shading181, and photometric stereo64, but noise is a major limitation. This complicated calculation amplifies the noise, leading to a degraded texture or profile in the re- covered shapes. Data-driven imaging is powerful for 3D imaging with a nonlinear modeling ability. Furthermore, this primarily depends on the semantic information of the image. Under the guidance of the physical model, this brings new possibilities for shapes from polarization. The shape of the polarization depends on the estim- ated azimuth and zenith angles. Ambiguity is a critical challenge. Regarding the azimuth angle, two phase angles with a π shift are derived from the period of the sinusoid function. For specular reflection, the azimuth angle would be retrieved with ± π/2 operation. However, the two zenith angles of the ambiguous solution are determ- ined for a given DoP in the specular reflection, which cannot be excluded without other information. These contributing factors result in high error rates and limita- tions in the generalization to mixed materials and light- Principles of polarization 3D shape reconstruction Therefore ηt =1, i.e. the refraction index of air, and ηi =n, i.e., the refraction index of diffuse surface. Eq. (23) can then be derived as: The Fresnel equations describe how incident light changes when propagating in media with different re- fractive indices. When specular reflection dominates, the DoP of the specular reflection is calculated using the Fresnel reflection coefficients: Pd = ( n −1 n )2 sin2θ 2 + 2n2 − ( n + 1 n )2 sin2θ + 4cosθ √ n2 −sin2θ . (23) Ps = R⊥−R∥ R⊥+ R∥ . (19) (19) Combined with the Fresnel function, it has the expres- sion in Eq.(20)177: (23) ϕ Given the azimuth angle and zenith angles θ, the normal vector of shape surface at any point could be ex- pressed as179: Ps = 2sin2θcosθ √ n2 −sin2θ n2 −sin2θ −n2sin2θ + 2sin4θ , (20) (20) lncident light Object Surface normal a b Object Microfacet Air Air Partially polarized light Incident light Surface normal Reflected light Reflected light light Subsurface scattering Unpolarized Fig. 13 | Polarization of specular reflection and diffuse reflection. (a) Specular reflection. (b) Diffuse reflection. 230042-18 lncident light Object Surface normal b Air Reflected light Subsurface scattering Surface normal Surface normal lncident light Reflected light Reflected light Fig. 13 | Polarization of specular reflection and diffuse reflection. (a) Specular reflection. (b) Diffuse reflection. 230042-18 Data-driven shape from polarization in single reflection In certain situations, the reflection can be purely specu- lar or diffuse. For instance, in human face recognition or clothed body reconstruction tasks, the skin, clothes, and other human tissues are diffuse surfaces, and specular re- flection is negligible. In transparent object reconstruc- tions, specular reflection dominates. For 3D clothed human shape reconstruction with clothing details, Zou et al. introduced polarization im- ages and two ambiguous normal maps into the designed Table 2 | Comparisons of specular reflection and diffuse reflection. Advantages Disadvantages Specular reflection ● Higher signal-to-noise ratio ● Lesser influence of the refraction index ● Suffer from more ambiguity problem ● Lower viewing angle ● Lesser application fields Diffuse reflection ● Monotony of degree of polarization about refraction index ● Wide viewing angle ● More application fields even for scene in wild ● More influence of noise ● Worse visual effects ● Stronger influence of refraction index 230042-19 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 human face reconstruction, Han et al. proposed a learn- ing-based method for passive 3D face reconstruction from polarization183, as shown in Fig. 15(a). Further- more, it derives the ambiguous normal of each microfa- cet over the face at the pixel level based on the polariza- tion of the diffuse reflection. The CNN-based 3D morph- able model (3DMM) generates a rough depth map of the face based on a directly captured polarization image, and is used to amend the ambiguous polarization normal and further reconstruct an accurate 3D face using Frankot–Chellappa 3D surface restoration functions. Figure 15(b) illustrates the final results, including a male face under indoor lighting, a male face under natural outdoor illumination, and an indoor plaster statue. The 3D rendering features fit well with the original appear- ance, and the lighting conditions had little influence. The experiments also demonstrate the benefits of introdu- cing deep learning into 3D polarization reconstruction. network65, as shown in Fig. 14. Specular reflection was omitted because of the rough surface of the clothing. Owing to the azimuth ambiguity problem, two possible maps were resolved and input into the network as phys- ical priors. The two ambiguous normal maps, n1 and n2 are classified into three categories: n1, n2, and back- ground. Each pixel point was classified as belonging to one of these three categories and then merged into n3 us- ing Eq. (26) with probabilities p0, p1 and p2. Data-driven shape from polarization in single reflection 230042-20 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 each segmented area. Moreover, this problem can also be solved by reconstructing each area separately; however, the segmentation algorithm and stitching of the 3D shape of an object are huge challenges. Neural networks provide solutions to fuse explainable or inexplicable fea- tures with mixed reflections, relying on their excellent nonlinear representation ability. network to reconstruct 3D shape-transparent objects from specular reflections81. However, transmitted light is often a diffuse reflection. Therefore, separating the trans- mitted light is critical. The AoLP features indicate stronger background noise in areas with higher transmit- tance. The closer it is to the center area, the stronger the noise, as shown in Fig. 16. The physical prior confidence concept is based on intrinsic faults in the AoLP maps of transparent objects. The first method combines deep learning and polariz- ation-reconstructed models; Ba et al. fed polarization im- ages and ambiguous normal maps into the network and trained the network to learn the effective inputs from training data automatically64. The inputs were four im- ages captured with a polarizer, and the ambiguous nor- mal maps consisted of one diffuse and two specular am- biguous maps. The proposed method achieved the low- est test error on the tested data under the three lighting conditions compared with conventional methods. ConfidenceK,m (i, j) = ∑ p∈Pi,j |p −¯pi,j| m max 0⩽x⩽W 0⩽y⩽H ∑ p∈Px,y |p −¯px,y| m , (27) (27) ¯px,y where pi,j represents the pixel values in the K×K neigh- borhood of the point (x, y), is the mean of the pixel values in this area, m is the smoothing exponential term. H and W denote the height and width of the map, re- spectively. This physics-based prior confidence map is then input into the network as an attention map to guide the fusion of the original-polar (DoLP and AoLP maps) and physics-based prior (four ambiguous maps). The proposed method achieves optimal performance and provides a new perspective for further transparent shapes from polarization research. Based on a polarimetric Bidirectional Reflectance Dis- tribution Function (pBRDF) model and real polarization scene rendering, Kondo et al. applied rendered polarized images to train a network for an accurate surface normal estimation71. A physics-based renderer was built to simu- late the polarization behavior of the rays based on the proposed pBRDF model for each material. Data-driven shape from polarization in single reflection Furthermore, it can correctly reproduce the polarization property, in- cluding the inter-reflection effect, in real-life scenes. Therefore, the synthetic-colored image and simulated polarization information, such as the phase and DoP, were fed into the CNN to estimate the surface normal. The detailed process and reconstruction results are shown in Fig. 17(a). Data-driven shape from polarization in single reflection n3 = (1 −p0) · p1n1 + p2n2 ∥p1n1 + p2n2∥2 . (26) (26) The final surface-normal prediction is refined using a denoising network. The smoothed normal concatenates the fused normal and raw polarization direction images as the input to accurately estimate the surface normal. Subsequently, the skinned multi-person linear (SMPL) representation182 and deformation stage were used to re- construct the refined 3D human shape with clothing de- tails rather than naked. The transparent objects exhibited typical specular re- flections. Shao et al. proposed a multibranch fusion Regarding diffuse-reflection-dominated cases, such as Ambiguous normal Classify net Fused normal Single polarization images Predicted normal Deformation ResNet 50 Joints Detailed body shape Fig. 14 | The proposed architecture of the Zou et al. method. Ambiguous normal Classify net Fused normal Single polarization images Predicted normal Deformation ResNet 50 Joints Detailed body shape Fig. 14 | The proposed architecture of the Zou et al. method. Improved 3DMM net 3D depth map Polarization- based 3D model Dimensional transformation Rough depth map High-precision 3D face a b I Imax Imin Fig. 15 | Han et al. passive 3D polarization face reconstruction method. (a) Overall schematic of the proposed method. (b) 3D polarization face reconstruction results. Figure reproduced with permission from ref.183, Multidisciplinary Digital Publishing Institute. 230042-20 Single polarization images Joints Joints Detailed body shape Detailed body shape Deformation Deformation Fig. 14 | The proposed architecture of the Zou et al. method. Improved 3DMM net 3D depth map Polarization- based 3D model Dimensional transformation Rough depth map High-precision 3D face a b I Imax Imin Fig. 15 | Han et al. passive 3D polarization face reconstruction method. (a) Overall schematic of the proposed method. (b) 3D polarization face reconstruction results. Figure reproduced with permission from ref.183, Multidisciplinary Digital Publishing Institute. 230042-20 Improved 3DMM net 3D depth map Polarization- based 3D model Dimensional transformation Rough depth map High-precision 3D face a b I Imax Imin Fig. 15 | Han et al. passive 3D polarization face reconstruction method. (a) Overall schematic of the proposed method. (b) 3D polarization face reconstruction results. Figure reproduced with permission from ref.183, Multidisciplinary Digital Publishing Institute. 230042-20 Improved 3DMM net High-precision 3D face High-precision 3D face Fig. 15 | Han et al. passive 3D polarization face reconstruction method. (a) Overall schematic of the proposed method. (b) 3D polarization face reconstruction results. Figure reproduced with permission from ref.183, Multidisciplinary Digital Publishing Institute. Data-driven shape from polarization in mixed reflections Mixed reflections stemming from the two primary con- ditions were prevalent in the natural scenarios. First, the reflectance of the surface determines the type of reflec- tion: specular reflection or diffuse reflection. For materi- als such as ceramics, plastics, and lacquers, specular re- flection dominates the highlighted areas, whereas diffuse reflection dominates the other areas. Second, objects made of different materials create reflections that vary in The proposed pBRDF model is described by the angle of incident light, plane, reflection angle, camera direc- tion, and half-vector, which allows accurate transmis- sion Mueller matrix modeling for arbitrary cameras and Mixed region Specular dominated region AoLP map Physics-based prior confidence map Fig. 16 | The physical-based prior confidence map according to the differences of polarization characteristics between transparent ob- ject and background. Physics-based prior confidence map Physics-based prior confidence map AoLP map Fig. 16 | The physical-based prior confidence map according to the differences of polarization characteristics between transparent ob- ject and background 230042-21 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 lighting positions. Specular and diffuse reflection mod- els were established separately. The Mueller matrix of specular reflection considers the rotation matrix of light in the incident plane, Fresnel elements, delay metrics, and the rotation matrix of light in the camera, as de- scribed by Eq. (28), R−χl 0 R+χlγc + R×γlχccosδ R×χcsinδ −R+χlχc + R×γlγccosδ R×γcsinδ −R×γlsinδ R×cosδ  , (28) Ms i,o =   R+ R−γl R−γc R+γlγc −R×χlχccosδ −R−χc −R+γlχc −R×χlγccosδ 0 R×χlsinδ (28) R+ = (R⊥+ R∥) /2 R−= (R⊥−R∥) /2 R× = √R⊥R∥ where , , and are Fresnel reflection coefficients. The other coefficients are elements of the rotation matrix. Accordingly, the Mueller matrix of diffuse reflection contains a rotation matrix of light into the incident plane, two Fresnel elements into and out of the surface, a depolarization matrix, and a rotation matrix of light into the camera, as denoted by Eq. (29), timization, all parameters can be calculated. Experi- ments show well-rendered results close to the real ones used to generate polarized images as synthetic datasets. This study guides the establishment of 3D reconstruc- tion of polarized datasets and encourages exploration to accurately transmit the interaction process between po- larized light and objects, even in the entire scenario. Similarly, Deschaintre et al. Data-driven shape from polarization in mixed reflections coupled polarimetric ima- ging with a CNN to estimate the 3D shape and calcu- lated the Spatially Varying Bidirectional Reflectance Dis- tribution Function (SVBRDF) using single-view polari- metric imaging under frontal flash illumination80, as shown in Fig. 17(b). U-Net contains three branched de- coders to generate the 3D shape: 1) surface normal and depth maps, 2) spatially varying reflectance properties as diffuse, and 3) specular albedo maps and specular rough- ness maps. Next, it was fed by the flash image, normal- ized diffuse color, and the Stokes map computed by the polarization image results, which were plausible, and the proposed method captured the real appearance of the in- puts. However, as the lighting or object becomes gradu- ally complex, for instance, multi-illumination, multiple objects with blurred details, etc., the methods based on Md i,o =   T+ o T+ i T+ o T− i βln T+ o T− i αln 0 T− o T+ i βnc T− o T− i βlnβnc T− o T− i αlnβnc 0 −T− o T+ i αnc −T− o T− i βlnαnc −T− o T− i αlnαnc 0 0 0 0 0  , (29) Md i,o =   T− o T+ i βnc T− o T− i βlnβnc T− o T− i αlnβnc 0 −T− o T+ i αnc −T− o T− i βlnαnc −T− o T− i αlnαnc 0 0 0 0 0  , (29) T+ = (T⊥+ T∥) /2 T−= (T∥−T⊥) /2 where and are Fresnel transmission coefficients. The final normalized Mueller matrix is the linear superposition of the reflec- tion matrix and depolarization matrix mentioned above, representing the diffraction and scattering characterist- ics of the light inside the materials. The linear superposi- tion process can effectively simulate the mixed reflec- tions. A generalized Lambertian reflection distribution function model was used to parameterize luminance and linear combination coefficients. Through the final op- Polarization characteristic measurement Polarization BRDF modeling Physical based polarization rendering Polarization information Surface normal normal/ diffuse/ roughness/ specular/ depth map Computed inputs Captured inputs Direct light 0 0 10 10 20 20 −0.15 −1 −1 1 1 0 0 0 0.01 0.02 0.03 −0.10 −0.05 0 a b Fig. 17 | The 3D shape reconstruction methods based on the pBRDF. (a) Y Kondo et al. method and the reconstruction examples. (b) V Des- chaintre et al. method and corresponding results. Reflection removal In summary, existing data-driven polarization 3D shape reconstruction methods are always end-to-end structures guided by a physical model. The basic struc- ture is input to the raw polarization images and other prior images, which are always ambiguous, to optimize the network, and the refined normal image is generated. In addition, other prior images are crucial in the corres- ponding methods for enhancing the reconstructed ef- fects. Different inputs may also result in various network architectural designs. Therefore, as shown in Fig. 18, the inputs in the 3D shape reconstruction task are visually displayed. The data-driven dataset for the ground truths was ob- tained in two ways. First, a Kinect depth camera is the most commonly used equipment for capturing a coarse depth map as the ground truth. Other operations may be conducted to refine the captured depth map, such as de- noising, exclusion of inaccurate values, and sparse point cloud67. Second, a simulation method based on the BRDF is adopted to generate the synthetic datasets. Plausible results could be produced, and other features such as roughness and depth maps80 could also be generated, which may be helpful for other computer vision tasks. However, robustness will decrease in a complicated environment. Mixed reflection dominated the outdoor scenario with multiple objects with different refractive indices. Lei et al. constructed the first real-world SfP dataset for complex scenes to train a network67. The proposed network input includes three parts: 1) four raw captured polarization images and 2) polarization feature images, including in- tensity, DoP, and encoded AoP by sine and cosine opera- tions. Encoded AoP solves the problem regarding raw AoP maps being similar at two given polarization angles with π difference; 3) The viewing encoding to account for non-orthographic projection in scene-level SfP. The introduction of viewing encoding effectively calibrates the polarization parameters influenced by the spatially varying viewing directions. Data-driven shape from polarization in mixed reflections 230042-22 Polarization information Surface normal Polarization characteristic measurement −1 −1 1 1 0 0 0 0.01 0.02 0.03 a Polarization BRDF modeling 0 0 10 10 20 20 −0.15 −0.10 −0.05 0 Physical based polarization rendering Direct light Polarization BRDF modeling Physical based polarization rendering Polarization characteristic measurement normal/ diffuse/ roughness/ specular/ depth map b normal/ diffuse/ roughness/ specular/ depth map normal/ diffuse/ roughness/ specula Captured inputs Captured inputs Computed inputs Computed inputs Fig. 17 | The 3D shape reconstruction methods based on the pBRDF. (a) Y Kondo et al. method and the reconstruction examples. (b) V Des- chaintre et al. method and corresponding results. 230042-22 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 BRDF71,80 will suffer poor recovery, and accurate simula- tion of mixed polarization remains an open challenge. Data-driven polarization reflection removal based on synthetic datasets The reflected light is polarized, and polarization has proven to be a feasible solution to this problem. Based on the Fresnel functions, the number of unknowns was sim- ilar to that of the input images. The reflection and trans- mission components can be separated by viewing the Brewster angle. Moreover, the closer to the Brewster angle, the better the reflection removal performance. Despite the incident angle having to be known, obtain- ing it in the real world is challenging. Additionally, the robustness and generalization of existing polarization- based methods cannot meet the requirements of real- world high-quality imaging, considering the various viewing angles, complex refractive indices, smoothness, and local curvature of the surface. Synthetic datasets are commonly used to train networks because they are hard-accessible real-world datasets. The traditional method directly sums the candidate image as a reflection and transmission using normalized weights. Real-world tests exhibit poor performance. Therefore, based on the polarimetric imaging model, synthetic data- sets of high quality and robustness were generated. T′ R′ By leveraging the properties of light polarization and residual representation, Wieschollek et al. presented the first deep learning approach to separate reflected and transmitted components73, as shown in Fig. 19(a1). The proposed network architecture uses three polarization orientation images to calculate the parallel and perpen- dicular components I∥, I⊥ as the input into the network. The output of network comprises the residual images , and the two single-channel weights ξ∥, ξ⊥. The final estimates of the reflection and transmission can be com- puted as follows: Deep learning methods based on CNN are excellent for extracting hidden features. Furthermore, it enables the prediction of potential prior information from cap- tured images and demonstrates good performance in the reflection removal task. Introducing polarization and imaging models into the network can improve removal performance and expand the diversity of datasets and ar- chitectures. However, acquiring the ground truth data- set is crucial. Thus, the generation of a synthetic dataset based on the proposed method and refined real dataset methods was proposed. Table 3 lists the elements that must be considered in synthetic and real-world dataset acquisition and their advantages and disadvantages. Ir = ξ⊥R′ + (1 −ξ⊥) I⊥and It = ξ∥T′ + ( 1 −ξ∥ ) I∥, (30) ∥ (30) where Ir and It denote the estimation of reflected and transmitted light, respectively, represented as predicted transmission and reflection in Fig. 19(a1). Limitations of reflection removal based on polarization Removal of reflection contamination is a challenging but critical and frequently encountered task because it may contaminate image quality. Several studies have been conducted based on diverse physical and image charac- teristics, but this remains an unsubstantial task14,184,185. Because the transmission image through the surface and the image reflected by the surface are simultaneously captured by a photographer, recovering two images from a single-mixture image is a highly ill-posed problem. 135° 90° 45° 0° Physical prior input Polar image input Diffuse Specular 1 Specular 2 RGB Phase DoP I45 IV IH I135 Normalized color Flash image Stokes map Computed inputs Captured inputs Iun ϕe ρ Imin Imax DoLP AoLP Physics-based prior input Physics-based prior confidence Original polar input Polarization representation P Viewing encoding V Polarization images Raw polarization image Calculate Ambiguous normal n1, n2 I0 Iπ/4 I3π/4 Iπ/2 a d e f g b c Fig. 18 | The inputs of network in 3D shape reconstruction task. (a) The raw polarization images as well as the diffuse and specular normal prior. (b) The raw polarization images and two diffuse ambiguous normal. (c) The DoFP image consists of four polarization sub-images, viewing encoding, intensity, DoP image and encoded AoP image. (d) The chromatic intensity, phase and DoP image. (e) The raw polarization images, flash image, normalized color and Stokes map. (f) The maximum and minimum polarization images. (g) The raw polarization images, physics- based prior confidence, DoLP and AoLP. 230042 23 Calculate Ambiguous normal n1, n2 b 135° 90° 45° 0° Physical prior input Polar image input Diffuse Specular 1 Specular 2 a Iun ϕe ρ Polarization representation P Viewing encoding V Polarization images Raw polarization image I0 Iπ/4 I3π/4 Iπ/2 c Calculate I45 IV IH I135 Normalized color Flash image Stokes map Computed inputs Captured inputs I45 d DoP RGB Phase DoP d Phase RGB Phase Fig. 18 | The inputs of network in 3D shape reconstruction task. (a) The raw polarization images as well as the diffuse and specular normal prior. (b) The raw polarization images and two diffuse ambiguous normal. (c) The DoFP image consists of four polarization sub-images, viewing encoding, intensity, DoP image and encoded AoP image. (d) The chromatic intensity, phase and DoP image. (e) The raw polarization images, flash image, normalized color and Stokes map. (f) The maximum and minimum polarization images. (g) The raw polarization images, physics- based prior confidence, DoLP and AoLP. Limitations of reflection removal based on polarization 230042-23 https://doi.org/10.29026/oes.2024.230042 Yang K et al. Opto-Electron Sci 3, 230042 (2024) Constraints are essential to solve this problem. Constraints are essential to solve this problem. Data-driven polarization reflection removal based on synthetic datasets An accurate synthetic data generation pipeline is in- troduced, including the simulation of realistic reflections, such as high-dynamic-range scenes, nonstatic scenes, and curved and nonideal surfaces, to enhance the robust- ness of the proposed method, as shown in Fig. 19(a2). First, because the world consists of high-dynamic-range elements, the light intensity naturally diminishes as it Therefore, artificial manipulations must be added to generate synthetic images or collect real-world images. In this section, based on the acquired aspects of the training datasets, we review the existing reflection re- moval methods using synthetic and real-world datasets. Table 3 | The comparison of synthetic and real-world datasets. Synthetic dataset Real-world dataset ● Elements should be considered in acquisition of datasets ● High dynamic range in the real-world. ● Disappearance or interruption of the reflection. ● Discontinuous scene of the reflection or transmission. ● Dynamic scenes. ● Semi-reflective surface. ● Curved or irregular surface. ● Rotating of reflection. ● Acquisition of ground truth. ● Dynamic scenes. ● Misalignment of refraction and transmission light. ● Luminousness and thickness of glass. ● Image registration with or without glass. ● Overexposure areas. ● Advantages ● Abundant samples. ● Easily generation. ● Real. ● Complicated enough to cover the real-world conditions. ● Disadvantages ● Illusory. ● Difficult to take fully account all conditions. ● Domain gap between synthesis and real-world data. ● Difficult capturing process. ● Limited samples. 230042 24 Table 3 | The comparison of synthetic and real-world datasets. ● Elements should be considered in acquisition of datasets Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 travels. Consequently, artificial adjustments match this phenomenon in real-world environments. The proposed method separately manipulates the dynamic range of the transmitted and reflected input images using a random factor. Second, for nonstatic scenes, such as cases where a swaying tree branch occurs during capture, local and nonrigid deformations are adopted by perturbing each grid over a patch. Third, for curved and non-ideal sur- faces, a parabola was utilized to simulate unconstrained surface curvatures with four variable parameters. where ϕpol denotes the polarization angle and ϕ denotes the azimuth angle. Data-driven polarization reflection removal based on synthetic datasets The unpolarized and polarized im- ages were then calculated using: Iunpol =ξ 2 · Ir + 2 −ξ 2 · It Ipol = ζ 2 · It + 2 −ζ 2 · It , (32) (32) where Iunpol, Ipol, ξ and ζ denotes the unpolarized, polar- ized images and weights for reflection and transmission, respectively. Next, the reflection and transmission im- ages can be computed as: Lyu et al. exploited the physical constraints from a pair of unpolarized and polarized images to separate reflec- tion and transmission74, as shown in Fig. 19(b). The coef- ficients of the glass plane are predicted by the semireflec- tor orientation module to compute the reflection and transmission based on the proposed physical image formation, denoted as: Ir =2 · (2 −ξ) · Ipol −(1 −ζ) · Iunpol 2ζ −ξ , It =2 · ζ · Iunpol −ξ · Ipol 2ζ −ξ . (33) (33) Finally, to close the gap between the physical model and real data, a refined module was adopted to improve the initial estimation. Additionally, the proposed captur- ing setup can potentially be integrated into smartphones ξ =R⊥+ R∥ ζ =R⊥cos2 ( ϕpol −ϕ ) + R∥sin2 ( ϕpol −ϕ ) , (31) a1 Produce image Parallel/vertical image a1 Produce image a1 b a2 Produce image Parallel/vertical image Residual images/ weights map Predicted transmission & reflection Transmission & reflection/ polarization sub-images Reflection physics Dealing with dynamic scenes Dynamic range manipulation Original data Unpolarized image Weight map Weight map Computed reflection Refined reflection Polarized image Computed transmission Refined transmission −1 +1 +1 +1 Ps f′|xp θ  Fig. 19 | The typical data-driven polarimetric methods of reflection removal. (a1) P Wieschollek et al. method. (a2) The image-based data generation procedure of (a1). (b) Y Lyu et al. method. 230042-25 Residual images/ weights map Predicted transmission & reflection Predicted transmission & reflection Produce image Parallel/vertical image Residual images/ weights map g weights map reflection Transmission & reflection/ polarization sub-images Reflection physics −1 +1 +1 +1 Ps f′|xp θ  reflection Transmission & reflection/ polarization sub-images weights map Reflection physics −1 +1 +1 +1 Ps f′|xp θ  Transmission & reflection/ polarization sub-images b Unpolarized image Weight map Weight map Computed reflection Refined reflection Polarized image Computed transmission Refined transmission Unpolarized image Polarized image Refined transmission Fig. 19 | The typical data-driven polarimetric methods of reflection removal. (a1) P Wieschollek et al. method. Data-driven polarization reflection removal based on synthetic datasets (a2) The image-based data generation procedure of (a1). (b) Y Lyu et al. method. al data-driven polarimetric methods of reflection removal. (a1) P Wieschollek et al. method. (a2) The image-based data re of (a1). (b) Y Lyu et al. method. https://doi.org/10.29026/oes.2024.230042 Yang K et al. Opto-Electron Sci 3, 230042 (2024) without affecting the original photography quality or achieving reliable results. The dataset was generated based on the PLACE2186 dataset, in which two random images were selected as the original reflection and trans- mission images. Reflection is blurred by Gaussian smoothing based on the assumption that people take photos of the transmitted light. eliminate the misalignment between these two elements, a loss of image similarity at the perceptual level, such as perceptual loss and contextual loss, was designed. However, the intensity delay and color distortion persist. The collected reflection-free images are not perfectly aligned with the input mixed images owing to glass re- fraction. To avoid misalignment issues, Lei et al. used a piece of black cloth to cover the back of the glass to block all transmissions for clear reflection53. This dataset in- cludes approximately 100 types of glass in the real world, which guides the proposed method to handle different types of reflections without introducing artifacts. The re- flection removal network uses multi-polarization direc- tion images as input. Furthermore, the calculated intens- ity, degree, and angle of polarization, and the overexpos- ure mask eliminating the overexposed areas, were com- bined into the network. Two stages were adopted to es- timate reflection and transmission. This design signific- antly improves the performance of the proposed method by a large margin. Pang et al. proposed a novel progressive polarization- based reflection removal network (P2R2Net) to generate a preliminary estimation of coarse transmission images be- fore guiding the final reflection removal187. The input im- ages for the network consisted of two parts: a reflection- obstructed image and high-column features from a pre- trained VGG19, which is a successful example of using pre-trained features as prior information. Reflection-ob- structed images are synthetic, based on physical func- tion Eq. (33). The high dynamic range in the real-world, considered as light intensity, is nonlinearly compressed in the captured image through the power function of gamma encoding73. Two independent parameters were used to simulate diverse practical imaging environments. Data-driven polarization reflection removal based on synthetic datasets Additionally, flat and parabolic surface models were ad- opted to simulate curved surfaces, which can be calcu- lated using the camera position (xc, yc) and incidence point (xp, yp): In summary, reflection removal is crucial because ob- taining analytical solutions to ill-posed problems is chal- lenging. The introduction of polarization information can guide reflection removal using Fresnel functions des- pite the unknown incident angle. Thus, combining deep learning to learn prior parameters is a feasible method. Acquiring datasets determines the effectiveness and ro- bustness of the parameter estimation of the proposed methods. In this study, synthetic and real-world meth- ods are proposed. In the future, more comprehensive en- vironments and complete theories must be developed to solve reflection-removal tasks effectively. θ = arccos xcyp −xpyc −xpyp √x2 p + y2 p √( x2 p −x2 c ) + ( y2 p −y2 c ) , (34) (34) where the θ is the angle of incidence. Additionally, ran- dom deformation, rotation, and wrapping expand the scale of the dataset and improve the robustness when synthesizing the reflected obstructed image. Target detection For target classification or detection, polarimetric data- driven methods can improve efficiency and do not re- quire manual extraction of image features compared to traditional methods. However, existing methods use only intensity information images, resulting in a reduction in the accuracy rate for low-light environments or camou- flaged targets188−193. The targets and backgrounds also dif- fer in their polarization characteristics. Polarimetric ima- ging can effectively reveal these differences and assist in target detection194,195. Therefore, we can expect positive results by introducing polarization into data-driven target detection. Despite synthetic datasets not being easy to obtain, they are often too ideal, and complex conditions cannot be fully considered in real-world environments. Real-world datasets are also crucial but challenging to obtain with the influence of glass and misalignment issues. Real-world datasets are typically collected using re- movable glass. Reflection-obstructed images were cap- tured using a camera with glass in front of the detector. The ground truth of the transmission was captured after the glass was removed. However, the difference between transmission and refractive transmission cannot be ig- nored. Intensity delay caused by attenuation and color distortion caused by colored glass are also common. To Fan et al. first proposed the use of polarization com- plementary to intensity-based information to improve 230042-26 https://doi.org/10.29026/oes.2024.230042 Yang K et al. Opto-Electron Sci 3, 230042 (2024) car detection accuracy54. A feature-selection process was performed to select the most informative polarization feature. Final detection is based on a fusion rule that takes the polarization-based model to confirm the color- based one. Gao et al. presented a similar work51. Blin et al. proved that polarimetric imaging is useful for target detection in road scenes52. Sun et al. adopted three-di- mensional convolutions to consider the relationship among S0, S1, and S2 images to improve the detection rate with limited polarimetric images59. Xie et al. used the Stokes vector to obtain four different configurations of polarization parameter image datasets: I, DoP, [I, DoP, AoP], and [S0, S1, S2] and trained different polarization image detection models, indicating that increased polar- ization information fusion enabled more learned target features and better target detection55. Tian et al. pro- posed a human face anti-spoofing method for real-life scenarios, which extracts and classifies the unique polar- ized features of faces using a CNN and an SVM together196. Experiments covering diverse face-spoofing attacks (print, replay, and mask) under uncontrolled in- door and outdoor conditions were conducted. Target detection Usmani et al. proposed unified polarimetric target detection and classification in degraded environments using 3D polari- metric integral imaging data197. 3D polarimetric images with deep neural networks can effectively detect and classify polarimetric targets under different low-light conditions and in the presence of occlusions. Shen et al. combined the advantages of polarimetric imaging and deep learning for rapid target detection of artificial tar- gets camouflaged in natural scenes198, as shown in Fig. 20. The color difference of each image is calculated to prove the proposed method can highlight the camou- flaged artificial targets to a greater extent. pathology. This section reviews the existing data-driven biomedical imaging and pathological diagnosis methods and applications. Next, we discuss the interpretation of physical properties of the network layers based on the distance-based learning classifier. Existing biomedical imaging methods Li et al. first presented a Mueller matrix imaging system to classify morphologically similar algae using a CNN60. Because of the low contrast in the polarimetric signals of algae based on previous measurements of the algal Mueller matrix, performing classification without high- precision instruments is challenging. The proposed methods compare the performances of various stacks of network layers to identify the number of convolution layers. The classifier network was trained to extract fea- tures from the Mueller matrix and achieved a classifica- tion accuracy of 97%. Subsequently, they introduced a distance metric learning method called the Siamese net- work, which aimed to learn good distance metrics of algal Mueller matrix images in low-dimensional feature spaces61. Compared to the convolutional CNN method, in the Siamese approach, data pairs are generated stochastically as inputs to train the network to determ- ine if they belong to the same category. The experiments demonstrated that the coupling of Mueller matrix ima- ging and CNN of the Siamese approach may be an effi- cient solution for the automatic classification of morpho- logically similar algae. Zhao et al. proposed a giant cell tumor bone detection method using Mueller matrix polarization microscopic imaging and a multi-parameter fusion network (MPFN) that combines three extracted polarimetric features: deep micro-Pol features, MMPD features, and MMT features62, as shown in Fig. 21. Wang et al. and Zhou et al. used polarized speckle images for in vivo skin cancer detection85 and polarized hyperspectral images for head and neck squamous cell carcinoma detection86. Yao et al. characterized the microstructures of endometrial samples at the typical proliferative and secretory phases using Müller matrix polar decomposition and a set of ro- tation-invariant parameters and their corresponding an- gular parameters87. In this study, polarimetric imaging was combined with a digital pathology technique to quantitatively study the microstructural features of endometrial samples. Furthermore, the incorporation of local image texture information through Local Binary Pattern (LBP) analysis improves the characterization Biomedical imaging and pathological diagnosis Biomedical imaging and pathological diagnosis methods based on Mueller matrix features, a typical polarization feature, are emerging label-free and noninvasive tech- niques suitable for characterizing the microstructures of biological tissues with anisotropic properties. Recently, results have been published based on Mueller matrix imaging for digital pathology25,28,29,199−207. However, achieving accurate pathological diagnosis by observing and evaluating stained pathological sections for interns is challenging. Furthermore, pathological diagnosis is a classification problem; therefore, learning-based meth- ods are crucial in achieving fast and accurate digital 230042-27 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 Splitting original polarization image Highlighting locations of camouflaged targets Input Output H,S, V channel images S0 stokes image Is image IE image S0 Is IE Tyo's method Wang's method Our method S1,S2 stokes images I0, I45, I90, I135 Decomposing S0 stokes image Extracting polarization signatures Enhancing S0 stokes image if necessary Calculating polarization parameter Is image Calculating stokes images DC=0.539 DC=0.445 DC=0.810 DC=0.599 DC=0.486 DC=0.849 DC=0.218 DC=0.248 DC=0.567 DC=0.620 DC=0.400 DC=0.452 a b a1 a2 a3 a4 a5 a6 b1 b2 b3 b4 b5 b6 c1 c2 c3 c4 c5 c6 d1 d2 d3 d4 d5 d6 Fig. 20 | Shen et al. method. (a) The flow chart of proposed method. (b) The detected results. Figure reproduced with permission from ref.198, In- stitute of Electrical and Electronics Engineers. Splitting original polarization image Input a Input Is S0 IE Fig. 20 | Shen et al. method. (a) The flow chart of proposed method. (b) The detected results. Figure reproduced with permission from ref.198, In- stitute of Electrical and Electronics Engineers. MMT MMPD Mueller matrix elements Hand crafted features Detected results Fig. 21 | The architecture and results of multi-parameters fusion network (MPFN) and the corresponding results. Fig. 21 | The architecture and results of multi-parameters fusion network (MPFN) and the corresponding results. architecture and results of multi-parameters fusion network ability of the polarization parameter images. The experi- ments demonstrated the feasibility of combining polari- metric imaging with digital pathological techniques in typical proliferative and secretory phases. main unclear. In data-driven polarimetric imaging, the Muller matrix provides the most comprehensive inform- ation representing the polarization information, and most decomposition methods that provide raw funda- mental parameters have been proposed. Thus, the Mueller matrix is crucial in exploring the interpretation of network layers. Yang K et al. Opto-Electron Sci 3, 230042 (2024) For example, complex cervical precancerous samples exhibit polariza- tion characteristics of various types of anisotropic super- positions. The depolarization ability of precancerous cer- vical samples changed with the development of lesions. In addition, changes in retardation and depolarization occur during the propagation and scattering of patholo- gical cervical samples at different stages. Therefore, the proposed method has high sensitivity and precision for the screening of cervical lesion pathological tissues, and may bring physical interpretability to the CNN. D =   xT 1 xT 2... xT N   N×M , X = Dω =   xT 1 · ω xT 2 · ω ... xT N · ω   N×1 , L (ω) = d(PNormal (X) , PCIN1 (X) ) −1 , (35) D =   xT 1 xT 2... xT N   N×M , X = Dω =   xT 1 · ω xT 2 · ω ... xT N · ω   N×1 , L (ω) = d(PNormal (X) , PCIN1 (X) ) −1 , (35) where xi is an M×1 vector representing PBPs elements. Yang K et al. Opto-Electron Sci 3, 230042 (2024) Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 In ref.61, the authors calculated the Pearson correla- tion coefficients between the elements of the algal Mueller matrix and features extracted by the CNN from f0 to f15. The experiments demonstrate that features f2, f3, f6, and f7 are positively correlated with depolarization-re- lated elements; however, f1, f4, f10, f12, and f13 are negat- ively correlated. In addition, the fast-axis-orientation-de- pendent periodic variations were preserved in f0, f5, f9, and f15. Dong et al. proposed a data-driven polarimetric imaging framework and constructed a dual-modality machine-learning framework for the quantitative dia- gnosis of cervical precancerous lesions63, as shown in Fig. 22. The U-net architecture was adopted to segment the epithelium in digitized cervical hematoxylin-eosin- stained images and mask the corresponding cervical sample’s polarimetry basis parameters (PBPs), which were decomposed based on the MMPD, MMT, and oth- er Mueller matrix rotation-invariant parameters. Fur- thermore, these masked parameters are processed by the designed statistical distance-based learning classifier for deriving a polarimetry feature parameter (PFP). The classifier of the negative class and those of the CIN1(mild dysplasia) samples can be expressed as: M and N are the number of PBPs and target pixels, re- spectively. X is an N×1 vector, calculated as a linear pro- jection of the input PBPs. PNormal(X) is the probability distribution of X from Normal cervical pathological tis- sues, whereas PCIN1(X) represents CIN1 tissues. ω rep- resents weight coefficients of PBPs. L(ω) is the energy distance between PNormal(X) and PCIN1(X) by energy dis- tance function d. PFP can be represented a simplified lin- ear combination of the PBPs, which is similar to the dis- tribution of specific microstructural variations. The dif- ferent weights indicate the significance of the elements of the PFP feature. The results demonstrate the physical interpretability of the polarimetry feature parameters. For example, complex cervical precancerous samples exhibit polariza- tion characteristics of various types of anisotropic super- positions. The depolarization ability of precancerous cer- vical samples changed with the development of lesions. In addition, changes in retardation and depolarization occur during the propagation and scattering of patholo- gical cervical samples at different stages. Therefore, the proposed method has high sensitivity and precision for the screening of cervical lesion pathological tissues, and may bring physical interpretability to the CNN. The results demonstrate the physical interpretability of the polarimetry feature parameters. Physical interpretation of network layers However, the physical properties of network layers re- 230042-28 Input and utilization of polarization information Input information is a crucial element in network train- ing with polarimetric imaging and deep learning. However, various inputs and polarization parameters ex- ist for different tasks. Three perspectives were con- sidered as network inputs: original polarization images (OPI), polarimetric parameter feature maps (PPFM), and associated parameter maps (APM), as shown in Fig. 25. Shaunak et al. transformed the information in an aug- mented dataset into a compact representation of polari- metric synthetic aperture radar data to classify and seg- ment urban areas82. The segmentation of road scenes is a typical application in which water hazards, transparent glass, and metallic surfaces are key challenges. Yang et al. proposed the prediction of polarization information from monocular RGB images as a complement to RGB- based pixel-wise semantic segmentation for applications in real-world wearable assistive navigation systems69, as shown in Fig. 23. Similarly, Zhang et al.56 and Blanchon et al.83 used different architectures to achieve the same goals: robust and accurate scene parsing of outdoor en- vironments paves the way for autonomous navigation and relationship inference. Focusing on transparent ob- ject segmentation, the polarization textures of transpar- ent objects provide extra but very different information than the background. Therefore, a polarized CNN frame- Original polarization images are among the most widely used inputs. OPI refers to images captured dir- ectly using a DoFP, camera, Mueller Matrix Polarization Microscope, or other equipment. Because of the rapid re- sponse and comprehensive polarization information captured in one shot, raw super-pixel images captured by DoFP are common input42−45. Other common inputs are polarization-oriented images, usually set to 0°, 45°, and 90°46−49,52. Furthermore, there are variants with the addi- tion of 135°50,51 or circular polarization information46. Parallel and perpendicular polarization components were used to train the dehazing network72. Polarization RGB image RGB & depth image Semantic segmentation/ polarization difference image Fig. 23 | Yang et al. method: proposed architecture and produced results of depth and segmented Semantic segmentation/ polarization difference image RGB & depth image Fig. 23 | Yang et al. method: proposed architecture and produced results of depth and segmented results. a b Class Mask Bbox lntensity & DoLP & AoLP Segmentation mask Ground truth Ground truth Intensity Mask R-CNN Intensity Mask R-CNN Polarization Mask R-CNN Polarization Mask R-CNN mAP=0.281 mAP=0.511 mAP=0.878 mAP=0.889 Fig. 24 | Transparent object segmentation. (a) The designed architecture. Semantic segmentation Semantic segmentation Segmentation is a popular topic for understanding the scene in remote sensing and automatic navigation fields. According to learning from different types of massive data, the data-driven segmentation method achieved where xi is an M×1 vector representing PBPs elements. where xi is an M×1 vector representing PBPs elements. a b c d g f e 500 μm 500 μm 500 μm 500 μm 500 μm Fixed χ Select ROI Caculate PBPs Moving γ Registration between the two images by transformation T m11 image Segmented by Mask f Mask M Select target pixels by mapping Mask M to PBPs Generate Mask M M=T·f T=χ·inv(γ) H & E image U-net for epithelium segmentation 0 0.0150.030 DL PL qL rL δ DL D PL qL rL δ Target pixels in PBPs Input data to designed classifier Analyze PFP PBPs 0.04 0.03 0.02 0.01 0 0.005 0.010 0.015 0.020 0.025 Probability Sets of PBPs as input data PFP's statistical features as diagnostic indicators Probility x Classification Normal | CIN1 | CIN2 | CIN3 A statistical distance-based machine learning classifier for deriving PFPs ε(X, Y)=2E|X−Y|d−E|X−X′|d−E|Y−Y′|d ε--Energy distance E|X|d<∞ E|Y|d<∞ Fig. 22 | Polarization-imaging-based ML framework for quantitative diagnosis of cervical precancerous lesions. Figure reproduced with permission from ref.174, Institute of Electrical and Electronics Engineers. 230042-29 Fig. 22 | Polarization-imaging-based ML framework for quantitative diagnosis of cervical precancerous lesions. Figure reproduced with permission from ref.174, Institute of Electrical and Electronics Engineers. 230042-29 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 work can be trained based on the intensity and polariza- tion information57, which will be helpful for applications in broad areas such as robotics, autonomous driving, and face authentication, as shown in Fig. 24. The mean average precision(mAP) is used to measure accuracy. good performance. However, intensity-based methods always suffer from degradation in scenes with similar colors, clutter, or reflective areas208−212. This choice is ori- ented towards polarimetric imaging, which provides the ability to distinguish and recover from changes in com- plex scenes. Several approaches have been proposed to achieve segmentation of remote sensing, road scenes, and transparent objects via polarimetric imaging and deep learning. Discussion Input and utilization of polarization information Datasets speckle images are another type of OPI66,85 captured by the detector after scattering the media. Owing to the demand for different tasks, the number of data-driven polarimetric imaging datasets has gradually increased, as listed in Table 5. Seven types of datasets were associated with the corresponding tasks described in previous section. Three strategies for building the datasets were considered. First, ground truths corres- ponding to the inputs exist; therefore, the real ground truth is captured directly to rectify the outputs. Second, the transfer function of the imaging system was con- sidered, and the generative process was simulated to gen- erate the ground truth. In addition, comparing different traditional methods and selecting the best results regard- ing the ground truth is another method that combines the advantages of existing methods. The polarimetric parameter feature maps were calcu- lated using the OPI. Based on the Stokes representation, [S0, S1, S2] is a common set widely used in polarization network training52,55,58,59. Furthermore, the DoP and AoP computed by the OPI or Stokes vector images are a type of new material for network training52−57. In biomedical diagnosis, Mueller matrix images are the most common input to the network91,95, and Mueller matrix parameters decomposed by Mueller matrix images can also be fed into network training62,208. Table 4 lists the existing de- composition elements of Mueller matrix methods. Associated parameter maps are images in which OPI, and PPFM are combined with other information or pre- processing for different tasks. Intensity information is the most common complement based on the OPI and PPFM53,55−57,66,70,74,196. Similarly, the spectrum86 and phase71,75 are general additions to the polarization in- formation. In 3D shape reconstruction tasks, there are different complements such as zenith and azimuth angle maps derived from specular and diffuse reflection64,65, viewing encoding, encoded AoP67, normalized color80, and physics-based prior confidence81 based on different conditions. The raw images were interpolated using the bicubic interpolation method in the demosaicing task68. An overexposure mask is used in the network input to avoid overexposed areas during reflection removal53. The scene segmentation network utilizes HSL color space representation by incorporating a polarizing pseudo-col- or image83. The overall datasets have a large gap between each other in terms of number and size, even in the same task, as shown in Table 5, which would not avoid the differ- ence in the extraction of features by the CNN. Datasets Further- more, researchers use self-collected training and test datasets; however, evaluating and comparing different methods is challenging. Therefore, authoritative datasets must be built for this task. Input and utilization of polarization information (b) The segmented results of intensity Mask R-CNN and polarized Mask R-CNN in several dataset. Fig. 24 | Transparent object segmentation. (a) The designed architecture. (b) The segmented results of intensity Mask R-CNN and polarized Mask R-CNN in several dataset. 230042-30 230042-30 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 Input and utilization of polarization information polarizer DoFP DoP DoLP DoCP AoP AoLP AoCP Polarization orientation images Circular polarization image Dual-rotating retarder images Polarization speckle images Stokes vectors MMT Rotation invariant parameters MMPD Convolutional neural network Mueller matrix images Preprocessing Polarizing pseudo-color image Physics- based prior confidence Normalized color Polarimetry feature parameters Viewing encoding Encoded AOP Diffuse reflection Phase Spectrum Intensity Specular reflection a b c Fig. 25 | Input and utilization of polarization information. (a) Original polarization images (OPI). (b) Polarimetric parameter feature maps (PPFM). (c) Associated parameters maps (APM). Rotation invariant parameters Convolutional neural network Fig. 25 | Input and utilization of polarization information. (a) Original polarization images (OPI). (b) Polarimetric parameter feature maps (PPFM). (c) Associated parameters maps (APM). Loss function Loss functions are critical elements, and their selection is crucial in guiding network training. Each loss function has advantages and disadvantages. Therefore, a specific loss function was adopted based on the given task and imaging environment. Table 6 lists the latest quality-loss functions for data-driven polarimetric imaging. The 230042-31 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 Yang K et al. Opto-Electron Sci 3, 230042 (2024) Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 Table 4 | Existing decomposition of Mueller matrix. Decomposition of Mueller matrix Physical meanings Mueller matrix polar decomposition (MMPD)101 M = MΔMRMD δ = arccos        [ (MR(2, 2) + MR(3, 3))2+ (MR(3, 2) + MR(2, 3))2 ] 1 2 −1        Linear retardation D = √ m2 12 + m2 13 + m2 14 (0 ⩽D ⩽1) Diattenuation Δ = 1 −|tr (MΔ −1)| 3 (0 ⩽Δ ⩽1) Depolarization θ = 0.5arctan ( r2 r1 ) Orientation of fast axis ψ = 1 2 arctan [ MR(3, 2) −MR(2, 3) MR(2, 2) −MR(3, 3) ] Optical rotation Mueller matrix transformation (MMT)102 t1 = √ (m22 −m33)2 + (m23 + m32)2 2 Anisotropy degree t2 = √ m2 21 + m2 31 2 b = m22 + m33 2 Polarizance A = 2b · t1 b2 + t2 1 ∈[0, 1] Normalized anisotropy t3 = √ m2 42 + m2 43 2 Linear retardation Rotation invariant parameters213−215 PL = √ m2 21 + m2 31 ∈[0, 1] Linear polarizance PC = m41 ∈[−1, 1] Circular polarizance DL = √ m2 12 + m2 13 ∈[0, 1] Linear diattenuation DC = m14 ∈[−1, 1] Circular diattenuation qL = √ m2 42 + m2 43 ∈[0, 1] Capability of transforming between linear and circular polarizations rL = √ m2 24 + m2 34 ∈[0, 1] αP = 1 2 arctan ( m31 m21 ) Orientation of maximum transmittance αD = 1 2 arctan ( m13 m12 ) αq = 1 2 arctan ( −m42 m43 ) Orientation of fast axis αr = 1 2arctan ( −m24 m34 ) Mueller matrix anisotropy coefficients103 α = √ 1 Σ [ (m01 + m10)2 + (m23 −m32)2] Σ = 3m2 00 − ( m2 11 + m2 22 + m2 33 ) + m01m10 + m02m20 + m03m30 −(m23m32 + m13m31 + m12m21) Horizontal linear anisotropy β = √ 1 Σ [ (m02 + m20)2 + (m13 −m31)2] 45° linear anisotropy γ = √ 1 Σ [ (m03 + m30)2 + (m12 −m21)2] Circular anisotropy 230042-32 Table 4 | Existing decomposition of Mueller matrix. Yang K et al. Opto-Electron Sci 3, 230042 (2024) Therefore, the measured information consists of polarimetric representations, such as DoP(DoLP), AoP(AoLP), Stokes vectors, Mueller matrix, spectrum, HSV color space, and their perceptual representations computed by a network model VGG. ˆy where y is the ground truth, and is the output of the network. Therefore, the measured information consists of polarimetric representations, such as DoP(DoLP), AoP(AoLP), Stokes vectors, Mueller matrix, spectrum, HSV color space, and their perceptual representations computed by a network model VGG. LSSIM = 1 − 2μyμˆy + c1 μ2 y + μ2 ˆy + c1 · 2σyˆy + c2 σ2 y + σ2 ˆy + c2 , (39) (39) μ∗ σ∗ σyˆy ˆy where and denote the mean and standard devi- ations of the image, respectively. is the cross-covari- ance computed from the images of y and . where c1 and c2 are constants. ii) The mean absolute error (MAE) is a widely used in- dicator. Compared to the MSE, the MAE has less blurri- ness and noise; however, it is more unstable. The utiliza- tion of the MAE loss function was similar to that of MSE because the range of AoLP is 0 to π that always maps in- to 0–177. However, 0 and 1 indicate the same physical meaning where the error is the largest. Therefore, the HSV spatial display rule was introduced to design a closer distance on the circle of the AoLP. The loss func- tion is defined as follows: Yang K et al. Opto-Electron Sci 3, 230042 (2024) Mueller matrix transformation (MMT)102 Mueller matrix transformation (MMT)102 t1 = √ (m22 −m33)2 + (m23 + m32)2 2 t2 = √ m2 21 + m2 31 2 b = m22 + m33 2 A = 2b · t1 b2 + t2 1 ∈[0, 1] t3 = √ m2 42 + m2 43 2 Capability of transforming between linear and circular polarizations Mueller matrix anisotropy coefficients103 230042-32 230042-32 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 Yang K et al. Opto-Electron Sci 3, 230042 (2024) Table 4 (Continued)   Decomposition of Mueller matrix Physical meanings Differential decomposition104 m = 16 ∑ n=1 mn =   κi κq + κ′ q κu + κ′ u κv + κ′ v κq −κ′ q κi −κ′ i,q ηv + η′ v ηu + η′ u κu −κ′ u −ηv + η′ v κi −κ′ i,u ηq + η′ q κv −κ′ v −ηu + η′ u −ηq + η′ q κi −κ′ i,v   Differential Mueller matrix for depolarizing anisotropic media Symmetric decomposition105 M = MD2MR2MΔdMT R1MD1 Symmetric decomposition of Mueller matrix M = MR2M′ D2MΔdM′ D1MT R1 M = MD2MR2MΔdM′ D1MT R1 M = MR2M′ D2MΔdMT R1MD1 Cloude decomposition106 mij = 1 2 Tr ( Tcη4i+j ) Cloude decomposition following is a detailed description of several functions that differ from the intensity loss functions. is different from the other information forms, ensuring that the CS loss becomes the most suitable indicator. The loss function is defined as follows: i) Mean squared error (MSE) is the most widely used indicator in deep learning, which standards the differ- ence between the output images and the ground truth. In data-driven polarimetric imaging, in addition to the dif- ference in intensity, the polarization parameters also en- sure the accuracy of the polarimetric representations. The loss can be expressed as: LCS = 1 − y · ˆy ∥y∥2∥ˆy∥2 . (38) (38) iv) SSIM is a widely used indicator of end-to-end net- works. SSIM focuses on the brightness, contrast, and structural similarity between two images. The SSIM im- proves as the value increases within the range of [0, 1], opposite to the goal of minimizing the similarity. The loss function is defined as follows: LMSE = ∥y −ˆy∥2 , (36) (36) ˆy where y is the ground truth, and is the output of the network. Future of data-driven polarimetric imaging The field of polarimetric imaging has been influenced by deep learning, which has recently become one of the most disruptive technologies. First, we analyze the trends in data-driven polarimetric imaging, focusing on the ap- plication of data-driven polarimetric imaging and re- view existing research achievements. Furthermore, the acquisition of high-accuracy polarization information is the foundation for subsequent imaging and semantic processing. Descattering, 3D shape reconstruction, re- flection removal, biomedical imaging, pathological dia- gnosis, target detection, and semantic segmentation are crucial in the application of data-driven polarimetric imaging. A comprehensive discussion is essential owing LMAE,AOLP = min ( ∥y −ˆy∥1, 1 −∥y −ˆy∥1 ) . (37) (37) iii) Cosine similarity (CS) is commonly used in the surface-normal estimation of 3D reconstructions based on polarimetric imaging. In the 3D polarimetric ima- ging task, the surface normal map is calculated using Fresnel's formula to generate the normal vectors, which iii) Cosine similarity (CS) is commonly used in the surface-normal estimation of 3D reconstructions based on polarimetric imaging. In the 3D polarimetric ima- ging task, the surface normal map is calculated using Fresnel's formula to generate the normal vectors, which 230042-33 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 to the input and utilization of polarization information, datasets and loss functions Strengths Data-driven polarimetric imaging represents a new   Table 5 | Summary of data-driven polarimetric imaging datasets. Task Data Dataset Paired or not No. Future of data-driven polarimetric imaging of images Size Synthetic/Real Section Restoration and enhancement of accurate polarization information 2020 Hu42 Paired 300000 64×64 Real 2022 LLCP50 Paired — 64×64 Real 2018 Zhang68 Paired 215 640×480 Synthetic 2019 Zeng43 Paired 76890 40×40 Synthetic 2021 Wu44 Paired — 64×64 Synthetic 2020 Li45 Paired 137000 64×64 Synthetic 2022 Yang78 Paired 337 1961×2381 Real 2022 Liu77 Paired 140000 64×64 Real 2021 Ting79 Paired 42228 512×512 Real 2022 Si58 Paired — 256×256 Real Section Polarimetric descattering 2020 Hu46 Paired 103000 64×64 Real 2021 Zhang70 Paired 2000 256×256 Real 2021 Ren148 Paired — — Real 2021 Zhou48 Paired — 240×240 Synthetic 2022 Shi72 Paired 60000 960×576 Synthetic 2022 Ding47 Paired 700 512×512 Real+Synthetic 2022 Li66 Paired 5000 256×256 Real+Synthetic Section Three-dimensional shape reconstruction 2019 Ba64 Paired 300 256×256 Real 2020 SURREAL+PHSPD65 Paired 312915 — Real 2020 Kondo71 Paired — — Real+Synthetic 2021 Lei67 Paired 522 1224×1024 Real 2021 Deschaintre80 Paired 100000 512×512 Synthetic 2022 TransSfP81 Paired 936 512×512 Real+Synthetic Section Reflection removal 2018 URD73 Paired — 128×128 Synthetic 2019 Lyu74 Paired — 256×256 Synthetic 2020 Lei53 Paired 100107 — Real Section Target detection 2018 Fan54 Paired 153 — Real 2019 Blin52 Paired 2730 — Real 2020 Sun59 Paired — 96×96 Real 2020 CASIA-DOLP196 Unpaired 10697 224×224 Real 2021 USMANI197 Paired 240 — Real 2022 Gao51 Paired 60 1224×1024 Real Section Biomedical imaging and pathological diagnosis 2017 Li60 Paired 10463 — Real 2018 Li61 Paired 12162 — Real 2020 Zhao62 Paired 100000 — Real 2021 Zhou86 Paired 4500 40×40 Real 2021 Yao87 Paired 400 64×64 Real 2021 Dong63 Unpaired 49870 128×128 Real Section Semantic segmentation 2018 Yang69 Paired 9736 320×240 Real 2019 POLABOT56 Paired 700 256×256 Real 2020 Kalra57 Paired 1000 — Real Table 5 | Summary of data-driven polarimetric imaging datasets. Table 5 | Summary of data-driven polarimetric imaging datasets. to the input and utilization of polarization information, datasets, and loss functions. to the input and utilization of polarization information, datasets, and loss functions. Yang K et al. Opto-Electron Sci 3, 230042 (2024) Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 Table 6 | Summary of loss functions. Loss function Applications Frobenius matrix norm45,46,68 Section Restoration and enhancement of accurate polarization information, Section Polarimetric descattering Weighted mean squared error (WMSE)70 Section Polarimetric descattering Preceptual loss53,70,77 Section Polarimetric descattering Mean squared error (MSE)46,48,49,61,65,71−74,76−78 Section Restoration and enhancement of accurate polarization information, Section Polarimetric descattering, Section Three-dimensional shape reconstruction, Section Reflection removal Mean absolute Error(MAE)42−44,47,48,50,66,75,79,80,148 Section Restoration and enhancement of accurate polarization information, Section Polarimetric descattering, Section Three-dimensional shape reconstruction Negative pearson correlation coefficient (NPCC)66 Section Polarimetric descattering Cosine similarity (CS)64,65,67,81 Section Three-dimensional shape reconstruction Perceptual normalized cross-correlation (PNCC)53 Section Reflection removal Structural similarity index (SSIM)43,79 Section Restoration and enhancement of accurate polarization information Total variation (TV)58,75 Section Restoration and enhancement of accurate polarization information Contrastive loss196 Section Target detection Logistic regression87 Section Biomedical imaging and pathological diagnosis Ridge loss87 Section Biomedical imaging and pathological diagnosis Focal loss69 Section Semantic segmentation Cross entropy loss (CEL)83 Section Semantic segmentation Table 6 | Summary of loss functions. representation domains. Applying the information rep- resented by the physical model and the deep network layers enables researchers to exploit the potential fea- tures embedded in all information-transmitted paths. ric imaging network, researchers must weigh the costs associated with data-driven polarimetric imaging and conventional approaches, which include the establish- ment of polarimetric imaging datasets, storage of data al- ways four times that of traditional datasets, and imaging systems. Because balancing these costs and benefits is not exact, some uncertainties are considered in this process. During the imaging process, the light source, trans- mitted media, imaging system, and image processing method influence visual performance. Moreover, model- ing these complex processes using physical functions or traditional methods is challenging. Network layers are high-order polynomials expressed by the convolution neural network. Therefore, nonlinear representations of the network layers may be promising for simulating the process. A fundamental element of data-driven polarimetric imaging is the availability of comprehensive datasets. Most data-driven polarimetric imaging methods have fo- cused on supervised deep learning. However, based on polarimetric imaging methods and imaging environ- ments, the corresponding ground truth is more challen- ging to capture than in an intensity-based network. Ad- ditionally, different polarimetric imaging techniques in- troduce various errors and influence the visual perform- ance of the network. Yang K et al. Opto-Electron Sci 3, 230042 (2024) For example, the division of time al- ways suffers from mismatching in dynamic scenes, and the division of the focal plane has a mosaicking problem in principle. Existing methods have established their datasets to respond to specific tasks. However, ensuring similar performance from other datasets is challenging; moreover, the existing dataset is insufficient to cover all conditions, and its generalization ability is insufficient. Real data changes over time, indicating increasing volume and improper handling of methods. The introduction of deep learning into conventional polarimetric imaging and pattern recognition generates more accurate coefficients for the physical model, simu- lating a complicated processing approach not modeled by physical functions. However, introducing a polari- metric imaging model into the deep network would add physical constraints and polarization information to guide the network training to achieve better perform- ance compared with the intensity network. Therefore, data-driven polarimetric imaging probably enables cap- abilities that cannot be realized using traditional methods. Weaknesses Strengths Data-driven polarimetric imaging represents a new paradigm that combines deep learning and traditional physical properties, including altering the patterns of physical properties to achieve better results and balan- cing the physical model and information extraction between the traditional physical and high-order nonlinear This section evaluates the future of data-driven polari- metric imaging based on its strengths, weaknesses, and opportunities. Furthermore, this approach is suitable for developing instructive strategies for further studies that combine deep learning and polarization information. 230042-34 The opportunities of methods The assistance of physical model: A synthetic dataset solves the scarcity of datasets. An accurate physical mod- el that simulates information transmission is crucial in dataset generation. With an additional physical con- straint on the CNN, fewer training data are required to achieve a more generalized result than conventional methods. In addition, to obtain a synthetic dataset, vari- ous parameters covering different imaging conditions are crucial. Thus, the development of conventional po- larimetric imaging methods is suitable for designing net- work architectures. Black boxes and their acceptance by applied research- ers are inherent drawbacks of deep learning methods by health professionals. Most researchers in practical ap- plied fields are wary because deep learning theories have not yet provided a complete and reasonable answer. In addition, further development and optimization would depend only on the performance of tasks without any guidance from theories, which results in indeterminacy in the study. Moreover, the legal implications of black box functionality could be another challenge. For ex- ample, who would be responsible if the results were in- correct in pathological diagnosis or target detection? In data-driven polarimetric imaging, the introduction of polarization information may be helpful for the inter- pretability of deep learning. Dong et al. attempted to use a linear projection of input PBPs to interpret their signi- ficance by learning the factors of each parameter. However, many studies have been conducted to achieve these goals63. Unsupervised or semi-supervised learning216−218: Ob- taining the ground truth for a large polarization dataset is challenging. Therefore, unsupervised or semi-super- vised learning is required to reduce the dependence on the ground truth. However, image enhancement or im- age processing is an end-to-end task; thus, existing learn- ing methods without ground truth achieve poor per- formance. A more comprehensive physical model must be established, and more effective loss functions de- signed to guide pipelines. In addition, the middle para- meter may be generated without the ground truth, which is also a feasible way to improve the performance. Transfer learning219,220: Transfer learning allows the optimized parameters for one dataset to train a new net- work as initialization values for another dataset, which is a feasible approach for reducing the dependence on data- sets in data-driven polarimetric imaging because the learned features can be promptly transferred from a trained network to a new network for another task. Weaknesses To achieve a better performance by training a polarimet- The loss function, which is crucial in guiding the The loss function, which is crucial in guiding the 230042-35 230042-35 https://doi.org/10.29026/oes.2024.230042 Yang K et al. Opto-Electron Sci 3, 230042 (2024) training of the network, is always similar to the intensity- based methods used in data-driven polarimetric imaging. General operators replace intensity images with polariza- tion parameters. However, the intensity and polarization information have entirely different optical properties. The intensity image describes the reflectivity and trans- missivity of the object; however, the polarization image describes the texture details, material properties, shape, shading, and roughness. These differences determine the disparate designs of loss functions. However, there are insignificant loss functions that focus on polarization parameters. ging or training theories to guide the optimization pro- cess. In addition, three broad application areas: descat- tering imaging, even high-scattering media, detection of camouflage, spoofing targets, and enhancement and fu- sion of information assess the potential of data-driven polarimetric imaging in future applications. The opportunities of methods The fine-tuning technique is a typical method used in trans- fer learning, which is faster and easier than training a network from scratch. Therefore, the extracted features were similar to the shallow layers; moreover, the shallow layers in a trained network can be copied to the new net- work for another task to reduce the cost of training time. The combination and utilization of polarization in- formation in deep learning are still in their infancy. In most existing methods, polarization parameter images are the only approach that uses polarization information as the input into the network. The extraction of polariza- tion features relies on the automatic processing of net- work layers, which remains a challenge to utilize polariz- ation information despite the unlimited opportunity for improvement yet to be explored. Opportunities Based on the weaknesses of data-driven polarimetric imaging, solutions have been proposed to address these gaps. Furthermore, many novel training methods and physical models exist, such as unsupervised or semi-su- pervised training, transfer learning, and computational imaging. Therefore, it must be combined with other ima- Multi-dimensional learning221,222: The introduction of polarization information that displays different physical properties into a traditional intensity network can provide more constraints and information sources to 230042-36 Yang K et al. Opto-Electron Sci 3, 230042 (2024) https://doi.org/10.29026/oes.2024.230042 For descattering imaging in high-scattering media, such as clouds, water, haze, smoke, smog, fog, mist in the air, soil particles, algae, and mineral salt, in underwater scenes, there are potential opportunities for data-driven polarimetric imaging. However, further development of physical models has amplified this ability. However, tra- ditional model functions are challenging to handle in complex imaging environments and always use simple assumptions to simulate real parameters. The introduc- tion of deep learning can model complicated conditions in nonlinear cases using convolutional neural layers. Fu- ture opportunities will arise from the development of more accurate parameters for forming improved ima- ging functions generated by deep learning. promote network inference. Similarly, the phase, spec- trum, and other physical properties can be embedded in- to a network to enhance performance. Phase is a repres- entative generation of the change in light, and the spec- trum describes the characteristics of the wavelength of light. These properties would fill the gap in which a single domain cannot fully represent the light domain for all physical properties. Federated learning223: To provide a practical training set for deep learning in polarimetric imaging applica- tions, obtaining various available datasets from different institutes or corporations may be a possible solution. There are several datasets for different tasks, as shown in Table 5; however, the datasets collected by different groups are not uniform because it is difficult to guaran- tee a similar performance from other datasets. Therefore, different datasets were beneficial, increasing the di- versity of the collected samples. In addition, different imaging systems, detectors, environments, and observa- tion directions are challenging to simulate using existing physical functions, which can improve the generaliza- tion ability of the network and avoid overfitting. There- fore, this is instructive for building and optimizing the evaluation criterion, focusing on polarization images. Spoofing targets are another opportunity for camou- flage detection. Opportunities Target detection is widely applied in po- larimetric imaging because the polarization information can describe the material of an object, which is suitable for camouflaging and spoofing targets of the same color that the intensity information cannot distinguish. Next, more comprehensive extraction of special features by the neural network may further improve the success rate of target detection. The material surface, texture, and contrast are the main characteristics described by polarization informa- tion for enhancing and fusing information. Polarization parameters are observable in low- or hard-light environ- ments because they are unaffected by intensity. Con- sequently, the fusion of polarization and other images can extend the feature domain of an object. The network generates a fusion based on data-driven polarization fu- sion, enhancing performance by extracting more fea- tures and providing more information on imaging ob- jects or scenes compared with artificial coefficients. Moreover, complementary features from various do- mains are advantageous for other computer vision tasks, such as object detection. Emergence of metasurface and metalens224−227: The util- ization of lenses and metasurfaces allows for tailored control over light with specific polarization states, achieved through deliberate design. This deliberate con- trol enables superior capture, separation, and analysis of polarized light signals, thereby significantly enhancing the sensitivity and accuracy of acquiring polarization datasets. 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In Proceedings of 2016 IEEE Conference on Computer Vision and Pattern Recognition 779–788 (IEEE, 2016); 191. Chen GH, Wang JY, Zhang AJ. Transparent object detection and location based on RGB-D camera. J Phys Conf Ser 1183, 208. 230042-43 230042-43 Competing interests Pan SJ, Yang Q. A survey on transfer learning. IEEE Trans Knowl Data Eng 22, 1345–1359 (2010). 219. The authors declare no competing financial interests. Scan for Article PDF 230042-44
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Baryogenesis in the two doublet and inert singlet extension of the Standard Model
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Download date: 24. Oct. 2024 aryogenesis in the two doublet and inert singlet extension of the Standard Mode Citation for pulished version (APA): Alanne, T., Kainulainen, K., Tuominen, K., & Vaskonen, V. (2016). Baryogenesis in the two doublet and inert singlet extension of the Standard Model. Journal of Cosmology and Astroparticle Physics, 2016(8), Article 57. https://doi.org/10.1088/1475-7516/2016/08/057 Citation for pulished version (APA): Alanne, T., Kainulainen, K., Tuominen, K., & Vaskonen, V. (2016). Baryogenesis in the two doublet and inert singlet extension of the Standard Model. Journal of Cosmology and Astroparticle Physics, 2016(8), Article 57. https://doi.org/10.1088/1475-7516/2016/08/057 Go to publication entry in University of Southern Denmark's Research Portal Terms of use This work is brought to you by the University of Southern Denmark. Unless otherwise specified it has been shared according to the terms for self-archiving. 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Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. Baryogenesis in the two doublet and inert singlet extension of the Standard Model This content has been downloaded from IOPscience. Please scroll down to see the full text. Download details: IP Address: 130.226.87.174 This content was downloaded on 26/05/2017 at 09:49 Please note that terms and conditions apply. View the table of contents for this issue, or go to the journal homepage for more JCAP08(2016)057 (http://iopscience.iop.org/1475-7516/2016/08/057) You may also be interested in: A strong electroweak phase transition from the inflaton field Tommi Tenkanen, Kimmo Tuominen and Ville Vaskonen Scalar representations in the light of electroweak phase transition and cold dark matter phenomenology Shehu S. AbdusSalam and Talal Ahmed Chowdhury Detectable gravitational waves from very strong phase transitions in the general NMSSM Stephan J. 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Sánchez Fermi Bubbles under Dark Matter Scrutiny Part II: Particle Physics Analysis Wei-Chih Huang, Alfredo Urbano and Wei Xue The electroweak phase transition in the Inert Doublet Model Nikita Blinov, Stefano Profumo and Tim Stefaniak Baryogenesis in the two doublet and inert singlet extension of the Standard Model JCAP08(2016)057 Tommi Alanne,a Kimmo Kainulainen,b,d Kimmo Tuominenc,d and Ville Vaskonenb,d Keywords: baryon asymmetry, cosmological phase transitions ArXiv ePrint: 1607.03303 doi:10.1088/1475-7516/2016/08/057 doi:10.1088/1475-7516/2016/08/057 Contents 1 Introduction 1 2 The model 2 2.1 Reparametrization invariance and tree-level vacuum stability 4 2.2 Spontaneus symmetry breaking 6 2.3 Finite-temperature potential 8 3 Results 8 3.1 Theoretical and experimental constraints 9 3.2 Dark matter abundance and direct detection limits 9 3.3 Electroweak phase transition 11 3.4 Electron EDM constraint 12 3.5 Baryogenesis 13 3.6 Bubble nucleation 15 4 Conclusions and outlook 17 A Diagonalization of mass matrices 19 B 1-loop beta functions for scalar couplings 20 C Singlet scalar annihilation cross sections 21 2 4 6 8 8 9 9 11 12 13 15 17 19 20 21 JCAP08(2016)057 C Singlet scalar annihilation cross sections Tommi Alanne,a Kimmo Kainulainen,b,d Kimmo Tuominenc,d and Ville Vaskonenb,d aCP3-Origins, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark bDepartment of Physics, University of Jyv¨askyl¨a, P.O. Box 35 (YFL), FI-40014 Jyv¨askyl¨a, Finland cDepartment of Physics, University of Helsinki, P.O. Box 64, FI-00014 Helsinki, Finland dHelsinki Institute of Physics, University of Helsinki, P.O. Box 64, FI-00014 Helsinki, Finland E-mail: alanne@cp3.sdu.dk, kimmo.kainulainen@jyu.fi, kimmo.i.tuominen@helsinki.fi, ville.vaskonen@jyu.fi aCP3-Origins, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark bDepartment of Physics, University of Jyv¨askyl¨a, P.O. Box 35 (YFL), FI-40014 Jyv¨askyl¨a, Finland cDepartment of Physics, University of Helsinki, P.O. Box 64, FI-00014 Helsinki, Finland dHelsinki Institute of Physics, University of Helsinki, P.O. Box 64, FI-00014 Helsinki, Finland E-mail: alanne@cp3.sdu.dk, kimmo.kainulainen@jyu.fi, kimmo.i.tuominen@helsinki.fi, ville.vaskonen@jyu.fi aCP3-Origins, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark bDepartment of Physics, University of Jyv¨askyl¨a, P.O. Box 35 (YFL), FI-40014 Jyv¨askyl¨a, Finland cDepartment of Physics, University of Helsinki, P.O. Box 64, FI-00014 Helsinki, Finland dHelsinki Institute of Physics, University of Helsinki, P.O. Box 64, FI-00014 Helsinki, Finland E-mail: alanne@cp3.sdu.dk, kimmo.kainulainen@jyu.fi, kimmo.i.tuominen@helsinki.fi, ville.vaskonen@jyu.fi E-mail: alanne@cp3.sdu.dk, kimmo.kainulainen@jyu.fi, kimmo.i.tuominen@helsinki.fi, ville.vaskonen@jyu.fi Received July 13, 2016 Accepted August 21, 2016 Published August 25, 2016 Abstract. We investigate an extension of the Standard Model containing two Higgs doublets and a singlet scalar field (2HDSM). We show that the model can have a strongly first-order phase transition and give rise to the observed baryon asymmetry of the Universe, consistent with all experimental constraints. In particular, the constraints from the electron and neutron electric dipole moments are less constraining here than in pure two-Higgs-doublet model (2HDM). The two-step, first-order transition in 2HDSM, induced by the singlet field, may lead to strong supercooling and low nucleation temperatures in comparison with the critical temperature, Tn ≪Tc, which can significantly alter the usual phase-transition pattern in 2HD models with Tn ≈Tc. Furthermore, the singlet field can be the dark matter particle. However, in models with a strong first-order transition its abundance is typically but a thousandth of the observed dark matter abundance. 1 Introduction The matter-antimatter asymmetry in the universe presents one of the major quests for particle cosmology. Due to cosmic inflation, such asymmetry cannot be an initial condition for the thermal history of the universe, but calls for a dynamical explanation. The Standard Model (SM) of elementary particle interactions fails in providing a successful mechanism for baryogenesis, and one must look at different extensions of the SM. In this paper we address these issues in the context of a 2HDSM featuring an extended scalar sector with two gauged Higgs doublets and an extra singlet. Generation of the matter-antimatter asymmetry in connection with the electroweak phase transition, i.e. electroweak baryogenesis, is a particularly appealing scenario due to the possibility of connecting it with the collider experiments. Generic 2HDMs have been studied earlier in connection with the electroweak baryogenesis problem [1–9]. They provide both a new source of CP violation arising from complex parameters in the 2HDM potential and a strong first-order phase transition arising from the one-loop effective potential. However, observational constraints are placing stringent limits also on 2HDMs [8]. Here we show that these constraints are alleviated when the model is further extended by a real scalar singlet field. A generic feature of 2HDM, also inherited by the 2HDSM, is the danger of generating large flavour changing neutral currents. To avoid these, one has to constrain the Higgs- fermion couplings in one way or the other. Here we choose to work in the context of universal – 1 – Yukawa alignment, which may be argued for by a requirement that the whole Lagrangian is invariant under the group GL(2,C) of linear reparametrization transformations in the doublet space. We also use the reparametrization invariance to develop an elegant way explore the vacuum stability and the phase-transition pattern in the model. In the 2HDM context large CP violation requires that scalar couplings have large com- plex phases and strong transition requires that couplings are large in magnitude. When combined, these requirements tend to give too large electron and neutron electric dipole mo- ments (EDMs). We will show that the presence of the additional scalar allows for a strong two-step electroweak phase transition, which does not rely on large radiative corrections to the effective potential. This alleviates the burden on the scalar self-couplings and significantly increases the phase space consistent with EDM constraints in the 2HDSM. 1 Introduction JCAP08(2016)057 The singlet scalar can also be a dark matter (DM) candidate when a discrete Z2 symme- try is imposed to stablize it. However, we will find that a strong first-order phase transition is not consistent with a dominant singlet scalar DM particle. The problem is that a strong two-step transition requires a large coupling between the singlet and doublet sectors and this implies so large annihilation rate for the DM that its relic abundance becomes too small to account for the full observed DM density. This conclusion is generic for all models of this type. We observe that two-step transitions may also give rise to too strong transitions. It is possible that fields get trapped in the metastable minimum so that electroweak symmetry remains unbroken. Also, the latent heat released in the transition may be so large that the transition walls necessarily become supersonic. However, we find also parameters for which walls may be subsonic, consistent with the electroweak baryogenesis scenario. Overall, we are able to find models that satisfy all observational and experimental constraints and can also give rise to a successful electroweak baryogenesis, accompanied by a subleading DM in the 2HDSM context. The structure of the paper is as follows: in section 2 we introduce the model and discuss the most general GL(2,C)-reparametrization invariant 2HDSM Lagrangian including Yukawa couplings. Here we also develop methods to study the vacuum stability and the phase-transition patterns in the theory. In section 3 we first go through the experimental constraints on the model and evaluate the DM relic abundance and the DM search limits on model parameters. We then evaluate the strength of the transition and compute the baryon asymmetry created in the electroweak phase transition. The section is concluded by a study of bubble nucleation in the 2HDSM and in the singlet extension of the SM. In section 4 we conclude and outline some directions for future research. 2 The model We start from the most general two-Higgs-doublet and inert-singlet extension of the SM with the scalar field Lagrangian: Lscalar = Zij(DµHi)†DµHj + 1 2(∂µS)2 −V (H1, H2, S) , (2.1) (2.1) – 2 – where Zij is an arbitrary Hermitian 2 × 2 matrix and the most general potential is given by V (H1, H2, S) = −m2 1|H1|2 −m2 2|H2|2 −  m2 12H† 2H1 + h.c.  −1 2m2 SS2 + λ1|H1|4 + λ2|H2|4 + λ3|H1|2|H2|2 + λ4(H† 1H2)(H† 2H1) +  λ5(H† 2H1)2 + λ6|H1|2(H† 2H1) + λ7|H2|2(H† 2H1) + h.c.  + 1 4λSS4+ 1 2λS1S2|H1|2+ 1 2λS2S2|H2|2+ 1 2λS12S2H† 2H1+h.c.  . (2.2) (2.2) JCAP08(2016)057 Both doublets Hi are assumed to be gauged under SU(2)L × U(1)Y , while the scalar S is a singlet under all SM gauge interactions. The singlet S is a crucial ingredient in the model because it will disentangle the source of a strongly first-order transition from that of sufficiently strong CP violation. The Lagrangian (2.1) is invariant under a reparametrization transformation Φ →Φ′ ≡ PΦ (and a simultaneous rescaling of S), where P is an element of the general linear group GL(2,C), and Φ is the Higgs hyperdoublet: Φ ≡(H1, H2)T . (2.3) (2.3) GL(2,C) is the semidirect product of special linear transformations SL(2,C) and multiplica- tive group of dilatations C×. We can always use the dilatation and a hyperbolic SL(2,C) transformation to bring the kinetic term into the canonical form, Zij →diag(1, 1), i.e. Zij(DµHi)†DµHj → |DµH1|2 + |DµH2|2 . The resulting Lagrangian is still invariant under elliptic SL(2,C) transformations, i.e. the usual SU(2) rotations of the doublets. The resulting Lagrangian is still invariant under elliptic SL(2,C) transformations, i.e. the usual SU(2) rotations of the doublets. A generic 2HDM gives rise to unacceptably large flavour-changing neutral currents (FCNCs) and the presence of a singlet does not change the situation. One way to avoid FCNCs is the Yukawa alignment [10], which assumes that both doublets couple to fermions with the same matrix structure (since S is a singlet under SM gauge interactions its couplings to charged SM fermions are excluded): (2.4) LYukawa = yuCi u ¯QL ˜HiuR + ydCi d ¯QLHidR + yℓCi ℓ¯LLHieR + h.c, (2.4) where ˜H2 ≡iσ2H∗ 2. Here ya are flavour matrices independent of the doublet index, and Ca i are doublet-index dependent complex numbers. 1This actually involves a rotation and a redefining of the scale of ya matrices. 2 The model In general the alignment may be different in different fermion sectors: Ca i ̸= Cb i . However, for simplicity, we choose to work in the special case of universal Yukawa alignment, where Ca i ≡Ci. In this case we can, without a further loss of generality, choose the basis where only the H2 field couples to fermions. This corresponds to setting C1 = 0 and C2 = 1,1 so that: (2.5) LYukawa = yu ¯QL ˜H2uR + yd ¯QLH2dR + yℓ¯LLH2eR + h.c . (2.5) – 3 – The choice of basis leading to (2.5) can be effected by an SU(2) rotation of Φ, and it exhausts our remaining freedom to perform elliptic SL(2,C)-reparametrization transformations after diagonalizing the kinetic term.2 Let us stress that while the Yukawa sector (2.5) appears to be of type-I 2HDM, we did not impose any discrete symmetry to derive it. This is why we have kept the λ6 and λ7 terms in the scalar potential. Note that renormalization does not change the form of the theory; while it both re-introduces a kinetic mixing between doublets and a coupling of H1 to fermions, these changes can be countered by another GL(2,C) transformation. Also, we point out that the universal Yukawa alignment can be argued for based on reparametrization invariance: only in the context of universal alignment is the complete Lagrangian including the 2HDM and Yukawa sectors invariant under GL(2,C) transformations. JCAP08(2016)057 Interestingly, the universal alignment structure arises as a low-energy effective theory in models of dynamical electroweak symmetry breaking [11–18]. In the bosonic technicolor [11– 13], the ultraviolet theory contains a new gauge theory responsible for dynamically breaking the electroweak symmetry and an elementary scalar doublet H, which communicates the symmetry breaking to the SM fields through its renormalizable Yukawa couplings. At low energies the strong technicolor dynamics is described in terms of an effective Lagrangian for a composite Higgs doublet, which couples with the elementary one through a Lagrangian of the form (2.1), including the non-trivial kinetic mixing. Only the elementary scalar couples to SM fermions, which naturally introduces Yukawa alignment. Moreover, when the kinetic mixing is removed by a non-unitary transformation, the Yukawa Lagrangian becomes naturally of the universally Yukawa-aligned form with Ca i = Ci. After a final SU(2) rotation, the model has a diagonal kinetic term, type-I Yukawa sector (2.5), and the most general potential of eq. (2.2). 2Note that in the case of general Yukawa alignment, where Ca i ̸= Cb i , one could still use the SU(2) rotation to set Cu 1 = 0, so that up-type quarks couple only to H2. Most of our subsequent analysis would hold also for this scenario, because it is mostly sensitive only to the large top-quark coupling. The only exception is the electron EDM, for which our analysis covers only a part of the full phase space available in the context of general alignment. † † g g 3Clearly r0 = P i |Hi|2 ≥0. Also rµrµ = 4(|H1|2|H2|2 −(H† 1H2)(H† 2H1)) ≥0 , by Schwartz inequality. 2.1 Reparametrization invariance and tree-level vacuum stability In this notation one can rewrite the Higgs potential (2.2) in a very compact form: V = −1 2m2 SS2 −1 2M2 µrµ + 1 4rµλµνrν + 1 4λSµrµS2 + 1 4λSS4 , (2.7) (2.7) where we defined mass and coupling four-vectors where we defined mass and coupling four-vectors M2 µ ≡ m2 1 + m2 2, 2m2 12R, −2m2 12I, m2 1 −m2 2  , λSµ ≡(λS1 + λS2, 2λS12R, −2λS12I, λS1 −λS2) (2.8) (2.8) and a symmetric coupling tensor JCAP08(2016)057 λµν ≡      λ1 + λ2 + λ3 λ6R + λ7R −λ6I + λ7I λ1 −λ2 λ6R + λ7R λ4 + 2λ5R −2λ5I λ6R −λ7R −λ6I + λ7I −2λ5I λ4 −2λ5R −λ6I −λ7I λ1 −λ2 λ6R −λ7R −λ6I −λ7I λ1 + λ2 −λ3     , (2.9) (2.9) where the subscripts R and I refer to the real and imaginary parts of the couplings, respec- tively. The reparametrization invariance is manifest in eq. (2.7), because V depends only on Lorentz-invariant products of vectors and tensors. This form is particularly suitable for a study of the vacuum stability and phase-transition patterns of the model. First consider the direction S = 0 in the potential (2.7). Here the term rµλµνrν must be bounded from below. In [19] it was shown that this is the case precisely when λµν is positive definite in the future light cone. That is, all stable potentials can be written as λµν ≡ΛµαλD αβΛβν, where Λµα is an SO(1,3)+ transformation and λD αβ = diag(λD 00, −λD 11, −λD 22, −λD 33), with λD 00 > 0 and λD 00 > λD ii . (2.10) (2.10) Note that the four parameters in λD αβ together with the six parameters in Λβν add up to the ten real degrees of freedom in the most general 2HDM potential. Second, if we set rµ = 0 (H1 = H2 = 0), we see that we must have λS > 0 . (2.11) (2.11) Finally, we have to consider the directions where both S and Hi are nonzero. First, if the vector λSµ of couplings which mix S and Hi lies in the future light cone, λSµ ∈LC+, i.e. λSµλµ S = 4 λS1λS2 −|λS12|2 > 0 and λ0 S = λS1 + λS2 > 0 , (2.12) (2.12) then the mixing term 1 4λSµrµS2 in the potential, eq. 2.1 Reparametrization invariance and tree-level vacuum stability The original Lagrangian with the most general potential, kinetic term, and the universally aligned Yukawa sector has 27 real parameters (not counting the parameters entering the Yukawa-flavour-mixing matrices). We removed the four arbitrary parameters from kinetic terms and three from the complex Yukawa coefficients Ci by the use of the GL(2,C) invariance of the theory. This still leaves us with 15 real couplings and five real mass parameters in the model potential V (H1, H2, S). Our next task is to find out which sets of these parameters correspond to physically viable models with a stable potential. We can use the reparametrization invariance to our advantage in constructing the stable potentials. To this end, it is convenient to rephrase the invariance in terms of Lorentz in- variance of the potential, written in terms of bilinears formed from hyperdoublets. Following the analysis of ref. [19–21], we define rµ ≡Φ†σµΦ , where σµ = (1, σi) . (2.6) (2.6) The bilinear four-vector rµ is positive definite.3 That is, rµ vectors span the future light cone, LC+, of a Minkowski space. Thus, in bilinear representation the elliptic and hyperbolic SL(2,C) basis transformations of fields Φ →Φ′ ≡PΦ correspond to proper orthochronous The bilinear four-vector rµ is positive definite.3 That is, rµ vectors span the future light cone, LC+, of a Minkowski space. Thus, in bilinear representation the elliptic and hyperbolic SL(2,C) basis transformations of fields Φ →Φ′ ≡PΦ correspond to proper orthochronous 2Note that in the case of general Yukawa alignment, where Ca i ̸= Cb i , one could still use the SU(2) rotation to set Cu 1 = 0, so that up-type quarks couple only to H2. Most of our subsequent analysis would hold also for this scenario, because it is mostly sensitive only to the large top-quark coupling. The only exception is the electron EDM, for which our analysis covers only a part of the full phase space available in the context of general alignment. g 3Clearly r0 = P i |Hi|2 ≥0. Also rµrµ = 4(|H1|2|H2|2 −(H† 1H2)(H† 2H1)) ≥0 , by Schwartz inequality. – 4 – Lorentz transformations rµ →r′µ = (ΛP)µ νrν, where (ΛP)µ ν ∈SO(1, 3)+. In this notation one can rewrite the Higgs potential (2.2) in a very compact form: Lorentz transformations rµ →r′µ = (ΛP)µ νrν, where (ΛP)µ ν ∈SO(1, 3)+. where Λµν ∈SO(1, 3)+. Let us comment on the role of the kinetic and Yukawa terms in the above construction of the potential. We implicitly assumed that kinetic term becomes diagonal, and the Yukawa term becomes of type-I form in the final frame, after the Lorentz transformation. Thus, they necessarily must be nontrivial in the original, diagonal frame. Indeed, the six degrees of freedom “missing” in the diagonal potential are in this frame evenly divided between the Ci coefficients in the Yukawa Lagrangian and the mixing parameters in the kinetic term, which in the bilinear notation can be written as Kµ(DαΦ)†σµ(DαΦ) . (2.16) (2.16) Here Kµ is some positive definite, but otherwise arbitrary four-vector of unit length (here α refers to the usual space-time indices and µ to potential indices).4 It is amusing to see that exactly a Lorentz boost (a hyperbolic SL(2,C) transformation on fields) is needed to bring an arbitrary Kµ into the canonical form: Kµ →(1; 0, 0, 0), after which the kinetic term is manifestly invariant under Lorentz rotations (elliptic SL(2,C) transformations on fields). These boosts and rotations into the canonical frame activate the whole SO(1, 3)+ group discussed above and thus create all physically viable Lagrangians with bounded potentials. 4In the most general case Kµ has four free parameters, but one parameter is here assumed to be removed by a dilatation, used to bring the length of Kµ to unity. 2.1 Reparametrization invariance and tree-level vacuum stability (2.7), is always positive, and no new conditions arise. However, if λSµ /∈LC+, there are always directions rµ ∈LC+ along which the product λSµrµ is negative. If we in such cases rewrite the quartic part of the potential as Vquartic = 1 4rµ  λµν − 1 4λS λSµλSν  rν + 1 4λS  S2 + λSµrµ 2λS 2 , (2.13) Vquartic = 1 4rµ  λµν − 1 4λS λSµλSν  rν + 1 4λS  S2 + λSµrµ 2λS 2 , (2.13) (2.13) we see that the potential is most negative as a function of S along direction 2λSS2 = −λSµrµ. In this subspace, the potential reduces to the form 4Vquartic = rµλS µνrν, with a new coupling matrix: λS µν ≡λµν − 1 4λS λSµλSν . (2.14) (2.14) – 5 – – 5 – The matrix λS µν is not in general diagonalizable by an SO(1, 3)+ rotation, and it may have also complex eigenvalues. Unfortunately we cannot restrict its properties like we did for λµν, because λS µν does not need not be positive definite in the entire future light cone, but only in the subset of LC+ where λSµrµ is negative. Instead of covering the full range of possibilities, we will require a sufficient (but not necessary) condition that λS µν is positive definite in the future light cone whenever λSµ /∈LC+. We now have the recipe to construct the space of stable potentials: we first choose a λD αα which satisfies eqs. (2.10). Then we generate a vector λD Sµ and check if it satisfies the positivity constraint (2.12), or if the matrix λS µν in eq. (2.14) is positive definite in the subset of LC+ where λSµrµ < 0. Having found an acceptable set, we generate a random Lorentz transformation and define JCAP08(2016)057 λµν ≡ΛµαλD αβΛβν and λµ S ≡Λµν(λD S)ν , (2.15) (2.15) 2.2 Spontaneus symmetry breaking Since we are interested in the cases where the singlet scalar S is a DM candidate, we restrict our considerations to the cases where the potential is unbroken in S direction and hence Z2 symmetric at low temperatures. However, to enhance the strength of the latter phase transition, we also need the S symmetry to be broken at high temperatures before the symmetry breaks in the doublet direction. To this end, we must have negative quadratic term in the S direction in the potential (m2 S > 0). This implies that there may be other minima away from the ⟨S⟩= 0 vacuum, and we must check that none of these minima is the global one at zero temperature. The extremization conditions are: ∂V ∂S = SζS = 0 , where ζS ≡−m2 S + λSS2 + 1 2λSµrµ , (2.17) (2.17) 4In the most general case Kµ has four free parameters, but one parameter is here assumed to be removed by a dilatation, used to bring the length of Kµ to unity. – 6 – and and ∂V ∂H† i = σµ ijHjζµ = 0 , where ζµ = −1 2  M2 µ −1 2λSµS2  + 1 2λµνrν . (2.18) ∂V ∂H† i = σµ ijHjζµ = 0 , where ζµ = −1 2  M2 µ −1 2λSµS2  + 1 2λµνrν . (2.18) (2.18) Eqs. (2.18) are complex, so we have five equations relating vacuum fields to the parameters of the theory. Our goal is to have an unbroken singlet and a broken neutral doublet vacuum at zero temperature. In [19] it was shown that the neutral and charged vacua cannot coexist in the pure 2HDM case. When S = 0, our potential reduces to the pure 2HDM case, and so the above statement applies here as well. Therefore, if we find a neutral vacuum, we know it is the global one in the doublet space and the charged extremum may at best be a saddle point. Moreover, even with S ̸= 0, the doublet-symmetry-breaking pattern is formally similar to the pure 2HDM case, only with an effective mass parameter JCAP08(2016)057 −m2 µ ≡−M2 µ + 1 2λSµS2. (2.19) (2.19) This implies that also any neutral minimum with S breaking is the lowest one in the doublet space. 2.2 Spontaneus symmetry breaking The most general neutral vacuum in 2HDM field space is given by ⟨H1⟩= 1 √ 2  0 v1eiθ1  , and ⟨H2⟩= 1 √ 2  0 v2eiθ2  . (2.20) (2.20) Of course the local SU(2)-gauge invariance guarantees that only the relative phase θ ≡θ2−θ1 is physical, and one could rotate for example θ1 →0. Explicitly we find that this corresponds to to rµ 0 = (v2 1 + v2 2, 2v1v2 cos θ, 2v1v2 sin θ, v2 1 −v2 2) , (2.21) (2.21) so that r2 0 = 0 as it should for a neutral vacuum [19]. This construction actually only ensures that the special point (2.20) is an extremum of the potential. To check that this extremum is a also a minimum, one needs to compute second derivative matrices of the potential corresponding to scalar field masses and require that there are no negative eigenvalues. For the mass of the physical excitation in the singlet direction, this corresponds to requiring that d2V dS2 vev = −m2 S + 1 2λSµrµ 0 ≡M2 S > 0 , (2.22) (2.22) where the left hand side is evaluated at the extremum ⟨S⟩= 0 and ⟨Hi⟩̸= 0 as defined in eq. (2.20). These requirements on the spectrum allow one to set the Lagrangian mass parameters in terms of physical masses and vacuum expectation values (vevs) of the fields. However, it still remains to check that the vacuum with S = 0 is the global minimum for any given set of parameters. It is straightforward to show that the value of the potential at the desired ⟨S⟩= 0, ⟨Hi⟩̸= 0 vacuum is V (⟨S⟩= 0, ⟨Hi⟩̸= 0) = −1 4rµ 0 λµνrν 0 . (2.23) (2.23) In the direction Hi = 0, the potential has at least a directional minimum at ⟨S⟩2 = m2 S/2λS, and the value of the potential in this directional minimum is V (⟨S⟩̸= 0, ⟨Hi⟩= 0) = −1 4λS  M2 S −1 2λSµrµ 0 2 . (2.24) (2.24) – 7 – We impose the condition that the minimum in eq. (2.23) is below that in eq. (2.24). Finally, there is an extremum where both ⟨S⟩̸= 0 and ⟨Hi⟩̸= 0, but this is a local maximum. 2.3 Finite-temperature potential JCAP08(2016)057 The final ingredient we need for our analysis is the effective potential at finite temperature. Here we only consider the leading corrections to the potential, which bring about the sym- metry restoration at high temperatures. In the high-temperature limit, these corrections are accounted for by the thermal masses: m2 a(T) = −m2 a + ca T 2 12 , (2.25) (2.25) (2.25) where (a = 1, 2, 12, S): c1 = cSM + 6λ1 + 2λ3 + λ4 + 1 2λS1 c2 = cSM + 6λ2 + 2λ3 + λ4 + 1 2λS2 c12 = cSM + 3λ6R + λ7R + 1 2λS12R −i  3λ6I −λ7I + 1 2λS12I  cS = 3λS + 2(λS1 + λS2) , (2.26) (2.26) and and cSM = 9 4g2 L + 3 4g2 Y + 3y2 t (2.27) (2.27) is the common SM contribution from the SU(2)L and U(1)Y gauge fields and the top quark. is the common SM contribution from the SU(2)L and U(1)Y gauge fields and the top quark. 2.2 Spontaneus symmetry breaking In addition to the massive scalar S, the physical spectrum contains the usual states arising from the two Higgs doublets: the three neutral scalars h0, H0 and A0, and two charged scalars H±. The diagonalization of the mass matrices is presented in detail in appendix A. The lightest neutral scalar state h0 is identified with the 125 GeV Higgs particle observed LHC, while the masses of the heavier neutral and charged scalar states are constrained to lie above the current limits. 3.1 Theoretical and experimental constraints The couplings between scalar fields tend to run strongly, which typically leads to a relatively low cut-offscale for the validity of the effective theory. To be specific, we demand that our model is consistent up to Λ = 1.5 TeV, i.e. we check that the vacuum is stable and that all couplings remain perturbative5 up to Λ. The 1-loop renormalization-group equations needed for this calculation are summarized in appendix B. Moreover, at zero temperature we impose |⟨H1⟩|2 + |⟨H2⟩|2 = v2 0, ⟨S⟩= 0 (3.1) (3.1) and check that this choice gives the global minimum of the potential as detailed in previous section. JCAP08(2016)057 Second, we address the current experimental constraints. We only accept points that give a light scalar of mass mh0 = 125 GeV, and for which the heavy scalars satisfy the mass limits from direct searches at LEP on charged particles, mH+ > 500 GeV [22], and at LHC on heavy neutral scalars, mH0, mA0 > 600 GeV [23]. For consistency, we also constrain the heavy scalar masses to be below the cut-offΛ = 1.5 TeV. We also take into account the electroweak precision data using the S and T parameters [24]. The necessary formulae for computing S and T can be extracted from [25], where the oblique parameters have been calculated for models with extra doublets and extra singlets. The current values of S and T parameters are S = 0.00 ± 0.08, T = 0.05 ± 0.07 with a correlation factor ρ = 0.90 [26]. We accept points that lie within the 2σ region in the (S, T) plane. Moreover we take into account constraints on the Higgs boson couplings to SM particles using the signal-strength data from the LHC and Tevatron experiments [27–29]. First, we impose the constraint from the invisible width of the Higgs boson from LHC at 2σ level: RI = ΓI ΓI + ΓSM, tot < 0.020, (3.2) (3.2) which here implies Γh0→SS ≲1.0 MeV. Second, we allow for modifications to the Higgs couplings and parametrize the deviations from the SM with parameters af and aV , Leff= aV 2M2 W v0 h0W +W −+ M2 Z v0 h0ZZ  −af X mψ v0 h0ψψ . 3.1 Theoretical and experimental constraints (3.3) (3.3) We accept points which are within 2σ from the best fit values aV = 0.993 , af = 0.968 , (3.4) (3.4) which we obtain by performing a χ2 fit to the Higgs boson signal strength data. We have neglected the imaginary part of af and the h0H+H−coupling in the fit, since we have checked that these couplings are very small.6 which we obtain by performing a χ2 fit to the Higgs boson signal strength data. We have neglected the imaginary part of af and the h0H+H−coupling in the fit, since we have checked that these couplings are very small.6 3 Results We now test our model against experimental constraints and for the consistency of its cos- mological predictions. We scan the parameter space by solving m2 1, m2 2 and m2 12 from the vacuum conditions and create Monte Carlo chains in the coupling constant space by the al- gorithm described in the previous section. We first subject the models to various theoretical and experimental constraints. For models that pass these constraints, we compute the DM abundance and the strength of the phase transition. We find that the model can provide either the observed DM abundance or a strong transition, but not both simultaneously. For the points providing a strong transition, we compute the prediction for the baryon-to-entropy ratio ηB. We show that the parameter space where ηB is sufficiently large is strongly con- strained but not excluded by the current experimental limit on the electric dipole moment of the electron. Finally, we compute the nucleation temperatures Tn and show that typically, and in particular for strong transitions, Tn is much smaller than the critical temperature Tc. – 8 – p p g g µ ≫ ; 5 V 6As a comparison, we have performed the χ2 fit of af and aV with maximum values of the imaginary part of af and h0H+H−coupling for out data points, and we find that the best-fit value does not change significantly. 5For perturbativity we use the upper limit (4π)2 on all scalar couplings. Over large portion of our data points the couplings will remain small also at larger scales µ ≫Λ; the value 1.5 TeV is chosen for concreteness. 6 2 5For perturbativity we use the upper limit (4π)2 on all scalar couplings. Over large portion of our data points the couplings will remain small also at larger scales µ ≫Λ; the value 1.5 TeV is chosen for concreteness. 6As a comparison, we have performed the χ2 fit of af and aV with maximum values of the imaginary part of af and h0H+H−coupling for out data points, and we find that the best-fit value does not change significantly. 3.2 Dark matter abundance and direct detection limits We compute the relic abundance of the S bosons for all models passing the experimental constraints described above. We assume that S is a thermal relic, i.e. we assume that at 5For perturbativity we use the upper limit (4π)2 on all scalar couplings. Over large portion of our data points the couplings will remain small also at larger scales µ ≫Λ; the value 1.5 TeV is chosen for concreteness. 6As a comparison, we have performed the χ2 fit of af and aV with maximum values of the imaginary part of 5For perturbativity we use the upper limit (4π)2 on all scalar couplings. Over large portion of our data points the couplings will remain small also at larger scales µ ≫Λ; the value 1.5 TeV is chosen for concreteness. 6 2 5For perturbativity we use the upper limit (4π)2 on all scalar couplings. Over large portion of our data points the couplings will remain small also at larger scales µ ≫Λ; the value 1.5 TeV is chosen for concreteness. 6As a comparison, we have performed the χ2 fit of af and aV with maximum values of the imaginary part of af and h0H+H−coupling for out data points, and we find that the best-fit value does not change significantly. – 9 – least some the portal couplings λS1, λS2 and λS12 are sufficiently large (larger than about 10−7 [30]). We then apply the standard freeze-out formalism [31], employing the accurate approximation scheme introduced in [32]. The relevant annihilation channels of our WIMP candidate are SS →h0h0, H0H0, A0A0, h0H0, h0A0, H0A0, H+H−, W +W −, ZZ, ¯ff. (3.5) (3.5) Cross-sections for all these processes are given in a very compact form in appendix C. To treat cases where S is a subdominant DM candidate, we define the ratio [33] frel = ΩSh2 0.12 , (3.6) JCAP08(2016)057 (3.6) which expresses how large fraction of the observed DM abundance is in form of S bosons. All annihilation channels are directly proportional to the couplings between the S boson and the Higgs fields, although the precise dependence is rather complicated (see appendix C). Since the relic abundance is roughly proportional to the inverse of the annihilation cross section, large (small) couplings corresponds to a small (large) relic abundance. Direct search limits for S follow from the bound on the spin-independent cross section for S scattering offnucleons. 3.2 Dark matter abundance and direct detection limits Shown is the LUX bound on a set of models passing all experimental constraints described in section 3.1 and for which frel ≥0.01. 3.2 Dark matter abundance and direct detection limits It is given by σSI = λ2 efff2 N 4π µm2 Nv2 0 m4 hM2 S , (3.7) (3.7) where mN = 0.939 GeV is the nucleon mass, µ = mNMS/(mN + MS) is the reduced mass of the nucleon-scalar system, and fN ≈0.30 [32] gives the strength of the Higgs-nucleon coupling: gh0N ¯ N = fNmN/v0. Finally, the effective SSh0 coupling is given by where mN = 0.939 GeV is the nucleon mass, µ = mNMS/(mN + MS) is the reduced mass of the nucleon-scalar system, and fN ≈0.30 [32] gives the strength of the Higgs-nucleon coupling: gh0N ¯ N = fNmN/v0. Finally, the effective SSh0 coupling is given by eff≡1 2 hRN44 cos β sin θ −RN42 cos β cos θ + RN41 sin β  λS12I + RN42 cos β sin θ −RN44 cos β cos θ −RN43 sin β  λS12R + cos β RN43 cos θ + RN41 sin θ  λS1 −sin βRN44λS2 i , (3.8) (3.8) where β is the vacuum mixing angle and θ the phase between the doublet vevs, and RNij are the components of the 4 × 4-mixing matrix between the neutral scalar fields given in eq. (A.8). Currently the most stringent limit for σSI come from the LUX experiment [34]. However, in the case of a subdominant DM, the LUX bound is not directly related to σSI shown in eq. (3.7). Instead, assuming that all DM components cluster similarly, the actual signal strength from S bosons is suppressed by the fraction frel. The relevant quantity to compare with the LUX limit then is σeff= frelσSI. (3.9) (3.9) We show the effect of this limit on the models passing all experimental bounds on figure 1. Note that the scatter in σeffas a function of mS is much larger here than in the singlet extension of the SM [33]. Note how also the models with relatively low abundance are constrained by the LUX data. – 10 – – 10 – XENON100 LUX 100 200 300 400 -46.0 -45.5 -45.0 -44.5 -44.0 -43.5 -43.0 MS (GeV) log10(σeff/cm2) log10(frel) -2.0 -1.5 -1.0 -0.5 0 Figure 1. Shown is the LUX bound on a set of models passing all experimental constraints described in section 3.1 and for which frel ≥0.01. XENON100 LUX 100 200 300 400 -46.0 -45.5 -45.0 -44.5 -44.0 -43.5 -43.0 MS (GeV) log10(σeff/cm2) JCAP08(2016)057 Figure 1. 3.3 Electroweak phase transition One of the necessary conditions for a successful baryogenesis scenario is the departure from thermal equilibrium. In EWBG this is provided by a strong first-order phase transition. In pure 2HDMs, with only leading thermal corrections (i.e. with thermal masses), the phase transition is of second order. While full one-loop effects may induce a first-order transition, it still tends to be rather weak [8, 9]. In a model with a singlet scalar, a strong transition may take place with just the leading corrections (2.25), given a specific two-step symmetry- breaking pattern [35]. This means that, when passing from high towards low temperatures, a minimum of the potential is generated first along the singlet direction. At lower temperature, then, the potential develops a minimum where the doublets have non-zero vevs while the singlet symmetry is restored, (⟨h1⟩, ⟨h2⟩; ⟨s⟩) = (0, 0; 0) →(0, 0; w(T)) →(v1(T), v2(T); 0), ensuing the actual electroweak phase transition. This pattern of minima should of course develop in such a way that the true ground state at T = 0 is given by eq. (3.1). Including the thermal mass corrections of eq. (2.25) to the potential, we find the tran- sition temperatures. In particular the electroweak transition temperature, Tc, where the two minima are degenerate is determined by V (0, 0, wc, Tc) = V (v1c, v2c, 0, Tc), (3.10) (3.10) where wc = w(Tc) and vic = vi(Tc). To determine when the transition is strong enough, we require, as usual, that vc/Tc ≥1 , (3.11) vc/Tc ≥1 , (3.11) where vc = p v2 1c + v2 2c. It turns out not to be possible to have simultaneously strong tran- sition and dominant DM candidate in the model with frel ≈1. This is because having a strong transition requires that at least some of the mixing couplings λSi are large. However, as we noted above, large mixing introduces large annihilation cross sections for S bosons and hence small relic abundances. Qualitatively the behaviour is the same as in the pure singlet model [32, 33]: a strong first-order EWPT and large frel ≈1 are realized in different portions of the parameter space. where vc = p v2 1c + v2 2c. It turns out not to be possible to have simultaneously strong tran- sition and dominant DM candidate in the model with frel ≈1. 3.3 Electroweak phase transition This is because having a strong transition requires that at least some of the mixing couplings λSi are large. However, as we noted above, large mixing introduces large annihilation cross sections for S bosons and hence small relic abundances. Qualitatively the behaviour is the same as in the pure singlet model [32, 33]: a strong first-order EWPT and large frel ≈1 are realized in different portions of the parameter space. – 11 – 100 150 200 250 300 350 400 0.5 1 2 5 MS(GeV) λeff 10-5 10-4 0.001 0.010 0.100 1 100 150 200 250 300 350 400 frel MS(GeV) Figure 2. Scanned data points which give a strong first-order EWPT. Yellow points are excluded by direct DM searches. 10-5 10-4 0.001 0.010 0.100 1 100 150 200 250 300 350 400 frel MS(GeV) 100 150 200 250 300 350 400 0.5 1 2 5 MS(GeV) λeff JCAP08(2016)057 Figure 2. Scanned data points which give a strong first-order EWPT. Yellow points are excluded by direct DM searches. Left panel of figure 2 shows the correlation between the DM particle mass MS and the effective coupling λefffor models with strong EWPT. Accepted values of λeffincrease as a function of MS because both quantities are linearly proportional to the mixing couplings λSi. Right panel of figure 2 shows the correlation between MS and the relative relic abundance frel for the same set. Note that for MS between the Higgs resonance and MW , both a strong transition and a relatively large DM abundance, frel ≈0.15 could be obtained, but this region is now excluded by LUX. The fact that LUX most strongly constrains models with small λeffis because frel is roughly inversely proportional to the square of λeff, and this to large extent cancels the direct λeffdependence in σSI. Larger mass region beyond the Higgs resonance is less constrained because the DM-number density falls with increasing mass. 3.4 Electron EDM constraint The non-observation of electric dipole moments (EDMs) of electrons, neutrons and atoms gives stringent bounds on CP-violating interactions in multi-Higgs models. As shown in [36], currently the most stringent bound for 2HDMs arises from the electron EDM, for which the ACME experiment gives an upper limit |de| < 8.7 × 10−29ecm , (3.12) (3.12) with 90% confidence level [37]. We calculate de for the points which give a strong first-order EWPT using the results from ref. [38], where Barr-Zee type contributions to fermionic EDM were calculated in 2HDM. These results are directly applicable here as well, because the singlet scalar S does not directly couple to gauge fields. In figure 3, we show the distribution of models passing all previous cuts as a function of de and the neutral scalar mixing matrix element RN42, which expresses the projection of h0 to complex part of the second doublet: RN42 ≡⟨H0 2I|h0⟩≡sin ∆CP . (3.13) (3.13) sin ∆CP is given in terms of the various mixing angles in eq. (A.8). The red region is excluded by the electron EDM constraint. Small de naturally correlates with small sin ∆CP, because the size of sin ∆CP is proportional to the size of the CP-violating mixing in the model. – 12 – 0.00 0.02 0.04 0.06 0.08 -3 -2 -1 0 1 2 |sinΔCP| log10(de/8.7×10-29ecm) 0.00 0.02 0.04 0.06 0.08 -3 -2 -1 0 1 2 |sinΔCP| log10(de/8.7×10-29ecm) Figure 3. Scatter plot of all models with strong enough EWPT as a function of the mixing parameter sin ∆CP and the electron EDM de. The red region is excluded by the eEDM limit. JCAP08(2016)057 Figure 3. Scatter plot of all models with strong enough EWPT as a function of the mixing parameter sin ∆CP and the electron EDM de. The red region is excluded by the eEDM limit. 3.5 Baryogenesis The actual baryogenesis mechanism in our model relies on CP-violating interactions of the top quark with the expanding phase-transition walls. The CP violation comes directly from the spatial evolution of the complex phases of the Higgs field H2, which renders the top mass a complex-valued function of the spatial coordinate across the wall. The first step for us is then to work out the evolution of the scalar fields over the bubble wall. We shall approximate the true phase-transition-wall profile in the usual way, by the stationary path that extremizes the Euclidean one-dimensional action Z dz  |DzH1| + |DzH2| + 1 2|∂zS| + V + . . .  , (3.14) (3.14) between two degenerate minima at critical temperature T = Tc, for which the condition (3.10) holds. The covariant derivatives involve the classical Zµ field: Dµ = ∂µ −ig/(2 cos θW)Zµ. We write the neutral components of the doublets as hjeiϕj and observe that the effective potential can depend only on the relative phase ϕ ≡ϕ1 −ϕ2. Following ref. [8], we work in the gauge Zµ = 0, whereby we need to account for four fields: h1, h2, S and ϕ, while solving the path. The relevant reduced action is S1 = Z dz X i 1 2(∂zhi)2 + 1 2(∂zS)2 + 1 2 h2 1h2 2 h2 1 + h2 2 (∂zϕ)2 + V (h1, h2, S, ϕ, Tc) ! . (3.15) (3.15) The invariance of the potential under the change of the total phase ϕ1 + ϕ2 implies a conservation law, which in the Zµ = 0 gauge allows us to work out the phase ϕ2 in terms of the relative phase ϕ [8]: The invariance of the potential under the change of the total phase ϕ1 + ϕ2 implies a conservation law, which in the Zµ = 0 gauge allows us to work out the phase ϕ2 in terms of the relative phase ϕ [8]: 2 ∂zϕ2 = − h2 1 h2 1 + h2 2 ∂zϕ . (3.16) (3.16) The complex, spatially-varying top mass can now be constructed from the phase ϕ2(x) and the modulus h2(z): The complex, spatially-varying top mass can now be constructed from the phase ϕ2(x) and the modulus h2(z): mt(z) = yt √ 2h2(z)eiϕ2(z). (3.17) (3.17) In fact, one does not need to solve for the top phase, since only its derivative, given by eq. 3.5 Baryogenesis (3.16), appears in the source term for the diffusion equations for chemical potentials: In fact, one does not need to solve for the top phase, since only its derivative, given by eq. (3.16), appears in the source term for the diffusion equations for chemical potentials: St = ξw K8,t(x2 t ϕ′ 2)′ −K9,tx2 t x2′ t ϕ′ 2  . (3.18) (3.18) – 13 – 0.00 0.02 0.04 0.06 0.08 -5 -4 -3 -2 -1 0 |sinΔCP| log10(ηB/8.7×10-11) -2.0 -1.5 -1.0 -0.5 0.0 -5 -4 -3 -2 -1 0 log10(de/8.7×10-29ecm) log10(ηB/8.7×10-11) Figure 4. Left: shown is the correlation between the baryon-to-entropy ratio ηB and the mixing matrix element sin ∆CP. Red dots correspond to models for which Tn cannot be found (in the thin- wall approximation). Right: the correlation between ηB and de. The red region is excluded by the eEDM limit and the black line shows the observed baryon-to-entropy ratio. 0.00 0.02 0.04 0.06 0.08 -5 -4 -3 -2 -1 0 |sinΔCP| log10(ηB/8.7×10-11) -2.0 -1.5 -1.0 -0.5 0.0 -5 -4 -3 -2 -1 0 log10(de/8.7×10-29ecm) log10(ηB/8.7×10-11) JCAP08(2016)057 Figure 4. Left: shown is the correlation between the baryon-to-entropy ratio ηB and the mixing matrix element sin ∆CP. Red dots correspond to models for which Tn cannot be found (in the thin- wall approximation). Right: the correlation between ηB and de. The red region is excluded by the eEDM limit and the black line shows the observed baryon-to-entropy ratio. Here ξw is the wall velocity, primes denote ∂zT and Kn,t are dimensionless functions of xt ≡|mt|/T arising from phase-space averaging of certain kinematic variables defined in [39]. Here ξw is the wall velocity, primes denote ∂zT and Kn,t are dimensionless functions of xt ≡|mt|/T arising from phase-space averaging of certain kinematic variables defined in [39]. Given the source, one can calculate chemical potentials µj(z) for top, bottom, anti-top and Higgs by solving a set of transport equations defined in [7] Finally the baryon to entropy Here ξw is the wall velocity, primes denote ∂zT and Kn,t are dimensionless functions of xt ≡|mt|/T arising from phase-space averaging of certain kinematic variables defined in [39]. Gi th l l t h i l t ti l ( ) f t b tt ti t Given the source, one can calculate chemical potentials µj(z) for top, bottom, anti-top and Higgs by solving a set of transport equations defined in [7]. 3.5 Baryogenesis Finally the baryon-to-entropy ratio ηB ≡nB/s is given by ηB = 405 4π2ξwg∗Tc Z ∞ 0 dz Γsph(z)µBL(z)e−45Γsph(z)z/4ξw. (3.19) (3.19) We take ξw = 0.1 for the wall velocity and g∗= 106.75 for the number of degrees of freedom in the plasma. The left-chiral baryon chemical potential is We take ξw = 0.1 for the wall velocity and g∗= 106.75 for the number of degrees of freedom in the plasma. The left-chiral baryon chemical potential is µBL = 1 2(1 + 4K1,t)µt + 1 2(1 + 4K1,b)µb −2K1,tcµtc. (3.20) (3.20) For the sphaleron rate we use a formula interpolating between the symmetric and the broken phase [8], 6 40 ( )/T Γsph(z) = min(10−6Tc, 2.4Tce−40v(z)/Tc), (3.21) (3.21) where v(z)2 = h1(z)2 + h2(z)2. where v(z)2 = h1(z)2 + h2(z)2. In the left panel of figure 4 we show how the baryon-to-entropy ratio relative to the observed value ηobs B = 8.7 × 10−11 [40] correlates with the CP-violation-sensitive parameter sin ∆CP defined in (3.13). Shown are only the points which survive the eEDM bound. As expected, the size of sin ∆CP correlates with the size of ηB. This trend is similar to the correlation between sin ∆CP and de shown in figure 3. However, a large ηB does not always imply a large de, as is clear from the right panel of figure 4, where we show the correlation between de and ηB, again for points that pass the EDM bound. Apparently, while both quantities are sensitive to the CP-violating parameters in the model, they can be sensitive to different linear combinations of them, so that large ηB may be obtained simultaneously with a small enough de. Figure 5 shows the distributions of various physical parameters in our parametric scan. Orange colour refers to models that pass all experimental cuts described in sections 3.1 and 3.2, and give a strong EWPT, blue to models that in addition satisfy EDM constraint and green to models which also give large baryon-to-entropy ratio ηB/ηobs B ∈[0.5, 2]. These plots – 14 – Figure 5. Shown are the frequency distributions of the vevs of the scalar fields and the critical temperature as well as scatter plots for the masses of the new heavy scalar particles in our parameter scan. For details see the text. JCAP08(2016)057 Figure 5. 3.5 Baryogenesis Shown are the frequency distributions of the vevs of the scalar fields and the critical temperature as well as scatter plots for the masses of the new heavy scalar particles in our parameter scan. For details see the text. must be interpreted with care, since our scans were partly tuned by hand. Nevertheless, we see that none of the vevs can be very large and in particular both v1 and v2 need to be nonzero. Also the critical temperature is bounded from above: Tc ≲100 GeV. Finally for the models with large ηB, the new scalar masses are in general bound from above: mH, mA0, mH± ≲ 1.4 TeV and mS ≲400 GeV, which is encouraging from the point of view of experimental verifiability of the model. 3.6 Bubble nucleation So far we have implicitly assumed that the bubble nucleation takes place at a temperature not too different from the critical temperature. This is typically the case in models where the first- order phase transition is effected by cubic corrections to potential from infrared modes, which leads to rather mild supercooling and small latent heat release. Here the situation is different, because the barrier between the degenerate minima is essentially due to a tree-level term. Thus a stronger supercooling and more latent heat release may be expected, or even a possibil- ity of a formation of a metastable vacuum where the electroweak breaking never takes place. We study the nucleation problem in the thin-wall limit [41]. The bubble nucleation rate is given by / is given by Γ ∼T 4 S3(T) 2πT 3/2 exp  −S3(T) T  , (3.22) (3.22) where S3(T) is the three-dimensional action for an O(3)-symmetric bubble. In the thin-wall limit, it is given by 3 S3(T) = 16π 3 σ3 ∆V (T)2 , (3.23) (3.23) where ∆V (T) is the potential energy difference between the electroweak-symmetric and electroweak-broken minima and σ is the surface tension, σ = Z dφ √ 2V , (3.24) (3.24) integrated along the path from the symmetric to the broken minimum at temperature T = Tc. The bubble nucleation temperature Tn is defined as the temperature at which creating at – 15 – 0.4 0.5 0.6 0.7 0.8 0.9 -5 -4 -3 -2 -1 0 Tn/Tc log10(ηB/8.7×10-11) 0.4 0.5 0.6 0.7 0.8 0.9 -3 -2 -1 0 1 Tn/Tc log10(α/αmax) Figure 6. Left: shown is the baryon asymmetry ηB as a function of the ratio of the nucleation and critical temperatures Tn/Tc. Right: shown is the bound for finding a deflagration wall. Only models below the line α/αmax = 1 are allowed. For yellow points the nucleation temperatures were rescaled by κ = 0.7. See text for details. We used αmax = 0.38, corresponding to ξw ≈0.1. 0.4 0.5 0.6 0.7 0.8 0.9 -5 -4 -3 -2 -1 0 Tn/Tc log10(ηB/8.7×10-11) 0.4 0.5 0.6 0.7 0.8 0.9 -3 -2 -1 0 1 Tn/Tc log10(α/αmax) JCAP08(2016)057 Figure 6. Left: shown is the baryon asymmetry ηB as a function of the ratio of the nucleation and critical temperatures Tn/Tc. Right: shown is the bound for finding a deflagration wall. Only models below the line α/αmax = 1 are allowed. 3.6 Bubble nucleation For yellow points the nucleation temperatures were rescaled by κ = 0.7. See text for details. We used αmax = 0.38, corresponding to ξw ≈0.1. least one bubble per horizon volume is of order one. This condition can be written as S3(Tn) Tn = −log 3 4π H(Tn) Tn 4 2πTn S3(Tn) 3/2! . (3.25) (3.25) We show the results of the nucleation-temperature calculation for our data set on the left panel of figure 6. Obviously, a large number of points displayed in figure 4 are missing in figure 6. The reason is that for these models, indicated by red dots in figure 4, no solution to eq. (3.25) was found. In these cases, in the thin-wall approximation, the fields were trapped in the false vacuum. Moreover, of the surviving models only four give large ηB. The situation is actually more dire than this: another implicit assumption in our baryon asymmetry calculation is that the transition walls are subsonic deflagrations, which is required for efficient diffusion of particle asymmetries across the bubble wall. However, with a large latent heat release the walls tend to be supersonic detonations instead. A full microscopic analysis of wall dynamics including a computation of the wall friction is beyond the scope of this paper. However, a deflagration wall must necessarily satisfy a condition [42] α ≡∆V (Tn) ρ(Tn) < 1 3(1 −ξw)−13/10 ≡αmax , (3.26) (3.26) where ρ(Tn) is the radiation energy density in the symmetric phase. We show this condition in the right panel of figure 6 (blue dots) for the set of models shown in the left panel. As ex- pected, for the models with the lowest nucleation temperatures, deflagrations are not possible. This applies in particular to all four surviving models with a large baryon-to-entropy ratio. There are two issues that ameliorate the situation. First, the validity of the thin-wall limit actually requires a small latent heat and/or a large surface tension, which is often not the case here. When not applicable, thin-wall limit tends to overestimate the action S3(T), and hence underestimate the nucleation rate and eventually Tn. Accurate calculation of the nucleation rate is quite complicated in the full model, however, and we do not pursue it here. Instead, we compare the nucleation temperatures found in the thin-wall limit and in the full calculation in the simpler, singlet extension of the SM, studied for example in [32, 33]. 3.6 Bubble nucleation In – 16 – 70 80 90 100 110 120 130 40 60 80 100 120 Tc(GeV) Tn(GeV) Figure 7. Shown are the nucleation temperatures in a scan over the parameters of the singlet extension of the SM. Blue dots correspond to the thin-wall temperature and the yellow dots to a full calculation. Gray lines connect pairs corresponding to the same physical parameters. The dotted line corresponds to Tn = Tc. 70 80 90 100 110 120 130 40 60 80 100 120 Tc(GeV) Tn(GeV) JCAP08(2016)057 Figure 7. Shown are the nucleation temperatures in a scan over the parameters of the singlet extension of the SM. Blue dots correspond to the thin-wall temperature and the yellow dots to a full calculation. Gray lines connect pairs corresponding to the same physical parameters. The dotted line corresponds to Tn = Tc. practice we minimize the action practice we minimize the action S3(T) = 4π Z r2dr 1 2 dh dr 2 + 1 2 dS dr 2 + VSSM(h, S, T) ! , (3.27) (3.27) where h is the SM-Higgs field, and VSSM(h, S, T) is the singlet-model potential. The results of this analysis are shown in figure 7. The blue dots show the nucleation temperature in the thin-wall approximation and the yellow dots the same quantity found from the solving minimizing the action (3.27) . As expected, the thin-wall approximation underestimates nucleation temperatures significantly. We find that the true nucleation tem- perature Tn and the thin-wall value T tw n are related by Tn = Tc −κ (Tc −T tw n ), where the coefficient κ to some extent depends on MS and λS, but is less than 0.7. We anticipated this result in the deflagration limit shown in the right panel of figure 6, where the yellow dots were found by redefining all thin-wall nucleation temperatures by the above equation with κ = 0.7. We believe that this scaling conservatively represents the effect of going beyond thin-wall approximation in the full 2HDM and singlet model, and hence shows that most parameter sets may in fact be deflagrations. We can also tune our search to prefer models with a higher critical temperatures. Indeed, as is clearly seen from figure 7, the nucleation temperature approaches the critical tempera- ture when Tc gets higher in the singlet extension of the SM and this feature persists also in the full 2HDSM. 3.6 Bubble nucleation Hence, we made a new parametric scan, where we accept only models with Tc > 80 GeV. The result of this scan is seen in figure 8, where the left panel again shows the baryon- to-entropy ratio and in the right panel the deflagration bound. We now found more points with large asymmetry and, in particular after one rescales the thin-wall nucleation tempera- tures as explained above, these points are now well below the deflagration bound eq. (3.26). 4 Conclusions and outlook We have studied the viability of a two-Higgs-doublet and inert-singlet model for EWBG and for DM, taking into account also all existing observational and collider constraints. Our model – 17 – 0.5 0.6 0.7 0.8 0.9 -2.0 -1.5 -1.0 -0.5 0.0 Tn/Tc log10(ηB/8.7×10-11) 0.5 0.6 0.7 0.8 0.9 -3 -2 -1 0 Tn/Tc log10(α/αmax) Figure 8. Left: shown is the baryon asymmetry ηB as a function of Tn/Tc for the new scan with Tc > 80GeV (here Tn is the thin-wall nucleation temperature). Right: the deflagration bound for the set displayed in the left plot as a function of Tn/Tc. We again used αmax = 0.38. 0.5 0.6 0.7 0.8 0.9 -2.0 -1.5 -1.0 -0.5 0.0 Tn/Tc log10(ηB/8.7×10-11) 0.5 0.6 0.7 0.8 0.9 -3 -2 -1 0 Tn/Tc log10(α/αmax) JCAP08(2016)057 Figure 8. Left: shown is the baryon asymmetry ηB as a function of Tn/Tc for the new scan with Tc > 80GeV (here Tn is the thin-wall nucleation temperature). Right: the deflagration bound for the set displayed in the left plot as a function of Tn/Tc. We again used αmax = 0.38. is based on the maximal GL(2,C) reparemetrization symmetry. This implies a universal Yukawa-alignment scheme, where both Higgs fields couple similarly to all fermions and there are no FCNCs. Exploiting the GL(2,C) symmetry, the the model can, in a particular basis, be written with a type-I Yukawa sector combined with the most general CP-violating potential. Following [19–21] we implemented a novel way to construct potentials with a tree- level stability and to study the symmetry breaking patterns at finite temperatures. This construction was based on the Lorentz symmetry induced by the reprametrization symmetry on bilinears formed from Higgs doublets. These techniques are applicable to all 2HDM models, and they proved extremely useful when performing large-scale parametric scans over the multidimensional phase space of the model. Dark matter and the strength of the electroweak phase transition in the model follow a similar pattern to the pure singlet extension: in accordance with [33], we find that strong two- step phase transitions are easily found, but they are consistent only with a subleading DM. Likewise, we find that experimental and observational constraints are fairly easy to avoid, with the outstanding exception of the electron-EDM bound, which strongly constrains the CP-violating parameters on the model. 4 Conclusions and outlook EDM constraints are particularly important because creating a large baryon asymmetry during the electroweak phase transition requires large CP-violating parameters; we found that the electron EDM indeed strongly constrains the phase space consistent with baryogenesis. Yet the bounds are not as strong here as in the pure 2HDM case [8], and we found a number of models consistent with all requirements. Finally, we observed that two-step transitions may suffer from an unexpected problem of providing a too strong phase transition. We found that fields may get trapped in the metastable minimum, and transition walls may not be subsonic as required by a successful baryogenesis scenario. However, our analysis in the full model was restricted to the thin-wall approximation. We then studied the bubble nucleation in full generality in the case of a pure singlet extension of the SM. While the generic problem of too strong transitions remained, we found that thin-wall limit tends to overestimate the strength of the transition. Based on these results we argued that, when corrected for the thin-wall bias, the walls may well remain subsonic in the full 2HDSM. In a revised scan concentrating to models with a large critical temperature, we found many models potentially consistent with all available constraints. While our results are not a definite proof, they provide a strong indication of a success of baryognesis in the 2HDM and an inert singlet model. Settling the issue beyond any doubt – 18 – would require two significant improvements. First is a detailed analysis of the bubble wall dynamics, including a microscopic computation of the friction on the wall. Second is going beyond the Tc-bounce solution, when solving the scalar field profiles over the bubble wall to compute the top quark mass profile and eventually the baryogenesis source. These are both very interesting topics that deserve to be studied in detail in the future. Acknowledgments We thank Jim Cline for useful comments and discussions and collaboration at the early stages of this work. KK would like to thank Stephan Huber, Mikko Laine and Jose M. No for useful discussions during the 2016 MIAPP programme “Why is there more Matter than Antimatter in the Universe?”. This work was financially supported by the Academy of Finland projects 278722 and 267842. The CP3-Origins centre is partially funded by the Danish National Research Foundation, grant number DNRF90. VV is supported by the Magnus Ehrnrooth foundation. TA acknowledges partial funding from a Villum foundation grant. JCAP08(2016)057 A Diagonalization of mass matrices In this appendix we show in detail the diagonalization of the scalar mass matrices. Follow- ing [43], we write (A.1) rµ = φxΣµ xyφy , (A.1) where φ = H+ 1I, H+ 2I, H+ 1R, H+ 2R, H0 1I, H0 2I, H0 1R, H0 2R  , (A.2) (A.2) where the subscripts R and I again denote the real and imaginary parts, respectively, and the matrices Σµ are block diagonal matrices consisting of 4 × 4 elements where the subscripts R and I again denote the real and imaginary parts, respectively, and the matrices Σµ are block diagonal matrices consisting of 4 × 4 elements Σ0 = 14 , Σ1 =  σ1 0 0 σ1  , Σ2 =  0 −iσ2 iσ2 0  , Σ3 =  σ3 0 0 σ3  . (A.3) (A.3) Now the (a, b) element of the mass matrix is Now the (a, b) element of the mass matrix is Now the (a, b) element of the mass matrix is Now the (a, b) element of the mass matrix is ∂2V ∂φa∂φb φ=⟨φ⟩ = −Σµ abMµ + Σµ abλµν⟨rν⟩+ 2Σµ ax⟨φx⟩λµν⟨φy⟩Σν yb , (A.4) (A.4) and the minimum conditions are and the minimum conditions are ∂V ∂φa φ=⟨φ⟩ = (−Σµ axMµ + Σµ axλµν⟨rν⟩) ⟨φx⟩= 0 . (A.5) (A.5) Let us consider the neutral part of the mass matrix. The eigenstate corresponding to eigen- value zero is G0 = NGΣ5⟨φ0⟩, where Σ5 = Σ0Σ1Σ2Σ3 and NG is the normalization factor. Now Let us consider the neutral part of the mass matrix. The eigenstate corresponding to eigen- value zero is G0 = NGΣ5⟨φ0⟩, where Σ5 = Σ0Σ1Σ2Σ3 and NG is the normalization factor. Now G0 = cos β cos θ H0 1R + sin β H0 1I −cos β sin θ H0 2R , (A.6) (A.6) where β = arctan v2 v1 . A Diagonalization of mass matrices (A.7) (A.7) – 19 – – 19 – Hence we can diagonalize the neutral mass matrix by Hence we can diagonalize the neutral mass matrix by RN =     1 0 0 0 0 cycz −cysz sy 0 sxsycz + cxsz cxcz −sxsysz −sxcy 0 sxsz −cxsycz cxsysz + sxcz cxcy         cβcθ sβ −cβsθ 0 −sβcθ cβ sβsθ 0 sθ 0 cθ 0 0 0 0 1    , (A.8) (A.8) where we have used the short-hand notations sα ≡sin α, cα ≡cos α, and the neutral mass eigenstates are where we have used the short-hand notations sα ≡sin α, cα ≡cos α, and the neutral mass eigenstates are JCAP08(2016)057 JCAP08(2016)057     G0 A0 H0 h0    = RN     H0 1I H0 2I H0 1R H0 2R    . (A.9) (A.9) On the charged sector there are two eigenstates with eigenvalue zero: G1 = NG1Σ5⟨φ0⟩ and G2 = NG2⟨φ0⟩. Now On the charged sector there are two eigenstates with eigenvalue zero: G1 = NG1Σ5⟨φ0⟩ and G2 = NG2⟨φ0⟩. Now G1 = cos β sin θ H+ 1R + cos β cos θ H+ 1I + sin β H+ 2I , G2 = cos β cos θ H+ 1R −cos β sin θ H+ 1I + sin β H+ 2R . (A.10) (A.10) Hence charged Goldstone bosons are G± = cos βe±iθH± 1 + sin βH± 2 . The charged mass matrix in the basis {H+ 1 , H+ 2 } can be diagonalized by RC =  cβeiθ sβ −sβeiθ cβ  , (A.11) (A.11) and charged mass eigenstates are and charged mass eigenstates are  G+ H+  = RC  H+ 1 H+ 2  . A Diagonalization of mass matrices (A.12) (A.12) B 1-loop beta functions for scalar couplings For completeness we show the 1-loop beta functions, βλi ≡ dλi d ln µ, (B.1) βλi ≡ dλi d ln µ, (B.1) – 20 – we used to compute the perturbativity limits on the model parameters: 16π2βλ1 = 24λ2 1 + 2λ2 3 + 2λ3λ4 + λ2 4 + 4|λ5|2 + 12|λ6|2 + 1 2λ2 S1 −3 3g2 L + g2 Y  λ1 + 3 8 3g4 L + 2g2 Lg2 Y + g4 Y  , (B.2) 16π2βλ2 = 24λ2 2 + 2λ2 3 + 2λ3λ4 + λ2 4 + 4|λ5|2 + 12|λ7|2 + 1 2λ2 S2 −3 3g2 L + g2 Y  λ2 + 3 8 3g4 L + 2g2 Lg2 Y + g4 Y  + 12y2 t λ2 −6y4 t , (B.3) 16π2βλ3 = (λ1 + λ2)(12λ3 + 4λ4) + 4λ2 3 + 2λ2 4 + 8|λ5|2 + 4|λ6|2 2 (B.2) (B.3) JCAP08(2016)057 (B.4) −3 3g2 L + g2 Y  λ3 + 3 4 3g4 L −2g2 Lg2 Y + g4 Y  + 6y2 t λ3, 16π2βλ4 = 4(λ1 + λ2)λ4 + 8λ3λ4 + 4λ2 4 + 32|λ5|2 + 10 |λ6|2 + |λ7|2 + 4Re (λ∗ 6λ7) + |λS12|2 −3 3g2 L + g2 Y  λ4 + 3g2 Lg2 Y + 6y2 t λ4, (B.5) 16π2βλ5 = (4λ1 + 4λ2 + 8λ3 + 12λ4)λ5 + 5λ2 6 + 2λ6λ7 + 5λ2 7 + 1λ2 S12 −3 3g2 L + g2 Y  λ5 + 6y2 t λ5, (B.6) 3 3gL + gY  λ3 + 4 3gL 2gLgY + gY  + 6yt λ3, 16π2βλ4 = 4(λ1 + λ2)λ4 + 8λ3λ4 + 4λ2 4 + 32|λ5|2 + 10 |λ6|2 + |λ7|2 + 4Re (λ∗ 6λ7) + |λS12|2 −3 3g2 L + g2 Y  λ4 + 3g2 Lg2 Y + 6y2 t λ4, (B.5) 16π2βλ5 = (4λ1 + 4λ2 + 8λ3 + 12λ4)λ5 + 5λ2 6 + 2λ6λ7 + 5λ2 7 (B 6) (B.5)  16π2βλ5 = (4λ1 + 4λ2 + 8λ3 + 12λ4)λ5 + 5λ2 6 + 2λ6λ7 + 5λ2 7 + 1 2λ2 S12 −3 3g2 L + g2 Y  λ5 + 6y2 t λ5, (B.6) + 1 2λ2 S12 −3 3g2 L + g2 Y  λ5 + 6y2 t λ5, (B.6) 2 16π2βλ6 = (24λ1 + 6λ3 + 8λ4)λ6 + (6λ3 + 4λ4)λ7 + (20λ∗ 6 + 4λ∗ 7)λ5 + λS1λS12 −3 3g2 L + g2 Y  λ6 + 3y2 t λ6, (B.7) 2 16π2βλ6 = (24λ1 + 6λ3 + 8λ4)λ6 + (6λ3 + 4λ4)λ7 + (20λ∗ 6 + 4λ∗ 7)λ5 + λS1λS12 −3 3g2 L + g2 Y  λ6 + 3y2 t λ6, (B.7) 2 16π2βλ6 = (24λ1 + 6λ3 + 8λ4)λ6 + (6λ3 + 4λ4)λ7 + (20λ∗ 6 + 4λ∗ 7)λ5 + λS1λS12 −3 3g2 L + g2 Y  λ6 + 3y2 t λ6, (B.7) 16π2βλ7 = (6λ3 + 4λ4)λ6 + (24λ2 + 6λ3 + 8λ4)λ7 + (4λ∗ 6 + 20λ∗ 7)λ5 + λS2λS12 −3 3g2 L + g2 Y  λ7 + 9y2 t λ7, (B.8) 16π2βλ7 = (6λ3 + 4λ4)λ6 + (24λ2 + 6λ3 + 8λ4)λ7 + (4λ∗ 6 + 20λ∗ 7)λ5 + λS2λS12 −3 3g2 L + g2 Y  λ7 + 9y2 t λ7, (B.8) 16π2βλS = 18λ2 S + 2λ2 S1 + 2λ2 S2 + 4|λS12|2, (B.9) 16π2βλS1 = (12λ1 + 6λS)λS1 + (4λ3 + 2λ4)λS2 + 4λ2 S1 + 12Re (λ∗ 6λS12) + 4|λS12|2 −3 2 3g2 L + g2 Y  λS1, (B.10) 16π2βλS2 = (4λ3 + 2λ4)λS1 + (12λ2 + 6λS)λS2 + 4λ2 S2 + 12Re (λ∗ 7λS12) + 4|λS12|2 −3 2 3g2 L + g2 Y  λS2 + 6y2 t λS2, (B.11) 16π2βλS12 = (2λ3 + 4λ4 + 6λS + 4λS1 + 4λS2)λS12 + 12λ5λ∗ S12 + 6λ6λS1 + 6λ7λS2 −3 2 3g2 L + g2 Y  λS12 + 3y2 t λS12. A Diagonalization of mass matrices (B.12) 18λS + 2λS1 + 2λS2 + 4|λS12| , (B.9) = (12λ1 + 6λS)λS1 + (4λ3 + 2λ4)λS2 + 4λ2 S1 + 12Re (λ∗ 6λS12) + 4|λS12|2 −3 2 3g2 L + g2 Y  λS1, (B.10) C Singlet scalar annihilation cross sections Here we give compact expressions for singlet pair annihilation rates used in the computation of the DM abundances. In the mass eigenbasis, the relevant Lagrangian may be written as L = X j,k c0S2HjHk + X i cijkHiHjHk ! + X i ciS2Hi + X i,f yifHifRfL + h.c.  + X i gWiW +W −Hi + gZiZZHi  , (C.1) L = X j,k c0S2HjHk + X i cijkHiHjHk ! + X i ciS2Hi + X i,f yifHifRfL + h.c.  X +  (C.1) – 21 – where H = (A0, H0, h0, H+) . Now the singlet scalar annihilation cross section to scalars is given by: where H = (A0, H0, h0, H+) . Now the singlet scalar annihilation cross section to scalars is given by: σ(SS →HjHk) = 1 16πs2vs A2 jkI0,j,k + 2AjkBjkI1,j,k + B2 jkI2,j,k  . C Singlet scalar annihilation cross sections (C.2) (C.2) The rate into fermion-antifermion pairs is given by: The rate into fermion-antifermion pairs is given by: σ(SS →ff) = I0,f,f 4πs2vs X i,j cicj (s −m2 i )(s −m2 j) × ×  (yifRyjfR −yifIyjfI) s 2 −2yifRyjfRm2 f  , (C.3) ×  (yifRyjfR −yifIyjfI) s 2 −2yifRyjfRm2 f  , (C.3) (C.3) JCAP08(2016)057 nd the annihilation to vector bosons is: σ(SS →V V ) = I0,V,V 4πs2vs X i,j cicjgV igV j (s −m2 i )(s −m2 j)  3 + s(s −4M2 V ) 4M2 V   1 −δV,Z 2  , (C.4) (C.4) where I0,j,k = q s2 + (m2 j −m2 k)2 −2s(m2 j + m2 k) , I1,j,k = 4I0,j,k m2 j + m2 k −s , I2,j,k = 2I0,j,k m2 jm2 k −M2 S(2m2 j + 2m2 k −s) + M2s s (m2 j −m2 k)2 + 8I0,j,k (m2 j + m2 k −s)2 , (C.5) I0,j,k = q s2 + (m2 j −m2 k)2 −2s(m2 j + m2 k) , I1,j,k = 4I0,j,k m2 j + m2 k −s , 2I 8I (C.5) ,j, q j k j k I1,j,k = 4I0,j,k m2 j + m2 k −s , I2,j,k = 2I0,j,k m2 jm2 k −M2 S(2m2 j + 2m2 k −s) + M2s s (m2 j −m2 k)2 + 8I0,j,k (m2 j + m2 k −s)2 , (C.5) d Ajk = 2(1 + δjk)c0 + 2 X i (1 + δij + δjk + δki + 2δijδjk) cicijk s −m2 i , Bjk = 4cjck . 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Peer Review #1 of "Mir-421 in plasma as a potential diagnostic biomarker for precancerous gastric lesions and early gastric cancer (v0.1)"
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Mir-421 in plasma as a potential diagnostic biomarker for precancerous gastric lesions and early gastric cancer Jianlin Chen Corresp., Equal first author, 1 , Lihua Wu Equal first author, 2 , Yifan Sun Corresp., 1 , Qi Yin 1 , Xianhua Chen 1 , Siqun Liang 1 , Qingyan Meng 1 , Haihua Long 2 , Fangying Li 2 , Changjun Luo 3 , Xiaorong Xiao 4 1 Department of Clinical Laboratory, Affiliated Liutie Central Hospital of Guangxi Medical University, Liuzhou, Guangxi, China 2 Department of Digestive internal medicine, Affiliated Liutie Central Hospital of Guangxi Medical University, Liuzhou, Guangxi, China 3 Department of Internal Medicine-Cardiovascular, Affiliated Liutie Central Hospital of Guangxi Medical University, Liuzhou, Guangxi, China 4 Department of Science and Education, Affiliated Liutie Central Hospital of Guangxi Medical University, Liuzhou, Guangxi, China Corresponding Authors: Jianlin Chen, Yifan Sun Email address: cjl801900@163.com, sunyifan13@126.com Objective. MicroRNA (miR)-421 plays a key role in cancer progression. It has been reported that circulating miR-421may be a potential tumor marker for the diagnosis of several cancers. However, the role of miR-421 in plasma as a potential biomarker in the diagnosis of precancerous gastric lesions (Pre) and early-stage gastric cancer (GC) remains poorly understood. In this study, we investigated miR-421 in plasma as a novel potential biomarker for the detection of precancerous gastric lesions and early-stage (GC). Objective. MicroRNA (miR)-421 plays a key role in cancer progression. It has been reported that circulating miR-421may be a potential tumor marker for the diagnosis of several cancers. However, the role of miR-421 in plasma as a potential biomarker in the diagnosis of precancerous gastric lesions (Pre) and early-stage gastric cancer (GC) remains poorly understood. In this study, we investigated miR-421 in plasma as a novel potential biomarker for the detection of precancerous gastric lesions and early-stage (GC). Materials & Methods. The miRNA content was determined by quantitative real-time polymerase chain reaction (qRT-PCR). MiR-421 content in all subjects was normalized by endogenous miRNA (miR-16). The diagnostic value of miR-421 for Pre and GC was assessed by comparing receiver operating characteristic (ROC) analysis with traditional tumor markers, including CEA, CA125, CA153, CA211 and CA50. The correlation between the expression of miR-421 and the pathological characteristics of Pre and GC was analyzed. Results. Elevated expression of miR-421 in plasma can robustly distinguish the normal population from Pre and GC cases, especially in the early stages of gastric cancer cases (all p <0.05). The ROC analyses showed that the area under the ROC curve (AUC), sensitivity, accuracy and Youden index of miR-421 were superior to traditional tumor markers (CEA, CA125, CA153, CA211, and CA50) in GC diagnosis, while its specificity was higher than CEA, CA153 and CA50 (all p <0.05). Manuscript to be reviewed Mir-421 in plasma as a potential diagnostic biomarker for precancerous gastric lesions and early gastric cancer Jianlin Chen Corresp., Equal first author, 1 , Lihua Wu Equal first author, 2 , Yifan Sun Corresp., 1 , Qi Yin 1 , Xianhua Chen 1 , Siqun Liang 1 , Qingyan Meng 1 , Haihua Long 2 , Fangying Li 2 , Changjun Luo 3 , Xiaorong Xiao 4 MiR-421 in plasma had higher AUC value than AFP, CA153, CA211 and CA50 in the diagnosis of Pre (all p<0.05), while specificity, accuracy and Youden index of miR-421 was only lower than CA211. The efficiency of miR-421 in the diagnosis of GC was significantly higher than that of CA211 and CA50, and it was significantly higher than CA153, CA211 and CA50 in the diagnosis of Pre (all p<0.05). In addition, up-regulation of miR-421 occurred initially in precancerous gastric lesions as well as in the early stage of GC. Conclusions. Overexpression of plasma PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) 2 precancerous gastric lesions and early gastric can 3 Jianlin Chen1,*, Lihua Wu2, Yifan Sun3, Qi Yin4, Xianhua Chen5, Siqun Liang6, Qingyan Meng7, 4 Haihua Long8, Fangying Li9, Changjun Luo10, Xiaorong Xiao11 5 Department of Clinical Laboratory1,3,4,5,6,7, Department of Digestive internal medicine2,8,9, 6 Department of Internal Medicine-Cardiovascular10, d Department of Science and Education11, 7 Affiliated Liutie Central Hospital of Guangxi Medical University, Liuzhou, Guangxi, China 8 Corresponding Author: Jianlin Chen, Yifan Sun 9 14 Fei-e Road, Liuzhou, Guangxi, 545007, China 10 Email address: cjl801900@163.com; sunyifan13@126.com 3 Jianlin Chen1,*, Lihua Wu2, Yifan Sun3, Qi Yin4, Xianhua Chen5, Siqun Liang6, Qingyan Meng7, 4 Haihua Long8, Fangying Li9, Changjun Luo10, Xiaorong Xiao11 5 Department of Clinical Laboratory1,3,4,5,6,7, Department of Digestive internal medicine2,8,9, 6 Department of Internal Medicine-Cardiovascular10, d Department of Science and Education11, 7 Affiliated Liutie Central Hospital of Guangxi Medical University, Liuzhou, Guangxi, China 8 Corresponding Author: Jianlin Chen, Yifan Sun 9 14 Fei-e Road, Liuzhou, Guangxi, 545007, China 10 Email address: cjl801900@163.com; sunyifan13@126.com 3 Jianlin Chen1,*, Lihua Wu2, Yifan Sun3, Qi Yin4, Xianhua Chen5, Siqun Liang6, Qingyan Meng7, 4 Haihua Long8, Fangying Li9, Changjun Luo10, Xiaorong Xiao11 5 Department of Clinical Laboratory1,3,4,5,6,7, Department of Digestive internal medicine2,8,9, 6 Department of Internal Medicine-Cardiovascular10, d Department of Science and Education11, 7 Affiliated Liutie Central Hospital of Guangxi Medical University, Liuzhou, Guangxi, China 8 Corresponding Author: Jianlin Chen, Yifan Sun 9 14 Fei-e Road, Liuzhou, Guangxi, 545007, China 10 Email address: cjl801900@163.com; sunyifan13@126.com 10 Email address: cjl801900@163.com; sunyifan13@126.com Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed miR-421 is a novel biomarker for the detection of precancerous lesions and early gastric cancer. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed 30 CA211 and CA50 in the diagnosis of Pre (all p<0.05), while specificity, accuracy and Youden 31 index of miR-421 was only lower than CA211. The efficiency of miR-421 in the diagnosis of 32 GC was significantly higher than that of CA211 and CA50, and it was significantly higher than 33 CA153, CA211 and CA50 in the diagnosis of Pre (all p<0.05). In addition, up-regulation of miR- 34 421 occurred initially in precancerous gastric lesions as well as in the early stage of GC. 35 Conclusions. Overexpression of plasma miR-421 is a novel biomarker for the detection of 36 precancerous lesions and early gastric cancer. 30 CA211 and CA50 in the diagnosis of Pre (all p<0.05), while specificity, accuracy and Youden 31 index of miR-421 was only lower than CA211. The efficiency of miR-421 in the diagnosis of 32 GC was significantly higher than that of CA211 and CA50, and it was significantly higher than 33 CA153, CA211 and CA50 in the diagnosis of Pre (all p<0.05). In addition, up-regulation of miR- 34 421 occurred initially in precancerous gastric lesions as well as in the early stage of GC. 35 Conclusions. Overexpression of plasma miR-421 is a novel biomarker for the detection of 36 precancerous lesions and early gastric cancer. 33 CA153, CA211 and CA50 in the diagnosis of Pre (all p 0.05). In addition, up regulation of miR 34 421 occurred initially in precancerous gastric lesions as well as in the early stage of GC. 35 Conclusions. Overexpression of plasma miR-421 is a novel biomarker for the detection of 36 precancerous lesions and early gastric cancer. 37 INTRODUCTION 38 Gastric cancer (GC) is one of the most common malignant tumors in China(Zheng et al. 2019). 39 Due to the lack of tumor markers and specific symptoms and signs, most tumors have 40 metastasized by the time of diagnosis. Studies have shown that patients with advanced GC have 41 a poor prognosis with a 5-year survival rate of less than 25%(Chan et al. 2001). However, if 42 intervention and treatment are performed in the pre- or early stages of gastric cancer, the five- 43 year survival rate will rise to 95%(Craanen et al. 1991). Therefore, it is very important to detect 44 and treat gastric cancer at an early stage. 45 46 Gastric cancer originates from the malignant and aggressive proliferation of gastric mucosal 47 cells. 11 ABSTRACT 12 Objective. MicroRNA (miR)-421 plays a key role in cancer progression. It has been reported 13 that circulating miR-421may be a potential tumor marker for the diagnosis of several cancers. 14 However, the role of miR-421 in plasma as a potential biomarker in the diagnosis of 15 precancerous gastric lesions (Pre) and early-stage gastric cancer (GC) remains poorly 16 understood. In this study, we investigated miR-421 in plasma as a novel potential biomarker for 17 the detection of precancerous gastric lesions and early-stage (GC). 18 Materials & Methods. The miRNA content was determined by quantitative real-time 19 polymerase chain reaction (qRT-PCR). MiR-421 content in all subjects was normalized by 20 endogenous miRNA (miR-16). The diagnostic value of miR-421 for Pre and GC was assessed by 21 comparing receiver operating characteristic (ROC) analysis with traditional tumor markers, 22 including CEA, CA125, CA153, CA211 and CA50. The correlation between the expression of 23 miR-421 and the pathological characteristics of Pre and GC was analyzed. 24 Results. Elevated expression of miR-421 in plasma can robustly distinguish the normal 25 population from Pre and GC cases, especially in the early stages of gastric cancer cases (all p 26 <0.05). The ROC analyses showed that the area under the ROC curve (AUC), sensitivity, 27 accuracy and Youden index of miR-421 were superior to traditional tumor markers (CEA, 28 CA125, CA153, CA211, and CA50) in GC diagnosis, while its specificity was higher than CEA, 29 CA153 and CA50 (all p <0.05). MiR-421 in plasma had higher AUC value than AFP, CA153, 24 Results. Elevated expression of miR-421 in plasma can robustly distinguish the normal 25 population from Pre and GC cases, especially in the early stages of gastric cancer cases (all p 26 <0.05). The ROC analyses showed that the area under the ROC curve (AUC), sensitivity, 27 accuracy and Youden index of miR-421 were superior to traditional tumor markers (CEA, 28 CA125, CA153, CA211, and CA50) in GC diagnosis, while its specificity was higher than CEA, 29 CA153 and CA50 (all p <0.05). MiR-421 in plasma had higher AUC value than AFP, CA153, PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed 62 MicroRNAs (miRNAs) are a class of endogenous non-coding RNA molecules (length of 18–25 63 nucleotides) that exert their roles by base pairing between the seed region of miRNA or 3′-un- 64 translated regions (3′-UTR) of the target gene(Lee & Ambros 2001). Studies have confirmed that 65 miRNAs’ expression are frequently de-regulated in human tumors(Hernando 2007; Lu et al. 66 2005). Recently, circulating miRNAs (miRNAs present in blood) have attracted attention due to 67 their stability and ease of measurement. Many circulating miRNAs have been quantified and 68 identified by qRT-PCR as tumor markers to detect GC, such as miR-223, miR-21, miR-218(Li et 69 al. 2012; Zhou et al. 2015), and miR-18a(Tsujiura et al. 2015) in plasma, and miR-20a(Yang et 70 al. 2017) in serum. However, there is limited research into circulating miRNA markers for the 71 diagnosis of precancerous gastric lesions and early-stage gastric cancer, and most of these studies 72 have not compared the value of these miRNAs with traditional tumor markers in the diagnosis of 73 gastric cancer and precancerous lesions. 75 MicroRNA-421 (miR-421) is a molecule that has been studied extensively. It has been 76 investigated and found to be expressed aberrantly in various types of cancer(Li et al. 2018; Wang 77 et al. 2018; Zhou et al. 2016). Previous studies showed that miR-421 rises notably in tissue and 78 gastric juice, as well as in blood mononuclear cells and serum of gastric cancer(Liu et al. 2015; 79 Zhang et al. 2012a; Zhao et al. 2015), suggesting that miR-421 seems to be an ideal biomarker 80 for gastric cancer. However, most of these studies failed to point out at which stage in the 81 evolution of gastric cancer miR-421 can be detected in the peripheral blood, and an optimal 82 circulating miRNA should have the ability to distinguish different phases in the course of cancer 83 development. Some critical issues remain unresolved. Firstly, the expression of miR-421 in the 84 plasma of precancerous patients has not been fully investigated. More importantly, the value and 85 significance of miR-421 in the early diagnosis of gastric cancer are still not clearly stated. The 86 present study aimed to clarify the diagnostic value of miR-421 in the early stage of gastric cancer 87 and in the precancerous lesions of gastric cancer and to explore its relation with the progression 88 of GC. Manuscript to be reviewed Most are developed and evolved by the chronic inflammation of gastric mucosa under the 48 influence of multiple factors, mainly in four stages (chronic superficial gastritis → chronic 49 atrophic gastritis → intestinal metaplasia and dysplasia → gastric cancer). Chronic atrophic 50 gastritis is currently recognized as a precancerous disease, intestinal metaplasia and dysplasia is a 51 precancerous lesion closely related to the development of gastric cancer. Currently, Gastroscopy 52 combined with biopsy is the gold standard for diagnosis of gastric cancer. However, it may not 53 be appropriate to carry out large-scale screening and early detection in most countries including 54 China due to acceptability, availability, cost and other reasons. GC related tumor markers such 55 as, cancer embryo antigen (CEA), pepsinogen (PG), carbohydrate antigen 199(CA199) and 56 carbohydrate antigen 724(CA724), gastrin-17(G17) have been used clinically for many years, 57 but many do not have sufficient sensitivity and specificity for GC screening(He et al. 2013; 58 Pectasides et al. 1997; Yang et al. 2014). Studies on their role in monitoring early gastric cancer 59 have rarely been reported. Therefore, there is a need for reliable and non-invasive biomarkers for 60 early detection and mass screening of GC. 61 61 PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) 90 Clinical specimens 91 Plasma samples from 90 GC patients, 89 patients with precancerous (Pre) lesions and 45 normal 92 healthy controls (NC) were collected at Affiliated Liutie Central Hospital of Guangxi Medical 93 University in Guangxi province, China and the levels of miR-421 were screened by qRT-PCR. 94 GC patients were confirmed by gastroscopy combined with biopsy. Tumor type and stage were PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed 95 identified according to the Union of International Cancer Control (UICC) tumor-node-metastasis 96 (TNM) system, 7th edition. The histology of all patients was evaluated according to the World 97 Health Organization (WHO) criteria. Patients who had received Radiation therapy or 98 chemotherapy were excluded from the study. The pathological outcomes of GC and Pre patients 99 were obtained by two experienced pathologists. In addition, 45 healthy people without a previous 100 history of diabetes, heart disease, hypertension, and cancer were chosen as normal controls (NC). 101 We compared the plasma levels of miR-421 in patients with gastric cancer, and precancerous 102 lesions, and in normal controls to assess the feasibility of miR-421 as a novel non-invasive 103 biomarker for early GC detection. The expression levels of conventional tumor markers in GC, 104 Pre, and normal controls were analyzed to evaluate the specificity and sensitivity of miR-421 for 105 early diagnosis of gastric cancer. This study was subject to approval by the Ethics Committee of 106 the Affiliated Liutie Central Hospital of Guangxi Medical University and written informed 107 consent was issued by all study participants. 108 Plasma preparation and storage 109 8~10ml of venous blood of each participant was collected using ethylenediaminetetraacetic acid 110 (EDTA) anticoagulative tubes immediately. Cell-free plasma was separated within 2h after 111 collection by 3000rpm,5min centrifugation to prevent contamination by cellular nucleic acids. 112 Plasma transferred into RNase/DNase-free tubes was stored at -80℃ until MicroRNA extraction. 113 Plasma samples for conventional tumor markers and Helicobacter pylori antibody determination 114 were separated and kept at -20˚C until assayed 113 Plasma samples for conventional tumor markers and Helicobacter pylori antibody determination 114 were separated and kept at -20˚C until assayed. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed 124 SDS 2.0 software (Applied Biosystems, Foster City, CA, USA). Samples with Ct values greater 125 than 30 were excluded. The levels of miR-421 in different samples were calculated by the 2−∆Ct 126 method, in which ∆Ct=Ct (miR-421)–Ct (miR-16). 124 SDS 2.0 software (Applied Biosystems, Foster City, CA, USA). Samples with Ct values greater 125 than 30 were excluded. The levels of miR-421 in different samples were calculated by the 2−∆Ct 126 method, in which ∆Ct=Ct (miR-421)–Ct (miR-16). 127 Conventional tumor markers and Helicobacter pylori antibodies 115 MicroRNA extraction and qRT-PCR assays 116 MicroRNAs were isolated from 200μl plasma utilizing Blood (Serum/Plasma) MicroRNA 117 Extraction and Purification Kit (spin column) (Novland Co., Ltd, Shanghai, China) following the 118 manufacturer’s protocol. The concentrations of miRNA from plasma samples were quantified 119 using a NanoQ Micro-volume Spectrophotometer, (CapitalBio, Beijing, China). The initial 120 template for qRT-PCR was 2ul. Circulating miRNAs expression was determined using one step 121 Stemaim-it miR qRT-PCR Kit Quantitation (Taqman probes) (Novland Co., Ltd, Shanghai, 122 China), while miR-16 served as a reference miRNA. Expression levels of target miRNAs were 123 performed on the ABI-7500 PCR system and calculated by the cycle threshold (Ct) values with 116 MicroRNAs were isolated from 200μl plasma utilizing Blood (Serum/Plasma) MicroRNA 117 Extraction and Purification Kit (spin column) (Novland Co., Ltd, Shanghai, China) following the 118 manufacturer’s protocol. The concentrations of miRNA from plasma samples were quantified 119 using a NanoQ Micro-volume Spectrophotometer, (CapitalBio, Beijing, China). The initial 120 template for qRT-PCR was 2ul. Circulating miRNAs expression was determined using one step 121 Stemaim-it miR qRT-PCR Kit Quantitation (Taqman probes) (Novland Co., Ltd, Shanghai, 122 China), while miR-16 served as a reference miRNA. Expression levels of target miRNAs were 123 performed on the ABI-7500 PCR system and calculated by the cycle threshold (Ct) values with PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed 127 Conventional tumor markers and Helicobacter pylori antibodies 128 Conventional tumor markers were tested by electrochemiluminescence imm 128 Conventional tumor markers were tested by electrochemiluminescence immunoassay according 129 to the standard procedure of Roche Company's kit and Roche E170 automatic immunity 130 analyzer. Helicobacter pylori antibodies were assayed following the Anti-helicobacter pylori 131 antibody detection kit (ELISA) (Beier, biological engineering co. Ltd, Beijing, China). 128 Conventional tumor markers were tested by electrochemiluminescence immunoassay according 129 to the standard procedure of Roche Company's kit and Roche E170 automatic immunity 130 analyzer. Helicobacter pylori antibodies were assayed following the Anti-helicobacter pylori 131 antibody detection kit (ELISA) (Beier, biological engineering co. Ltd, Beijing, China). 132 Statistical analysis 133 All data were analyzed using MedCalc statistical software (v18.2.1) and GraphPad Prism 7.0. 134 The levels of miR-421and conventional tumor markers in plasma among Pre, GC patients, and 135 health cases were established by the Mann Whitney test. Receiver operating characteristic curves 136 (ROC) and the AUC were constructed to evaluate the diagnostic values of each tumor markers. 137 The optimal cut-off thresholds were determined by using the highest Youden index. The Chi- 138 squared test was utilized to analyze the associations between the expression level of miR-421 139 and the clinicopathological factors. All differences were examined statistically significant at p < 140 0.05. 141 Results 142 Expression of miR-421 and tumor markers in plasma of GC patients, Pre patients, 143 and normal controls 144 This study recruited 90 GC patients, 89 Pre patients, and 45 normal healthy subjects as controls. 145 Plasma miR-421 levels were detected by qRT-PCR and analyzed using miR-16 as a reference 146 miRNA. To verify whether miR-16 was adaptable to be a reference miRNA in our system, the 147 expression levels of miR-16 were detected. As shown in Fig. 1A, there was no statistical 148 difference in the expression of miR-16 between the normal and gastric cancer group, or the 149 normal and precancerous group, which implied that miR-16 was a suitable reference control for 150 detecting miR-421 in plasma samples. The qRT-PCR results showed that miR-421 was 151 significantly up-regulated in GC and Pre patients compared with the control group (all p<0.001, 152 Figs. 1B,1C). The concentrations of CEA, CA125, CA153, CA211, and CA50 in the GC group 157 miR-421 in plasma had higher diagnostic value as a non-invasive biomarker than 158 traditional tumor markers for gastric cancer 159 To further evaluate the diagnostic value of miR-421 for GC, receiver-operating characteristic 160 (ROC) curve analyses were performed (Fig. 2A and Table 2). According to the ROC curve, 161 when the cut-off of 3.23, the optimal sensitivity and specificity of miR-421 were 96.67% and 162 95.56% respectively [AUC =0.981 (0.942 - 0.997)]. To further clarify the diagnostic efficiency 163 of miR-421 in gastric cancer, the diagnostic values of traditional tumor markers were compared 164 to those of miR-421. Fig. 2B shows that the AUC, sensitivity, accuracy and Youden index of 165 miR-421 were higher than all five conventional biomarkers, while the specificity was higher than 166 CEA, CA153, and CA50. Table 3 shows the direct comparison of the ROC curves of circulating 167 miR-421 with five conventional tumor markers. The diagnostic efficacy of miR-421 was 168 significantly higher than CEA, CA153, CA211, and CA50 (all p<0.05). In order to determine 169 whether the plasma level of miR-421 has clinical value for GC diagnosis, the false positive rate 170 (FPR) and false negative rate (FNR) and diagnosis efficiency were calculated. As shown in 171 Table 2, the results indicate that the FNR of miR-421 was lower than all five conventional 172 biomarkers, while the FPR was lower than CEA, CA153, and CA50, which indicated that miR- 173 421 has a high diagnostic value for GC. These results indicate that the plasma miR-421 has 174 greater diagnostic value for GC than five conventional tumor markers and can be used as a non- 175 invasive diagnostic marker for gastric cancer. 142 Expression of miR-421 and tumor markers in plasma of GC patients, Pre patients, 144 This study recruited 90 GC patients, 89 Pre patients, and 45 normal healthy subjects as controls. 145 Plasma miR-421 levels were detected by qRT-PCR and analyzed using miR-16 as a reference 146 miRNA. To verify whether miR-16 was adaptable to be a reference miRNA in our system, the 147 expression levels of miR-16 were detected. As shown in Fig. 1A, there was no statistical 148 difference in the expression of miR-16 between the normal and gastric cancer group, or the 149 normal and precancerous group, which implied that miR-16 was a suitable reference control for 150 detecting miR-421 in plasma samples. The qRT-PCR results showed that miR-421 was 151 significantly up-regulated in GC and Pre patients compared with the control group (all p<0.001, 152 Figs. 1B,1C). The concentrations of CEA, CA125, CA153, CA211, and CA50 in the GC group PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed 153 were significantly higher than in the healthy controls (all p<0.05, Table 1). Moreover, the 154 concentration of CA211 and CA50 in the Pre group was significantly higher than the healthy 155 controls (all p<0.05, Table 1). Of note, compared with the precancerous group, the concentration 156 of AFP and CA153 was significantly higher in the normal group (all p<0.05, Table 1). 157 miR-421 in plasma had higher diagnostic value as a non-invasive biomarker than 158 traditional tumor markers for gastric cancer 159 To further evaluate the diagnostic value of miR-421 for GC, receiver-operating characteristic 160 (ROC) curve analyses were performed (Fig. 2A and Table 2). According to the ROC curve, 161 when the cut-off of 3.23, the optimal sensitivity and specificity of miR-421 were 96.67% and 162 95.56% respectively [AUC =0.981 (0.942 - 0.997)]. To further clarify the diagnostic efficiency 163 of miR-421 in gastric cancer, the diagnostic values of traditional tumor markers were compared 164 to those of miR-421. Fig. 2B shows that the AUC, sensitivity, accuracy and Youden index of 165 miR-421 were higher than all five conventional biomarkers, while the specificity was higher than 166 CEA, CA153, and CA50. Table 3 shows the direct comparison of the ROC curves of circulating 167 miR-421 with five conventional tumor markers. The diagnostic efficacy of miR-421 was 168 significantly higher than CEA, CA153, CA211, and CA50 (all p<0.05). In order to determine 169 whether the plasma level of miR-421 has clinical value for GC diagnosis, the false positive rate 170 (FPR) and false negative rate (FNR) and diagnosis efficiency were calculated. As shown in 171 Table 2, the results indicate that the FNR of miR-421 was lower than all five conventional 172 biomarkers, while the FPR was lower than CEA, CA153, and CA50, which indicated that miR- 173 421 has a high diagnostic value for GC. These results indicate that the plasma miR-421 has 174 greater diagnostic value for GC than five conventional tumor markers and can be used as a non- 175 invasive diagnostic marker for gastric cancer. 176 miR-421 in plasma could be used to distinguish early-stage GC patients from 177 healthy controls 153 were significantly higher than in the healthy controls (all p<0.05, Table 1). Moreover, the 154 concentration of CA211 and CA50 in the Pre group was significantly higher than the healthy 155 controls (all p<0.05, Table 1). Manuscript to be reviewed Of note, compared with the precancerous group, the concentration 156 of AFP and CA153 was significantly higher in the normal group (all p<0.05, Table 1). 176 miR-421 in plasma could be used to distinguish early-stage GC 178 To evaluate the potential of the differentially expressed miR-421 to diagnose early-stage gastric 179 cancer patients, we further analyzed the expression levels of miR-421 in GC patients at an early 180 stage (TNM stages I-II). The results showed that the expression levels of circulating miR-421 in 181 healthy controls were significantly lower than those in early-stage gastric cancer patients PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed 182 (p<0.0001, Fig.3A). The AUC values of miR-421 were 0.9907 (95% confidence interval (CI) = 183 0.931 to 1.0, sensitivity = 100% and specificity = 95.56%, Fig. 3B). These results indicate that 184 miR-421 could be invoked as a novel biomarker for early diagnosis of gastric cancer. 185 186 miR-421 had higher diagnostic value than traditional tumor markers for 187 precancerous gastric lesions 188 Based on the verified difference of miR-421 between the Pre and NC group (Fig. 1B,1C), ROC 189 curves were constructed to further assess the role of miR-421 expression in the diagnosis of 190 precancerous lesions. According to the different concentration of traditional tumor markers 191 between the Pre group and the NC group (Figs. 4A,4B), their diagnostic values with miR- 192 421were further compared. ROC analyses showed that up-regulation of plasma miR-421 could 193 discriminate Pre patients well from control subjects, with an AUC value of 0.873 (sensitivity = 194 66.29%, specificity = 95.56%, cutoff value: 3.23) (Fig.4C). Furthermore, as shown in table 4 and 195 Figs. 4C & 4D, the AUC of miR-421 was higher than CA153, CA211, and CA50, the sensitivity 196 was higher than CA50, while the specificity and Youden index were higher than CA153, and 197 CA50. Moreover, the FNR of miR-421 was lower than CA50, while the FPR was lower than 198 CA153, and CA50. According to the comparison of the ROC curves, the diagnostic efficacy of 199 miR-421 was considerably higher than CA153, CA211 and CA50 (all p<0.05, Table 5). These 200 results indicate that miR-421 has high diagnostic value for precancerous patients. 201 Up-regulation of miR-421 occurred initially in the precancerous diseases as well 202 as the early GC 203 We found that up-regulation of miR-421 not only distinguishes early-stage gastric cancer well, 204 but can also well distinguish precancerous lesions from normal controls. However, it is not clear 205 at what stage miR-421 begins to be up-regulated throughout the development of GC. Therefore, 206 expressions of miR-421 in precancerous disease, and different clinical stages (I, II, III and IV) 187 precancerous gastric lesions 188 Based on the verified difference of miR-421 between the Pre and NC group (Fig. 1B,1C), ROC 189 curves were constructed to further assess the role of miR-421 expression in the diagnosis of 190 precancerous lesions. According to the different concentration of traditional tumor markers 191 between the Pre group and the NC group (Figs. Manuscript to be reviewed 4A,4B), their diagnostic values with miR- 192 421were further compared. ROC analyses showed that up-regulation of plasma miR-421 could 193 discriminate Pre patients well from control subjects, with an AUC value of 0.873 (sensitivity = 194 66.29%, specificity = 95.56%, cutoff value: 3.23) (Fig.4C). Furthermore, as shown in table 4 and 195 Figs. 4C & 4D, the AUC of miR-421 was higher than CA153, CA211, and CA50, the sensitivity 196 was higher than CA50, while the specificity and Youden index were higher than CA153, and 197 CA50. Moreover, the FNR of miR-421 was lower than CA50, while the FPR was lower than 198 CA153, and CA50. According to the comparison of the ROC curves, the diagnostic efficacy of 199 miR-421 was considerably higher than CA153, CA211 and CA50 (all p<0.05, Table 5). These 200 results indicate that miR-421 has high diagnostic value for precancerous patients. Manuscript to be reviewed 211 gradually up-regulated from the NC to the Pre and stage I cases, and finally, the maximum 212 expression value appeared in stage I cases (Table 6). The marked up-regulation of miR-421 213 occurred initially in precancerous disease as well as early-stage GC. 211 gradually up-regulated from the NC to the Pre and stage I cases, and finally, the maximum 212 expression value appeared in stage I cases (Table 6). The marked up-regulation of miR-421 213 occurred initially in precancerous disease as well as early-stage GC. 214 Association of miR-421 with clinicopathological features 214 Association of miR-421 with clinicopathological features 215 In order to determine whether miR-421 levels in plasma were correlated with clinicopathological 216 features in patients with GC and precancerous lesions, the clinical data of 89 Pre and 90 GC- 217 proven cases were collected and analyzed. The high and low expression groups of each 218 parameter were calculated by the median of the relative quantity of miR-421. Correlation 219 analysis showed no statistically significant association of miR-421 expression with GC patients’ 220 age (p = 0.0736), gender (p = 0.3265), TNM stage (p= 0.3531), differentiation degree (p= 221 0.5105), and histological type (p = 0.5079). Similarly, we found that there were no apparent 222 correlations of miR-421 expression with Pre patients’ age (p = 0.5896), gender (p = 0.5934), 223 histological type (p = 0.6939), and Helicobacter pylori infection status (p = 0.3095) (Table 7). 224 Discussion 201 Up-regulation of miR-421 occurred initially in the precancerous diseases as well 202 as the early GC 201 Up-regulation of miR-421 occurred initially in the precancerous diseases as well 202 as the early GC 203 We found that up-regulation of miR-421 not only distinguishes early-stage gastric cancer well, 204 but can also well distinguish precancerous lesions from normal controls. However, it is not clear 205 at what stage miR-421 begins to be up-regulated throughout the development of GC. Therefore, 206 expressions of miR-421 in precancerous disease, and different clinical stages (I, II, III and IV) 207 were further analyzed and compared. The results showed that the expressions of miR-421 were 208 statistically different between the Pre cases and GC stages I, II, III and IV (p<0.05, Fig.5). 209 However, there was no difference of miR-421 expression between the GC stages II, III and IV 210 and GC cases stage I. Moreover, compared to the expression in Pre cases, the biomarker was PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed 240 diagnostic value in cancer. However, these researches are based on invasive, patients may not be 241 willing to accept, is bound to affect its promotion. Moreover, our study showed that miR-421 242 could achieve a satisfactory diagnostic efficiency in distinguishing GC patients from healthy 243 controls with an AUC of 0.981 (sensitivity = 96.67% and specificity = 95.56%), higher than 244 miR-21, miR-20a and miR-378 tumor markers in previous studies (Liu et al. 2012; Mirzaei et al. 245 2016). In our study, we compared its diagnostic efficiency with that of traditional markers, 246 further clarifying its diagnostic value as a new tumor marker. 247 248 It is particularly important to improve the early diagnosis and prompt treatment of patients with 249 gastric cancer. Researchers have illuminated the potential roles of several miRNAs in the early 250 detection of cancers, such as lung carcinoma(Pan et al. 2018), colorectal neoplasm(Liu et al. 251 2018), and early-stage breast carcinoma(An et al. 2018). However, to date only a few miRNAs 252 have been found effective as non-invasive biomarkers for the early detection of GC. Although 253 the study such as the five-miRNA panel presented a high diagnostic value for the early stage of 254 GC(Liu et al. 2012), the study did not include gastric precancerous lesions, and the subtle 255 changes and performances of the miRNAs were not further elaborated. The miR-17-92 cluster 256 was considered as a circulating biomarker for early detection of GC, but there is no ROC data to 257 support miRNA recognition of early stage cancer(Li et al. 2017). In our study, based on the 258 detailed TNM stage characteristics of the GC sample, we explored the diagnostic value of 259 plasma miR-421 in early gastric cancer. The results showed that the expression level of miR-421 260 could well distinguish early gastric cancer from normal controls, suggesting that miR-421 may 261 have significant clinical value in the early diagnosis of gastric cancer. 262 Precancerous changes in the stomach can increase the risk of gastric cancer six-fold. Previous 263 studies have reported that precancerous lesions are regulated by miRNAs(Li et al. 2014; Rotkrua 264 et al. 2011). 265 Due to the absence of clinical manifestations of gastrointestinal metaplasia and atypical 266 hyperplasia, there are few means for clinical non-invasive detection. Several circulating 240 diagnostic value in cancer. 224 Discussion 225 To date, there is no reliable non-invasive blood-based biomarker for the detection of 226 precancerous lesions and early-stage gastric cancer. We studied the diagnostic value of miR-421 227 in plasma in precancerous gastric lesions and GC patients. We found that the plasma expression 228 levels of miR-421 were statistically significantly higher in early-stage GC and precancerous 229 gastric lesions patients than in healthy controls. ROC analyses demonstrated that miR-421 has 230 high sensitivity and specificity in discriminating early-stage GC and precancerous gastric lesions 231 patients from healthy controls. Our results indicated that miR-421 in plasma presented 232 statistically significant diagnostic efficacy in detecting early GC as well as precancerous gastric 233 lesions patients. 234 235 High-throughput analysis showed that miR-421 is localized on human chromosome1p34.23, and 236 is one prominent member of the miR-200 family(Liu et al. 2015). Liu et al reported that miR-421 237 elevation in GC tissue(Liu et al. 2015; Zhao et al. 2015)and gastric juice(Zhang et al. 238 2012b)probably had implications in GC diagnosis. These results are consistent with our findings 239 that miR-421 was highly expressed in the plasma of gastric cancer might have the potential PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) 235 High-throughput analysis showed that miR-421 is localized on human chromosome1p34.23, and 236 is one prominent member of the miR-200 family(Liu et al. 2015). Liu et al reported that miR-421 237 elevation in GC tissue(Liu et al. 2015; Zhao et al. 2015)and gastric juice(Zhang et al. 238 2012b)probably had implications in GC diagnosis. These results are consistent with our findings 239 that miR-421 was highly expressed in the plasma of gastric cancer might have the potential 238 2012b)probably had implications in GC diagnosis. These results are consistent with our findings 239 that miR-421 was highly expressed in the plasma of gastric cancer might have the potential PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed However, these researches are based on invasive, patients may not be 241 willing to accept, is bound to affect its promotion. Moreover, our study showed that miR-421 242 could achieve a satisfactory diagnostic efficiency in distinguishing GC patients from healthy 243 controls with an AUC of 0.981 (sensitivity = 96.67% and specificity = 95.56%), higher than 244 miR-21, miR-20a and miR-378 tumor markers in previous studies (Liu et al. 2012; Mirzaei et al. 245 2016). In our study, we compared its diagnostic efficiency with that of traditional markers, 246 further clarifying its diagnostic value as a new tumor marker. 248 It is particularly important to improve the early diagnosis and prompt treatment of patients with 249 gastric cancer. Researchers have illuminated the potential roles of several miRNAs in the early 250 detection of cancers, such as lung carcinoma(Pan et al. 2018), colorectal neoplasm(Liu et al. 251 2018), and early-stage breast carcinoma(An et al. 2018). However, to date only a few miRNAs 252 have been found effective as non-invasive biomarkers for the early detection of GC. Although 253 the study such as the five-miRNA panel presented a high diagnostic value for the early stage of 254 GC(Liu et al. 2012), the study did not include gastric precancerous lesions, and the subtle 255 changes and performances of the miRNAs were not further elaborated. The miR-17-92 cluster 256 was considered as a circulating biomarker for early detection of GC, but there is no ROC data to 257 support miRNA recognition of early stage cancer(Li et al. 2017). In our study, based on the 258 detailed TNM stage characteristics of the GC sample, we explored the diagnostic value of 259 plasma miR-421 in early gastric cancer. The results showed that the expression level of miR-421 260 could well distinguish early gastric cancer from normal controls, suggesting that miR-421 may 261 have significant clinical value in the early diagnosis of gastric cancer. 262 Precancerous changes in the stomach can increase the risk of gastric cancer six-fold. Previous 263 studies have reported that precancerous lesions are regulated by miRNAs(Li et al. 2014; Rotkrua 264 et al. 2011). 262 Precancerous changes in the stomach can increase the risk of gastric cancer six-fold. Previous 263 studies have reported that precancerous lesions are regulated by miRNAs(Li et al. 2014; Rotkrua 264 et al. 2011). Manuscript to be reviewed This study provides us with a new insight into the role of miR- 291 421 in gastric cancer These results could pave the way for the development of a novel biomarker 278 The occurrence and development of gastric cancer is a multi-stage process, and miR-421 may be 279 associated with a certain stage or the whole process. In this study, we further analyzed the trend 280 of the up-regulation of miR-421 with the progression of GC. Results showed that the expression 281 of plasma miR-421in GC cases at stage I is no different from stages II, II, and IV, but plasma 282 miR-421in precancerous cases can be distinctly distinguished from GC at each stage, indicating 283 that miR-421 occurred initially in precancerous lesions as well as early GC. Therefore, we 284 speculate that the up-regulation in the miR-421 may be triggered by risk factors, especially 285 chronic inflammation in the premalignant stomach, suggesting that a detailed investigation is 286 needed. Manuscript to be reviewed 269 and of GC progression. However, the comparative value of these miRNAs and traditional tumor 270 markers in the diagnosis of gastric cancer and precancerous lesions has not been clarified, and an 271 optimal circulating miRNA biomarker should have better specificity and sensitivity than 272 traditional tumor markers. In the current study, we found that plasma miR-421 could well 273 distinguish precancerous lesions of gastric cancer patients from healthy controls with an AUC of 274 0.872 (sensitivity = 66.29% and specificity = 95.56%). Furthermore, the diagnostic efficacy of 275 miR-421 was markedly higher than traditional tumor markers, such as CA153, CA211, and 276 CA50. 277 269 and of GC progression. However, the comparative value of these miRNAs and traditional tumor 270 markers in the diagnosis of gastric cancer and precancerous lesions has not been clarified, and an 271 optimal circulating miRNA biomarker should have better specificity and sensitivity than 272 traditional tumor markers. In the current study, we found that plasma miR-421 could well 273 distinguish precancerous lesions of gastric cancer patients from healthy controls with an AUC of 274 0.872 (sensitivity = 66.29% and specificity = 95.56%). Furthermore, the diagnostic efficacy of 275 miR-421 was markedly higher than traditional tumor markers, such as CA153, CA211, and 276 CA50. 277 278 The occurrence and development of gastric cancer is a multi-stage process, and miR-421 may be 279 associated with a certain stage or the whole process. In this study, we further analyzed the trend 280 of the up-regulation of miR-421 with the progression of GC. Results showed that the expression 281 of plasma miR-421in GC cases at stage I is no different from stages II, II, and IV, but plasma 282 miR-421in precancerous cases can be distinctly distinguished from GC at each stage, indicating 283 that miR-421 occurred initially in precancerous lesions as well as early GC. Therefore, we 284 speculate that the up-regulation in the miR-421 may be triggered by risk factors, especially 285 chronic inflammation in the premalignant stomach, suggesting that a detailed investigation is 286 needed. 287 288 Conclusions 289 Our findings demonstrate that plasma miR-421 may be a potential marker for precancerous 290 lesions and early gastric cancer. Manuscript to be reviewed 265 Due to the absence of clinical manifestations of gastrointestinal metaplasia and atypical 266 hyperplasia, there are few means for clinical non-invasive detection. Several circulating 267 miRNAs, especially microRNA-196a (Chen et al. 2018), miR-16-5p, and miR-19b-3p(Zhang et 268 al. 2015), have been screened out for early markers of gastric cancer and precancerous lesions 265 Due to the absence of clinical manifestations of gastrointestinal metaplasia and atypical 266 hyperplasia, there are few means for clinical non-invasive detection. Several circulating 267 miRNAs, especially microRNA-196a (Chen et al. 2018), miR-16-5p, and miR-19b-3p(Zhang et 268 al. 2015), have been screened out for early markers of gastric cancer and precancerous lesions PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) 298 REFERENCES 299 An X, Quan H, Lv J, Meng L, Wang C, Yu Z, and Han J. 2018. Serum microRNA as potential biomarker to detect 300 breast atypical hyperplasia and early-stage breast cancer. Future Oncol. 10.2217/fon-2018-0334 301 Chan AO, Wong BC, and Lam SK. 2001. Gastric cancer: past, present and future. Can J Gastroenterol 15:469-474. 302 Chen TH, Lee C, Chiu CT, Chu YY, Cheng HT, Hsu JT, Tsou YK, Wu RC, Chen TC, Chang NC, Yeh TS, and Lin 303 KH. 2018. Circulating microRNA-196a is an early gastric cancer biomarker. Oncotarget 9:10317-10323. 304 Craanen ME, Dekker W, Ferwerda J, Blok P, and Tytgat GN. 1991. Early gastric cancer: a clinicopathologic study. J 305 Clin Gastroenterol 13:274-283. 306 He CZ, Zhang KH, Li Q, Liu XH, Hong Y, and Lv NH. 2013. Combined use of AFP, CEA, CA125 and CAl9-9 307 improves the sensitivity for the diagnosis of gastric cancer. BMC Gastroenterol 13:87. 308 Hernando E. 2007. microRNAs and cancer: role in tumorigenesis, patient classification and therapy. Clin Transl Oncol 309 9:155-160. 310 Lee RC, and Ambros V. 2001. An extensive class of small RNAs in Caenorhabditis elegans. Science 294:862-864. 311 Li BS, Zhao YL, Guo G, Li W, Zhu ED, Luo X, Mao XH, Zou QM, Yu PW, Zuo QF, Li N, Tang B, Liu KY, and 312 Xiao B. 2012. Plasma microRNAs, miR-223, miR-21 and miR-218, as novel potential biomarkers for gastric 313 cancer detection. PLoS One 7:e41629. 10.1371/journal.pone.0041629 314 Li H, Wu Q, Li T, Liu C, Xue L, Ding J, Shi Y, and Fan D. 2017. The miR-17-92 cluster as a potential biomarker for 315 the early diagnosis of gastric cancer: evidence and literature review. Oncotarget 8:45060-45071. 316 10.18632/oncotarget.15023 317 Li T, Lu YY, Zhao XD, Guo HQ, Liu CH, Li H, Zhou L, Han YN, Wu KC, Nie YZ, Shi YQ, and Fan DM. 2014. 318 MicroRNA-296-5p increases proliferation in gastric cancer through repression of Caudal-related homeobox 319 1. Oncogene 33:783-793. 10.1038/onc.2012.637 320 Li Y, Cui X, Li Y, Zhang T, and Li S. 2018. Upregulated expression of miR-421 is associated with poor prognosis in 321 non-small-cell lung cancer. Cancer Manag Res 10:2627-2633. 320 Li Y, Cui X, Li Y, Zhang T, and Li S. 2018. 288 Conclusions 289 Our findings demonstrate that plasma miR-421 may be a potential marker for precancerous 290 lesions and early gastric cancer. This study provides us with a new insight into the role of miR- 291 421 in gastric cancer. These results could pave the way for the development of a novel biomarker 292 for the early diagnosis of gastric cancer. Used this biomarker may allow a reliable indicator of 293 gastric cancer at a much lower cost than methods currently available. The limitations of this 294 study are that our normal control samples were relatively small, and most cancer samples were at 295 an advanced stage. The exact use of miR-421 as an early plasma biomarker in clinical diagnosis 296 of precancerous lesions and gastric cancer should be further explored in studies that use larger 297 samples and in clinical trials. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed 338 Pectasides D, Mylonakis A, Kostopoulou M, Papadopoulou M, Triantafillis D, Varthalitis J, Dimitriades M, and 339 Athanassiou A. 1997. CEA, CA 19-9, and CA-50 in monitoring gastric carcinoma. Am J Clin Oncol 20:348- 340 353. 38 Pectasides D, Mylonakis A, Kostopoulou M, Papadopoulou M, Triantafillis D, Varthalitis , y , p , p p , , , , 339 Athanassiou A. 1997. CEA, CA 19-9, and CA-50 in monitoring gastric carcinoma. Am J Clin Oncol 20:348- 340 353. 341 Rotkrua P, Akiyama Y, Hashimoto Y, Otsubo T, and Yuasa Y. 2011. MiR-9 downregulates CDX2 expression in 342 gastric cancer cells. Int J Cancer 129:2611-2620. 10.1002/ijc.25923 , y , , , g p 342 gastric cancer cells. Int J Cancer 129:2611-2620. 10.1002/ijc.25923 43 Tsujiura M, Komatsu S, Ichikawa D, Shiozaki A, Konishi H, Takeshita H, Moriumura R, N 344 Hirajima S, Arita T, Fujiwara H, Okamoto K, and Otsuji E. 2015. Circulating miR-18a in plasma contributes 344 Hirajima S, Arita T, Fujiwara H, Okamoto K, and Otsuji E. 2015. Circulating miR-18a in plasma contributes 345 to cancer detection and monitoring in patients with gastric cancer. Gastric Cancer 18:271-279. 45 to cancer detection and monitoring in patients with gastric cancer. Gastric Cancer 18 346 Wang Y, Liu Z, and Shen J. 2018. MicroRNA-421-targeted PDCD4 regulates breast cancer cell proliferation. Int J 347 Mol Med. 348 Yang AP, Liu J, Lei HY, Zhang QW, Zhao L, and Yang GH. 2014. CA72-4 combined with CEA, CA125 and CAl9- 349 9 improves the sensitivity for the early diagnosis of gastric cancer. Clin Chim Acta 437:183-186. 350 10.1016/j.cca.2014.07.034 g , , , g Q , , g , 349 9 improves the sensitivity for the early diagnosis of gastric cancer. Clin Chim Acta 437:183-186. 350 10.1016/j.cca.2014.07.034 351 Yang R, Fu Y, Zeng Y, Xiang M, Yin Y, Li L, Xu H, Zhong J, and Zeng X. 2017. Serum miR-20a is a promising 352 biomarker for gastric cancer. Biomed Rep 6:429-434. 353 Zhang J, Song Y, Zhang C, Zhi X, Fu H, Ma Y, Chen Y, Pan F, Wang K, Ni J, Jin W, He X, Su H, and Cui D. 2015. 354 Circulating MiR-16-5p and MiR-19b-3p as Two Novel Potential Biomarkers to Indicate Progression of 355 Gastric Cancer. Theranostics 5:733-745. 355 Gastric Cancer. Theranostics 5:733-745. 298 REFERENCES Upregulated expression of miR-421 is associated with poor prognosis in 321 ll ll l C M R 10 2627 2633 322 Liu H, Gao Y, Song D, Liu T, and Feng Y. 2015. Correlation between microRNA-421 expression level and prognosis 323 of gastric cancer. Int J Clin Exp Pathol 8:15128-15132. 324 Liu H, Zhu L, Liu B, Yang L, Meng X, Zhang W, Ma Y, and Xiao H. 2012. Genome-wide microRNA profiles identify 325 miR-378 as a serum biomarker for early detection of gastric cancer. Cancer Lett 316:196-203. 326 Liu X, Xu T, Hu X, Chen X, Zeng K, Sun L, and Wang S. 2018. Elevated circulating miR-182 acts as a diagnostic 327 biomarker for early colorectal cancer. Cancer Manag Res 10:857-865. 10.2147/cmar.s158016 28 Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, Peck D, Sweet-Cordero A, Ebert BL, 328 Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, Peck D, Sweet-Cordero A, Ebert BL, Mak RH, Ferrando AA, 329 Downing JR, Jacks T, Horvitz HR, and Golub TR. 2005. MicroRNA expression profiles classify human 330 cancers. Nature 435:834-838. 10.1038/nature03702 331 Mirzaei H, Khataminfar S, Mohammadparast S, Sales SS, Maftouh M, Mohammadi M, Simonian M, Parizadeh SM, 332 Hassanian SM, and Avan A. 2016. Circulating microRNAs as Potential Diagnostic Biomarkers and 333 Therapeutic Targets in Gastric Cancer: Current Status and Future Perspectives. Curr Med Chem 23:4135- 334 4150. 335 Pan J, Zhou C, Zhao X, He J, Tian H, Shen W, Han Y, Chen J, Fang S, Meng X, Jin X, and Gong Z. 2018. A two- 336 miRNA signature (miR-33a-5p and miR-128-3p) in whole blood as potential biomarker for early diagnosis 337 of lung cancer. Sci Rep 8:16699. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed 356 Zhang X, Cui L, Ye G, Zheng T, Song H, Xia T, Yu X, Xiao B, Le Y, and Guo J. 2012a. Gastric juice microRNA- 357 421 is a new biomarker for screening gastric cancer. Tumour Biol 33:2349-2355. 10.1007/s13277-012-0497- 358 x 359 Zhang Y, Gong W, Dai S, Huang G, Shen X, Gao M, Xu Z, Zeng Y, and He F. 2012b. Downregulation of human 360 farnesoid X receptor by miR-421 promotes proliferation and migration of hepatocellular carcinoma cells. 361 Mol Cancer Res 10:516-522. 10.1158/1541-7786.mcr-11-0473 362 Zhao G, Xu L, Hui L, and Zhao J. 2015. Level of circulated microRNA-421 in gastric carcinoma and related 363 mechanisms. Int J Clin Exp Pathol 8:14252-14256. 364 Zheng RS, Sun KX, Zhang SW, Zeng HM, Zou XN, Chen R, Gu XY, Wei WW, and He J. 2019. Report of cancer 365 epidemiology in China, 2015. Zhonghua Zhong Liu Za Zhi 41:19-28. 366 Zhou S, Wang B, Hu J, Zhou Y, Jiang M, Wu M, Qin L, and Yang X. 2016. miR-421 is a diagnostic and prognostic 367 marker in patients with osteosarcoma. Tumour Biol. 368 Zhou X, Ji G, Chen H, Jin W, Yin C, and Zhang G. 2015. Clinical role of circulating miR-223 as a novel biomarker 369 in early diagnosis of cancer patients. Int J Clin Exp Med 8:16890-16898. 370 Manuscript to be reviewed PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed Figure 1(on next page) MiR-421 expression level between different groups MiR-421 expression level between different groups (A) Differential expression levels of reference miR-16( #p >0.05). (B) The ΔCt values of miR-421 between different groups (****p < 0.0001). (C) The levels (2 -ΔCt) of miR-421 between different groups (****p < 0.0001). The ΔCt values denote the normalized Ct value by subtracting the Ct value of miR-16 from that of miR-421. The lower ΔCt value means the higher level of miR-421 expression. The Mann- Whitney U test was used to determine statistical significance at the level of p <0.05. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) GC NC Pre 0 10 20 30 # # Normalized Ct value GC NC Pre -16 -8 0 8 **** **** **** GC NC Pre 0.01 0.1 1 10 100 1000 10000 100000 The levels (2-ΔCt) of miR-421 **** **** **** A B C PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed GC NC Pre # # Normalized Ct value GC NC Pre -16 -8 0 8 **** **** **** GC NC Pre 0.01 0.1 1 10 100 1000 10000 100000 The levels (2-ΔCt) of miR-421 **** **** **** B C PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed GC NC Pre 0 10 20 30 The raw Ct value of miR-16 # # Normalized Ct value GC NC Pre -16 -8 0 8 **** **** **** GC NC 0.01 0.1 1 10 100 1000 10000 100000 The levels (2-ΔCt) of miR-421 **** **** A B C PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed Normalized Ct value GC NC Pre -16 -8 0 8 **** **** **** GC NC Pre 0.01 0.1 1 10 100 1000 10000 100000 The levels (2-ΔCt) of miR-421 **** **** **** B C Manuscript to be reviewed Normalized Ct value GC NC Pre -16 -8 0 8 **** **** **** B Manuscript to be reviewed GC NC Pre 0.01 0.1 1 10 100 1000 10000 100000 The levels (2-ΔCt) of miR-421 **** **** **** C GC NC Pre 0 10 20 30 The raw Ct value of miR-16 # # A 8B Normalized Ct value Figure 2 The ROC curves of miR-421 and traditional tumor markers for GC (A) The AUC of miR-421was 0.981 (p <0.05). (B)The AUC of CEA, CA50, CA125, CA153 and CA211 were 0.672, 0.754, 0.645, 0.656 and 0.799, respectively (all p <0.05). Manuscript to be reviewed Figure 3 MiR-421 could distinguish early stage gastric cancer patients from healthy controls (A) MiR-421 was up-regulated in early stages (TNM stage Ⅰ-Ⅱ) gastric cancer patients compared to healthy controls (****p < 0.0001). (B) The ROC analysis for detection of early- stages gastric cancer patients from health controls using miR-421. *Note: Auto Gamma Correction was used for the image. This only affects the reviewing manuscript. See original source image if needed for review. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Note: Auto Gamma Correction was used for the image. This only affects the reviewing manuscript. See original source *Note: Auto Gamma Correction was used for the image. This only affects the reviewing manuscript. See original source image if needed for review. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed Figure 4 miR-421 had higher diagnostic value than traditional tumor markers for precancerous patients. miR-421 had higher diagnostic value than traditional tumor markers for precancerous patients. Differential expression levels of CA50(A) and CA211(B) between precancerous patients and healthy controls (*p < 0.01, **** p < 0.0001). The ROC analysis for detection of precancerous using miR-421(C) and traditional tumor markers CA50 and CA211(D). *Note: Auto Gamma Correction was used for the image. This only affects the reviewing manuscript. See original source image if needed for review. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed Manuscript to be reviewed Table 1(on next page) Table 1(on next page) Figure 5(on next page) Figure 5(on next page) MiR-421 expression levels in plasma of GC cases with different TNM stages, PLGC cases and NC controls. Mann-Whitney U test was used to determine statistical significance, p < 0.05 was accepted as statistically significant. * p < 0.05, * *** p < 0.0001, # p >0.05. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) NC Pre I II III,IV 0.01 0.1 1 10 100 1000 10000 100000 The levels (2-ΔCt) of miR-421 GC **** * **** **** # # PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed 0.01 0.1 1 10 100 1000 10000 100000 The levels (2-ΔCt) of miR-421 GC Mann-Whitney U test was used to determine other categorical variables with statistical significance at the level of p <0.05. ann-Whitney U test was used to determine other categorical variables with statistical significan t For these categorical variables, the p value were calculated by Student’s t-test. used to determine other categorical variables with statistical significance at the level of p <0.05 Manuscript to be reviewed Manuscript to be reviewed Table 1(on next page) Differential expression levels of traditional tumor markers in plasma of GC, Pre and health controls t For these categorical variables, the p value were calculated by Student’s t-test. Mann- Whitney U test was used to determine other categorical variables with statistical significance at the level of p <0.05. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed Table 1. Differential expression levels of traditional tumor markers in plasma of GC, Pre and health con GC Pre Pre vs NC NC GC vs NC n mean ± SD n mean ± SD p value n mean ± SD p value AFP 85 (26.14±133.56) 66 (3.32±1.72) 0.0505 45 (3.69±1.25) 0.3002 CEA 85 (32.23±128.76) 66 (1.82±1.39) 0.3122t 45 (2.08±1.20) 0.0011 Ferritin 80 (281.08±383.42) 61 (270.11±302.39) 0.1270t 45 (185.67±237.46) 0.9908 CA125 68 (59.22±92.72) 53 (12.50±7.78) 0.9363 45 (11.07±4.05) 0.0091 CA153 67 (13.44±17.70) 50 (10.41±5.45) 0.0065t 45 (13.67±5.83) 0.0049 CA199 83 (107.03±251.90) 57 (13.95±12.37) 0.1071 45 (14.58±8.22) 0.0705 CA211 62 (16.15±44.84) 40 (2.96±1.40) <0.0001 45 (2.09±0.48) 0.0002 CA242 66 (19.17±39.24) 45 (4.92±4.8) 0.2976t 36 (6.14±5.48) 0.1425 CA50 77 (38.01±99.02) 48 (8.18±5.97) 0.0101t 34 (4.91±4.46) 0.0089 CA724 75 (15.12±38.23) 44 (2.49±2.87) 0.2347t 26 (3.32±2.59) 0.2049 t For these categorical variables, the p value were calculated by Student’s t-test. Mann-Whitney U test was used to determine other categorical variables with statistical significance at the level of p <0.05. vels of traditional tumor markers in plasma of GC, Pre and health controls al expression levels of traditional tumor markers in plasma of GC, Pre and he Table 1. Differential expression levels of traditional tumor markers in plasma of GC, Pre and health controls Table 1. Differential expression levels of traditional tumor markers in plasma PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) mber of positive cases and negative cases. Manuscript to be reviewed Table 2(on next page) Table 2(on next page) The diagnostic value of miR-421 and tumor markers for GC AUC: Area under curve; FPR: False positive rate; FNR: False negative rate. Positive was defined as >cut-off value; Negative was defined as < cut-off value; The sensitivity, specificity, FPR, FNR, accuracy and Youden index were calculated based on the number of positive cases and negative cases. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed Manuscript to be reviewed Table 2. The diagnostic value of miR-421 and tumor markers for GC. Cut-off Sensitivity (%) Specificity (%) FPR (%) FNR (%) Accuracy (%) Youden index (%) AUC p miR-421 3.23 96.67 95.56 4.44 3.33 96.30 92.22 0.981(0.942 - 0.997) <0.0001 CEA 1.94 67.06 66.67 33.33 32.94 66.93 33.73 0.672(0.584 - 0.752) 0.0002 CA125 18.75 45.59 100.00 0 54.41 67.26 45.59 0.645(0.549 - 0.732) 0.0055 CA153 11.31 68.66 62.22 37.78 31.34 66.07 30.88 0.656(0.561 - 0.743) 0.0034 CA211 2.2 80.65 95.65 4.35 19.35 86.96 76.30 0.799(0.711 - 0.871) <0.0001 CA50 7.96 55.84 85.29 14.71 44.16 64.86 41.14 0.754(0.663 - 0.831) <0.0001 AUC: Area under curve; FPR: False positive rate; FNR: False negative rate. Positive was defined as >cut-off value; Negative was defined as < cut-off value; The sensitivity, specificity, FPR, FNR, accuracy and Youden index were calculated based on the number of positive cases and negative cases. Table 2. The diagnostic value of miR-421 and tumor markers for GC. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Table 3(on next page) Table 3(on next page) The comparison of ROC curves between miR-421 and tumor markers ROC curves comparisons were analyzed by using MedCalc statistical software with statistical significance at the level Of p <0.05. ROC curves comparisons were analyzed by using MedCalc statistical software with statistical significance at the level Of p <0.05. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed 1 Table 3. The comparison of ROC curves between miR-421 and tumor markers 2 ROC curves comparisons were analyzed by using MedCalc statistical software with statistical significance at 3 the level of p <0.05. CEA CA125 CA153 CA211 CA50 ROC curves comparison Z p Z p Z p Z p Z p miR-421 2.418 0.0156 1.896 0.0580 2.021 0.0432 3.975 0.0001 3.343 0.0008 1 Table 3. The comparison of ROC curves between miR-421 and tumor markers 2 ROC curves comparisons were analyzed by using MedCalc statistical software with statistical significance at 3 the level of p <0.05. 3 the level of p <0.05. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed Table 4(on next page) Table 4(on next page) The diagnostic value of miR-421 and tumor markers for Pre. The sensitivity, specificity, FPR, FNR, accuracy and Youden index were calculated based on the number of positive cases and negative cases. AUC, FPR and FNR, Positive and Negative as stated in Table 2. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed Manuscript to be reviewed Table 4. The diagnostic value of miR-421 and tumor markers for Pre. Cut-off Sensitivity (%) Specificity (%) FPR (%) FNR (%) Accuracy (%) Youden Index (%) AUC p miR-421 3.23 66.29 95.56 4.44 33.71 76.37 61.85 0.872(0.804 - 0.924) <0.0001 CA153 11.81 72.00 60.00 40.00 28.00 66.32 32.00 0.679(0.576 - 0.772) 0.0012 CA211 2.2 85.00 95.56 4.44 15.00 90.56 80.56 0.844(0.750 - 0.914) <0.0001 CA50 7.26 52.08 82.35 17.65 47.92 64.63 34.44 0.677(0.564 - 0.776) 0.0034 The sensitivity, specificity, FPR, FNR, accuracy and Youden index were calculated based on the number of positive cases and negative cases. AUC, FPR and FNR, Positive and Negative as stated in Table 2. Table 4. The diagnostic value of miR-421 and tumor markers for Pre. and negative cases. AUC, FPR and FNR, Positive and Negative as stated in Table 2. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Table 5(on next page) Table 5(on next page) The comparison of ROC curves among miR-421 and tumor markers for Pre ROC curves comparisons were analyzed by using MedCalc statistical software with statistical significance at the level of p <0.05. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed Table 6(on next page) Manuscript to be reviewed 1 Table 5. The comparison of ROC curves among miR-421 and tumor markers for Pre 2 3 4 5 ROC curves comparisons were analyzed by using MedCalc statistical software with 6 statistical significance at the level of p <0.05. 7 8 CA153 CA211 CA50 ROC curves comparison Z p Z p Z p miR-421 2.211 0.027 4.414 < 0.0001 1.997 0.0458 Table 5. The comparison of ROC curves among miR-421 and tumor markers for Pre PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Table 6(on next page) The levels (2 -ΔCt)of miR-421 between different groups ap represents the comparison of Pre between GC with Ⅰ,Ⅱ,ⅢandⅥ, respectively. bp represents the comparison of Ⅰ between GC withⅡ,ⅢandⅥ, respectively PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed Manuscript to be reviewed 1 Table 6 The levels (2-ΔCt)of miR-421 between different groups 2 3 4 5 6 7 8 9 10 aP represents the comparison of Pre between GC with Ⅰ,Ⅱ,ⅢandⅥ, respectively. 11 bP represents the comparison of Ⅰ between GC withⅡ,ⅢandⅥ, respectively n miR-421 in plasma Sd aP value bP value NC 45 0.45(0.16,37.0) 5.50 Pre 89 7.57(0.06,1674.25) 229.36 Ⅰ 15 59.97(8.54,702.0) 187.37 <0.0001 Ⅱ 9 53.60(3.3,1577.80) 510.76 0.0277 0.3786 ⅢandⅥ 63 40.72(0.95,22511.89) 3020.8 <0.0001 0.3325 1 Table 6 The levels (2-ΔCt)of miR-421 between different groups 10 aP represents the comparison of Pre between GC with Ⅰ,Ⅱ,ⅢandⅥ, respectively. 11 bP represents the comparison of Ⅰ between GC withⅡ,ⅢandⅥ, respectively 10 aP represents the comparison of Pre between GC with Ⅰ,Ⅱ,ⅢandⅥ, respectively. PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed Table 7(on next page) Table 7(on next page) Table 7(on next page) The relationships between the expression levels of miR-421(2 −ΔCt) in plasma and clinicopathological factors of patients with precancerous gastric lesions and gastric cancer PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019) Manuscript to be reviewed Manuscript to be reviewed Manuscript to be reviewed 1 Table 7 The relationships between the expression levels of miR-421(2−ΔCt) in plasma and 2 clinicopathological factors of patients with precancerous gastric lesions and gastric cancer 3 4 miR-421 level (2−ΔCt) Characteristics Case No. High Low χ2 test P value GC Total cases 90 45 45 Age(years) <60 ≥60 30 60 11 34 19 26 3.2 0.0736 Gender Male Female 68 22 32 13 36 9 0.9626 0.3265 TNM stage Ⅰ Ⅱ Ⅲ Ⅵ 16 9 23 40 11 5 12 17 5 4 11 23 3.261 0.3531 Diferentiation degree High and moderate Poor 25 46 11 24 14 22 0.433 0.5105 Histological type Adenocarcinoma(A) Mucinous carcinoma(M) Signet ring cell carcinoma(S) A with S 65 2 5 11 31 0 3 6 34 2 2 5 2.324 0.5079 Pre Total cases 89 45 44 Age(years) <60 ≥60 41 48 22 23 19 25 0.2916 0.5892 Gender Male Female 46 43 22 23 24 20 0.2851 0.5934 Histological type Intestinal metaplasia Atypical hyperplasia and other type 82 7 41 4 41 3 0.1316 0.6939 HP infection status HP (-) HP (+) 38 16 20 6 18 10 1.033 0.3095 Manuscript to Table 7 The relationships between the expression levels of miR-421(2−ΔCt) in plasma and 2 clinicopathological factors of patients with precancerous gastric lesions and gastric cancer 1 Table 7 The relationships between the expression levels of miR-421(2−ΔCt) in plas PeerJ reviewing PDF | (2018:10:31786:2:0:NEW 16 Apr 2019)